MICROBIAL ECOLOGY
AND
BIOTECHNOLOGY BRANCH
U.S. ENVIRONMENTAL PROTECTION AGENCY
SABINE ISLAND,
GULF BREEZE, FLORIDA
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PROGRAM
OF THE
ANNUAL BIOTECHNOLOGY RESEARCH REVIEW
JULY 8-9, 1986
U.S. Environmental Protection Agency
Microbial Ecology and Biotechnology Branch
Sabine Isand, Gulf Breeze, Florida
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CONTENTS
I. Participants And Observers
II. Schedule
III. Biotechnology Programs
Extramural
A. Alexander
B. Chakrabarty
C. Colwell
D. Miller
E. 01 sen
F. Sayler
G. Stahl
Inhouse
H. Barkay
I. Chatterjee
J. Cuskey
K. Devereux
L. Genthner
M. McCarthy
N. Walter
IV. Biodegradation Programs (0.)
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I.
BIOTECHNOLOGY RESEARCH REVIEW PARTICIPANTS AND OBSERVERS
PARTICIPANTS
Alexander, Martin
Barkay, Tamar
Bourquin, Al U.
Chatterjee, Oeb
Colwell, R1ta K.
Cuskey, Stephen
Genthner, Fred
Jones, Garth W.
Lenski, Richard E.
Miller, Robert V.
01 sen, Ronald H.
Paul, John
Pritchard, Hap P.
Sayler, Gary S.
Stahl, David A.
Walter, Ron
ORGANIZATION
Cornell University
Dept. of Agronomy
EPA/ERL
EPA/ERL
EPA/ERL
University of Maryland
Dept. of Microbiology
EPA/ERL
EPA/ERL
ADDRESS
708 Bradfield Hall
Ithaca, NY 14853
Sabine, Island
Gulf Breeze, FL 32561
Sabine, Island
GUlf Breeze, FL 32561
Sabine Island
Gulf Breeze, FL 32561
College Park, MD 20742
Sabine Island
Gulf Breeze, FL 32561
Sabine Island
Gulf Breeze, FL 32561
University of Michigan 6779 Medical Science Bldg. II
Microbiology & Immunology Dept. Ann Arbor, MI 48109
University of California
Dept. of Ecol. & Evol. Biology
Loyola University Medical Center
Dept. of Biochem. & Biophys.
University of Michigan
Dept. of Microbiology
University of South Florida
Irvine, California 92717
2160 S. First Avenue
Maywood, IL 60513
222 Research Adm, Bldg.
Ann Arbor, Mich. 48109
140 7th Avenue, South
Dept. of Marine Science & Biology St. Petersburg, Fl. 33701
EPA/ERL
University of Tennessee
Microbiology/Ecology Dept.
University of Illinois
Dept. of Vet. Pathobiology
EPA/ERL
Sabine Island
Gulf Breeze, Fl 32561
583 Old Dabney Harbor
Knoxville, TN 37996
2001 South Lincoln Ave.
Urbana, IL 61801
Sabine Island
Gulf Breeze, FL 32561
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I. Cont.
BIOTECHNOLOGY RESEARCH REVIEW PARTICIPANTS AND OBSERVERS
OBSERVERS
Chapman, Peter J.
Devereux, Richard
Frederick, Robert J.
Levin, Morris A.
McCarthy, Susan
MacDonell, Michael
Mueller, Len
Nelson, Michael
Schneider, William
Sharak Genthner, Barbara
ORGANIZATION
EPA/ERL
EPA/ERL
OTS
ORD
EPA/ERL
University of Maryland
Dept. of Mtcrob.tology
EPA/ERL
EPA/ERL
OPP
EPA/ERL
ADDRESS
Sabine Island
Gulf Breeze, Fl 32561
Sabine Island
Gulf Breeze, Fl 32561
401 M Street, S.W.,
Washington, DC 20460
401 M Street. S.W.,
Washington, DC 2Q46Q
Sabine Island
Gulf Breeze, Fl 32561
College Park, MD 20742
* Sabine Island
Gulf Breeze, Fl 32561
Sabine Island
Gulf Breeze, Fl 32561
401 M Street, S.W.,
Washington, DC 20460
Sabine Island
Gulf Breeze, Fl 32561
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II.
BIOTECHNOLOGY RESEARCH REVIEW SCHEDULE
July 7 Arrive Pensacola - Reservations Holiday Inn - Bay Beach (904 932-2214)
8:00 PM P.I.'s meet with Dr. Bourquin at Holiday Inn and go to Dinner.
July 8
8:00 AM Introduction by Al W. Bourquin
DETECTION. SURVIVAL. GENETIC STABILITY - MICROBIOLOGICAL APPROACHES
8:15 AM Colwell - Survival, Modification and Effects of Genetically Engineered
Microorganisms Released to the Aquatic Environment
9:15 AM Break
9:30 AM Alexander - Fate in Natural Environments of New Genotypes
Developed by Genetic Engineering'
.10:30 AM Miller - Genetic Transfer in Aquatic Environments
11:30 AM Genthner - Genetic Exchange Potential of Natural Aquatic
Bacterial Isolate?
12:15 PM Lunch
DETECTION. SURVIVAL. GENETIC STABILITY - MOLECULAR GENETIC APPROACHES
1:00 PM 01 sen - Development, Characterization and Utilization of
Bacterial Benchmark PlasmuTs
2:00 PM Chatterjee - An Approach to Assess the Fate and Effects of
Genetically Engineered Microorganisms in Simulated
Natural Environments
2:45 PM Break
3:00 PM Cuskey - The Effect of Conditional Lethal Genetic Determinants
on Cell Survival in Selected Environments
3:45 PM Walter - The construction of Bacillus Subtilus Strains Containing
a Marker Sequence allowing Quantitative Enumeration in
Environmental Cultures
4:30 PM Paul/Steward - The Role of Extracellular DNA in the Dissemination
of Recombinant DMA in Aquatic Environments
5:00. PM Jones - Plasmid Transmitsibility in Bacterial Populations of
the Air-Water Microfilm
5:30 PM Cocktails - Holiday Inn
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July 9
Ecological Effects Methods Research
8:00 AM Barkay - Fate and Effects of Mercury Reducing Bioengineered
Organisms in Estuarine Environments
9:00 AM
10:00 AM
11:00 AM
11:30 AM
12:00 PM
1:00 PM
Stahl - The use of rRNA Sequences to Characterize Natural
Microbial Populations
Sayler - Genetic Approaches for Determining Persistence and
Effects of Introduced Species
Lenski - Genetic Factors Influencing the Ecological Fate of
Two Model Recombinant Microorganisms
Pritchard - Development of Aquatic Test Systems for Assessing
Fate and Effects of GEM's
Lunch
Discussion
- Future directions
- Change in scope
- Year-end report
- Protocol development
3:00 PM Program Office
- Relationship o.f 1st years's research to P.O. needs
4:00 PM - Adjourn
July 10
Biodegradation Research Discussions
8:30 AM Informal discussions in Conference Room with Drs. Pritchard,
Chapman, Nelson, Sharak-Genthner, and other scientists from
GB/ERL.
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III.
BIOTECHNOLOGY RESEARCH PROGRAMS
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATS : 7/ 2/86
WORK PLAN CODE : Q283
OU CODE & TITLE : L194 Chemical Testing and Assessment
OBJECTIVE CODE & TITLE : G Develop 5, Validate Improved Risk Assess
PPA CODE & TITLE ' 02 Environmental Aspacts of Biotechnology
PROJECT CODE L TITLE : 24
WORK PLAN TYPE : COOP
INVESTIGATOR : A Bourquin PRINCIPAL INVESTIGATOR: M. Alexander
PHONE NO : FTS 636-9011
WORK PLAN TITLE : Fate in Natural Environment of New Genotypes Developed by
Genetic Engineering
WORK PLAN OBJECTIVE : a) Determine the potential for growth in natural waters
of bacteria likely to be important in genetic engineering.
b) Establish conditions that permit the survival and-or growth of bacteria
that do not usually survive or grow in natural waters.
c) Develop generalizations on the physiological traits and environmental
conditions that are responsible for, or are correlated with, the inability
of bacteria to persist or multiply in natural waters.
d) Develop a simple laboratory procedure for the prediction of survival
and growth of bacteria in natural environments and validate that procedure
by field trials.
WORK PLAN APPROACH : Bacterial strains anticipated to be bioengineered and
released in the environment will be studied. Antibiotic-resistant mutants
of these strains will be add-ed to simple experimental systems representing
aquatic environments. Test bacteria will be enumerated by selective
plating. The effects of starvation, competition, predation and parasitism
on survival and growth will ba examined. Variable aspects of these
phenomena will be investigated in depth to obtain understanding of
principles jjoverning survival and growth of GEMs.
STATUS : On Schedule
ACCOMPLISHMENTS : The survival and growth of bacteria in natural waters may be
controlled by many factors, including: (a) orotozoa, Cb) production of
toxins by organis/us during incubation, Cci 3dellovibrio» (d) lytic
organisns-lysis• Ce) bactariophagas, and (f) competition for nutrients.
Two survival patterns ware observed. The first was a rapid decline
followed by a pariod of slew or no decline. The second was a decline to
levels oelow detection limits within tha first 5 to 10 days. These results
indicate that survival uias affected by a factor which was removed by
autoclaving or filtering the water through 3.2 urn filters. Thus, toxins
are not responsible for the lack of survival. Sinca toxins have been
previously reported by us to be a factor potentially affecting survival of
bacteria in lake water (Klein and Alexander, in press), it was necessary tc
assess whether toxins produced by indigenous organisms during incubation
might have sean responsible for the decline. To test this hypothesis, lake
water was collected and incubated for 2 days at 30 degrees C in the dark or
a rotary shaker operating at 100 rpm. The incuoated water was then used tc
examine the survival of Pseudomonas sp. L2 and Klebsiella pneumoniae Kno.
The results obtained in this experiment also showad the absence of a role
for toxins. That survival was enhanced after filteration indicated that
bacteriophages were probably not responsible for the decline. The survival
of tha bacteria in buffer indicated that competition for nutrients was
unlikely because an organism losing in a competitive interaction would
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ENVIRONMENTAL RESEARCH LA3C3AT03Y
GULF BREEZE , FLORIDA 32361
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q283
decline because of its starvation. Bdellovibrio was not present in these
waters (data not shown)* so they do not cause the decline.
Protozoa mere the most important of the factors tasted. However* only son
of the organisms tested ware found to be sensitive to grazing pressure by ^ -•*
protozoa. McCambridge and McMeekin (1980) found that protozoa had tha
greatest affect on E. coli in estuarine mater during tha first 2 days. Tr~
organises uuhich were sensitive to graring pressure mere also the same
organisms that exhibited the second survival oattern.
Growth of all the organisms tested except B. subtilis was most apprec iably-
affacted by the addition of carbon. Growth of strain L2 was also enhancet
by tha inhibition of protozoa when nutrients were added to the lake watari
However* strain L2 was not affected by protozoa in the unamended lake
water. This indicates that growth or growth rate may be related to
suppression by protozoa. Inorganic nutrients mere limiting only whan
additional carbon was added.
The effect of seasonaTity was tested for strain L2. Ths only seasonal
difference observed was the concentration achieved in the "no deletion"
water*
These results indicate that competition for nutrients and predation ,_.
pressure exerted by protozoa are major factors that affect the growth and
survival of bacteria in natural waters. Although tha organisms tested were
able to survive up to 15 days in ouffer containing no added carbon source.
the nutriant conditions in lake water may be complicated by the presence f
other organises and-or the physical and chemical environment. Protozoa i_J
seem to be selectively grazing. Tha reason for this selectivity mill b?
investigatedfurther.
MILESTONES (DATES) : TARGET REVISED REVISED REVISED ACTU
Final Report on the Effects of 06/33
Biological Factors of the
Survival of GEM's in Aquatic
Ecosystems (74348)
Manuscript "Bacterial Inhibitors 06/36 06/r">
in Lake Water" LJ
-1— j
-l
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QUARTERLY REPORT
June 15, 1986
Martin Alexander, Cornell University
(Staff: P. Scheuerman, Ph.D, J. P. Schmidt, J. Ellis)
The factors affecting survival and growth of bacteria in natural
waters are not well understood. Interest in this subject has
increased because of. concern with the deliberate or inadvertent
release of genetically engineered organisms into natural ecosystems.
To date, nearly all studies on the survival of bacteria in natural
waters have been on bacteria of public health significance. Little
work has been performed on the factors governing survival and
growth of individual species in natural environments. Without an
understanding of such factors, it is difficult to assess the potential
problems and risks associated with the release of novel
microorganisms into the environment.
In this report, we describe studies initiated to identify the
factors that affect survival and growth of bacteria in samples from
aquatic environments.
MATERIALS AND METHODS
Microcosms. Experiments were performed using acid-
washed 250-ml Erlenmeyer flasks. The flasks were washed for a
minimum of 2 h in No-chromix (a metal-free oxidizer) and
subsequently rinsed three times in tap water and then three times in
distilled water. The flasks were covered with foam plugs, which
were then covered with foil and autoclaved for 30 min. Flasks
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containing 100 ml of test suspension (lake water or buffer) were
incubated in the dark at 30°C on a rotary shaker operating at 100
rpm.
Bacteria. Cultures of Escherichia coli C-3000, Bacillus subtilis
CU 155 (an asporogenous strain), Micrococcus flavus 731, Klebsiella
pneumoniae Kno 649, Pseudomonas sp. B4 (719), and Pseudomonas
sp. L2 were maintained on Trypticase soy agar slants at 4°C or room
temperature. Rhizobium phaseoli 574 was maintained on slants of
yeast extract-mannitol agar (YEM) at 4°C. Cultures were prepared
for experimental use by inoculation into 50 ml of Trypticase soy
broth (TSB) or YEM containing the appropriate antibiotics and
incubation of the cultures at 30°C until the culture reached the late
logarithmic phase. The cells were harvested by washing three times
in either autoclaved lake water, filter-sterilized lake water, or sterile
buffer. The washing solution was chosen as appropriate for the
experiment in which the organisms were to be used. Centrifugation
was performed at 8,000 rpm for 10 min at 4°C. The cells were then
inoculated the test water at 104 to 106 CFU/ml. Bacteria were
enumerated using the drop-plate technique (Hoben and
Somasegaran, 1982). The media were supplemented with the
appropriate concentration of antibiotics.
B. subtilis was grown on Trypticase soy agar (TSA, 15 g of
Trypticase soy broth and 15 g of agar (per liter) containing 50 ng of
rifampicin and 1000 ^g of streptomycin per ml. Pseudomonas sp. L2
was grown on TSA containing 1000 ng of streptomycin and 50 ng of
erythromycin per ml. Pseudomonas sp. B4 was grown on TSA
containing 1000 ng of streptomycin, 50 ng of erythromycin, and 10
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Hg of brilliant green per ml. M. flavus was grown on TSA containing
1000 h*g of spectinomycin and 100 Kg of erythromycin per ml. R.
phaseoli was grown on YEM containing 1000 ng of streptomycin and
50 n g of erythromycin per ml. All these media were also
supplemented with 250 ng of cycloheximide per ml. E. coli was
grown on eosin methylene blue agar without antibiotics.
Snore counts. Spores were counted by incubating the
appropriate dilutions at 80°C for 10 min, and subsequently plating
the resulting suspensions.
Water samples. Water samples were used within 1 h of
collection. Water was obtained from Beebe Lake, Ithaca, N.Y. The pH
of the lake water ranged from 7.4 to 8.0.
Survival experiments; The survival of bacteria was tested
in untreated, autoclaved, and filter-sterilized lake water. Water was
autoclaved 30 min at 121 °C. Water was filter sterilized through 8.0,
0.45, and 0.2 ^m membrane filters. The lake water samples were
assayed for total bacteria (by plate counts) and the test bacterium at
various intervals for 15 days. Protozoa were inhibited by the
addition of 250 Kg of cycloheximide and 30 pg of nystatin per ml to
the appropriate flasks.
Growth experiments. Nutrients were added to a final
concentration of 1 mg of glucose, 0.14 mg of K2HPO4, and 0.29 mg of
NH4NO3 per ml. Protozoa were inhibited as described above.
RESULTS
Survival
Figures 1 and 2 show the survival of Pseudomonas sp. L2,
Pseudomonas sp. B4, R. phaseoli, M. flavus, E. coli, and total bacteria
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in autoclaved, filter-sterilized, or nonsterile lake water. All the test
bacteria declined in the nonsterile lake water, but the cell densities
remained essentially constant in the autoclaved and filter-sterilized
lake water. M. flavus and E. coli were detectable at day 5 but not at
day 10. B. subtilis was not detectable after 1 day in any of the
treatments. Spores of B. subtilis were also not detected.
The survival patterns of strain L2 and Klebsiella pneumoniae
were not influenced by pre-incubation of the lake water (Figure 3).
In this instance, the lake water was incubated in flasks for 2 days
before adding the test species.
Of five organisms tested in buffer to which was added no
carbon source, only B. subtilis did not survive (Figure 4).
Figures 5, 6, and 7 show the effect of eucaryotic inhibitors on
the survival of Pseudomonas sp. L2, E. coli, and K. pneumoniae.
Included in these figures are the counts of active, trophic states of
protozoa (counted microscopically). The protozoa were eliminated by
the inhibitors within the first day of incubation in the waters
containing Pseudomonas sp. L2 or E. coli, and after the first day in
the waters containing K. pneumoniae. Pseudomonas sp. L2 did not
show any difference in survival pattern in the presence or absence of
protozoa. However, both E. coli and K. pneumoniae maintained
higher densities of cells in the inhibitor-amended waters, in which
the protozoa were markedly suppressed.
Growth
Figures 8 and 9 show the response of Pseudomonas sp. L2 and
Pseudomonas sp. B4 to the addition of nutrients (C, N, and P) to
Beebe Lake water. For both bacteria, a growth response in
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unamended lake water was evident. The amount of C added was
expected to support the growth of 108 to 109 cells per ml. This was
achieved with both bacteria. However, Pseudomonas sp. L2 actually
reached a density higher than expected, whereas Pseudomonas sp.
B4 only reached the minimum level expected.
Figures 10 and 11 show the response of Pseudomonas sp. L2
and Pseudomonas sp. B4 to the addition of eucaryotic inhibitors to
the nutrient-amended lake water. Pseudomonas sp. L2 grew more
extensively and declined later in the inhibitor-treated water than in
lake water with active protozoa. Pseudomonas sp. B4 populations
were the same with and without the inhibitors until the 7th day.
Deletion experiments. Figures 12 through 15 show the
response of Pseudomonas sp. L2, Pseudomonas sp. B4, R. phaseoli,
i
and B. subtilis in nutrient-amended (C, N, and P sources) inhibitor-
treated lake water. Water samples were also amended with the
complete mixture (inhibitors, C, N, and P) minus either carbon,
inorganic nutrients (N and P), or eucaryotic inhibitors. The response
of Pseudomonas sp. L2 in water collected during the winter and the
spring is presented in Figure 12. With no carbon addition,
Pseudomonas sp. L2 was unable to grow even when the protozoa
were suppressed. In the treatments which contained carbon,
nitrogen, and phosphorus, a decreased growth was evident if the
eucaryotic inhibitors were not added. Pseudomonas sp. B4
responded in a similar manner as strain L2, except that it did not
respond to the deletion of eucaryotic inhibitors from the waters
(Figure 13).
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R. phaseoli grew in lake water amended only with carbon
(Figure 14). It did not respond to the deletion of eucaryotic
inhibitors until after 2 days. In contrast, B. subtilis did not grow in
lake water receiving any of the treatments (Figure 15). A decrease
in the rate of its decline was evident in the nutrient-amended water.
The spore density remained constant at less than 100/ml for the first
2 days, and none was detectable at 3 days. Figure 16 shows the
response of B subtilis to the addition of 1 or 0.01 mg of glucose per
ml in a buffered mineral salts solution. These results indicate that B.
subtilis was able to use a concentration of glucose 100-fold lower
than that added to lake water. However, spores were only formed
after 5 days in the buffer-amended with 1 mg of glucose per ml.
DISCUSSION
The survival and growth of bacteria in natural waters may be
controlled by many factors, including: (a) protozoa, (b) production of
toxins by organisms during incubation, (c) Bdellovibrio, (d) lytic
organisms/lysis, (e) bacteriophages, and (f) competition for nutrients.
Two survival patterns were observed. The first was a rapid
decline followed by a period of slow or no decline. The second was a
decline to levels below detection limits within the first 5 to 10 days.
These results indicate that survival was affected by a factor which
was removed by autoclaving or filtering the water through 0.2-^m
filters. Thus, toxins are not responsible for the lack of survival.
Since toxins have been previously reported by us to be a factor
potentially affecting survival of bacteria in lake water (Klein and
Alexander, in press), it was necessary to assess whether toxins
produced by indigenous organisms during incubation might have
-------
been responsible for the decline. To test this hypothesis, lake water
was collected and incubated for 2 days at 30°C in the dark on a
rotary shaker operating at 100 rpm. The incubated water was then
used to examine the survival of Pseudomonas sp. L2 and Klebsiella
pneumoniae Kno. The results obtained in this experiment also
showed the absence of a role for toxins. That survival was enhanced
after filtration indicated that bacteriophages were probably not
responsible for the decline. The survival of the bacteria in buffer
indicated that competition for nutrients was unlikely because an
organism losing in a competitive interaction would decline because of
its starvation. Bdellovibrio was not present in these waters (data not
shown), so they do not cause the decline.
Protozoa were the most important of the factors tested.
However, only some of the organisms tested were found to be
sensitive to grazing pressure by protozoa. McCambridge and
McMeekin (1980) found that protozoa had the greatest effect on E.
coli in estuarine water during the first 2 days. The organisms which
were sensitive to grazing pressure were also the same organisms that
exhibited the second survival pattern.
Growth of all the organisms tested except B. subtilis was most
appreciably affected by the addition of carbon. Growth of strain L2
was also enhanced by the inhibition of protozoa when nutrients were
added to the lake water. However, strain L2 was not affected by
protozoa in the unamended lake water. This indicates that growth or
growth rate may be related to suppression by protozoa. Inorganic
nutrients were limiting only when additional carbon was added.
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8
The effect of seasonality was tested for strain L2. The only
seasonal difference observed was the concentration achieved in the
"no deletion" water.
These results indicate that competition for nutrients and
predation pressure exerted by protozoa are major factors that affect
the growth and survival of bacteria in natural waters. Although the
organisms tested were able to survive up to 15 days in buffer
containing no added carbon source, the nutrient conditions in lake
water may be complicated by the presence of other organisms
and/or the physical and chemical environment. Protozoa seem to be
selectively grazing. The reason for this selectivity will be
investigated further.
SUMMARY
The survival of several strains of bacteria was studied in
autoclaved, filter-sterilized, and untreated lake water. All species
tested (Micrococcus flavus, Bacillus subtilis, Klebsiella pneumoniae,
Escherichia coli, Rhizobium. phaseoli, and two Pseudomonas spp.)
declined in untreated lake water. Two survival patterns were
observed. The first was a rapid decline followed by a period of slow
or no decline. The second was a decline below detection limits within
the first 5 to 10 days. Only Bacillus subtilis declined in the
autoclaved or filter sterilized lake water, indicating that toxins,
bacteriophages, or other soluble components or nonfilterable agents
were not responsible for the decline in the untreated lake water.
Growth of all species except for Bacillus subtilis was enhanced by the
addition of nutrients. Survival and growth of some of the bacteria
were found to be affected by protozoa.
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REFERENCES
Hoben, H. L, and P. Somasegaran. 1982. Comparison of the pour,
spread, and drop plate methods for pre-sterilized peat. Appl.
Environ. Microbiol. 44:1246-1247.
McCambridge, L, and T. A. McMeekin. 1980. Relative effects of bac-
terial and protozoan predators on survival of Escherichia coli
in estuarine water samples. Appl. Environ. Microbiol. 40:907-
911.
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10
LIST OF FIGURES
Figure 1. Survival of Pseudomonas sp. L2, Pseudomonas sp. B4, and
R. phaseoli in Beebe Lake water, (a) Summer, (b) Winter.
Figure 2. Survival of M. flavus, E. coli, and total bacteria in Beebe
Lake water.
Figure 3. Survival of Pseudomonas sp. L2 and K. pneumoniae in
pre-incubated Beebe Lake water.
Figure 4. Survival of bacteria in buffered mineral salts solution
without an added carbon source.
Figure 5. The effect of protozoa on the survival of Pseudomonas sp.
L2 in Beebe Lake water.
Figure 6. The effect of protozoa on the survival of E. coli in Beebe
Lake water.
Figure 7. The effect of protozoa on the survival of K. pneumoniae in
Beebe Lake water.
Figure 8. Growth of Pseudomonas sp. L2 in nutrient-amended and
unamended Beebe Lake water.
Figure 9. Growth of Pseudomonas sp. B4 in nutrient-amended and
unamended Beebe Lake water.
Figure 10. The effect of protozoa on the growth of Pseudomonas sp.
L2 in nutrient-amended Beebe Lake water.
Figure 11. The effect of protozoa on the growth of Pseudomonas sp.
B4 in nutrient-amended Beebe Lake water.
Figure 12. Growth of Pseudomonas sp. L2 in Beebe Lake water
amended with nutrients and eucaryotic inhibitors and in
water in which carbon, inorganic nutrients, or inhibitors
were not added.
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11
Figure 13. Growth of Pseudomonas sp. B4 in Beebe Lake water
amended with nutrients and eucaryotic inhibitors and
in water in which carbon, inorganic nutrients, or inhibi-
tors were not added.
Figure 14. Growth of R. phaseoli in Beebe Lake water amended with
nutrients and eucaryotic inhibitors and in water in which
carbon, inorganic nutrients, or inhibitors were not added.
Figure 15. Growth of B. subtilis in Beebe Lake water amended with
nutrients and eucaryotic inhibitors and in water in which
carbon, inorganic nutrients, or inhibitors were not added.
Figure 16. Growth of Bacillus subtilis in buffered mineral salts solu-
tion amended with 1 or 0.01 mg of glucose per ml.
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Gulf Breeze Environmental Research Laboratory
Research Plan
Task Q
Title: Construction and Detection of Genetically Engineered Microorganisms
Investigator(s): A.M. Chakrabarty
Project Officer: A. Bourquin
Branch: Mlcrobial Ecology and Biotechnology
Project Code and Title: 24 Ecological Consequences of Genetically Engineered
Microorganisms
OU Code: L104 Toxics
APPROVALS: (signature) (Date)
Branch Chief
Lab Director
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RESEARCH PLAN
OBJECTIVE:
Develop appropriate bacterial strains and vectors that can be used
for determining the survival and fate of novel strains and their genotypes
in aquatic environments.
RATIONALE;
As we develop new strains, in EPA - Cincinnati laboratory, capable
of utilizing hazardous chemicals such as chlorinated dioxins or
trichloroethylene, their effectiveness as well as their survival will be
studied in Cincinnati in soil experiments as described for 2,4,5-T degrading
£. cepacia AC1100 (30). Such strains will be tested for their survival
and effectiveness in microcosm tests at the EPA Gulf Breeze laboratory.
EXPERIMENTAL DESIGN OR APPROACH:
In general, two types of programs will be pursued at Gulf Breeze:
(i) Looking for specific sequences that are part of 2,4,5-T degradative
genes in P_. cepacia AC1100 that are foreign to Pseudomonas and therefore
allow specific detection only in those Pseudomonas cells that harbor
them. Thus if P_. cepacia AC1100 with a unique sequence RS-1100-I is
released into the environment, and if RS-1100-I sequence is absent in any
other pseudomonads, then the proportion of AC1100 among all the other
Pseudomonas can be quickly determined by a single colony hybridization
experiment. If during environmental dissemination, this unique sequence
is mobilized to other bacteria, pseudomonads or non-pseudomonads, then
such bacteria can initially be recognized by colony hybridization and
later tested for 2,4,5-T growth or other characteristic genetic markers
present in AC1100 (such as chromosomal Nalr or Rifr mutation) to confirm
-------
if it is AC1100 or not. Characterization of such unique sequences should
therefore, permit screening for survival as well as detection of novel
genotypes in the environment. The other approach to be pursued at Gulf
Breeze would be to construct new plasmids which would contain strong
selectable markers as well as unique sequences (such as eukaryotic
sequences) that are normally absent in natural microbial community. The
selectable markers would be of two types: (i) antibiotic resistance
markers that are normally readily expressed in different microorganisms
and which would allow detection of a large number of microorganisms on
the primary selection plate; (11) the other type of selectable marker
will be the degradation of a chlorinated compound such as 3-chlorobenzoic
acid or 2,4-D which is not expressed in all microorganisms because of the
unique specificity of its promoters. This means that colonies that will
grow initially on antibiotic supplemented plate could be tested for growth
with the chlorinated substrate such as 2,4-D or 3-chlorocatechol. If the
antibiotic resistant colonies grow with either of the two substrates,
that will mean that the novel genotype-containing plasmid vector is
present in these cells. If the antibiotic resistant colonies do not grow
with the chlorinated compounds, that may mean that either these colonies
do not harbor the novel vector plasmid, or they harbor the vector plasmid
but cannot express the 2,4-D degradatlve genes because of lack of
recognition of the promoter. This can be resolved by conducting a colony
hybridization experiment using a segment of the 2,4-D or chlorocatechol
genes as a probe. If the vector plasmid is absent, there would be no
hybridization. Presence of the vector plasmid along with the 2,4-D
degradative genes will demonstrate positive hybridization with the 2,4-D
gene segment as a probe. Additional evidence for the presence of the
-------
vector plasmid can then be obtained by hybridization with other probes
such as the rabbit immunoglobulin or SV40 sequences that are also
incorporated into the versatile vector plasmid.
-------
-------
ENVIRONMENTAL RESEARCH LABORATORY
3ULF BREEZE , FLORID* 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q284
3U CODE & TITLE : L104 Chemical Testing & Assessment
OBJECTIVE CODE I TITLE : S Develop & Validate Improved Risk Assess Meth
?PA CODE & TITLE : 02 Envriomantal Aspects of Biotechnology
PROJECT CODE t TITLE : 24 Ecol Consequences of Gen Eng Microorgs
WORK. PLAN TYPE : COOP
INVESTIGATOR : A Bourquin PRINCIPAL INVESTIGATOR: R C
PHONE NO :
PLAN TITLE : Survival* Mod & Eff of Gan Eng Microorg Rel to the Aq E
nv
WORK PLAN OBJECTIVE : Tha objectives of this prooosal are: CD to develop
accurate, precise* and cost effective methods for detecting and tracking
genetically engineered microorganisms in aquatic environments; (II) to
examine possible modification* including continued metabolic functions, of
genatically engineered microorganisms surviving release to the aquatic
environment? and (III)" to determine affects on the environment of release
of genetically engineered organisms.
v^ORK PLAN APPROACH : The most pertinent part of the project is the comparative
development of methods for the detection of GEMs in aquatic environments.
Several methods mill be tested: plasmid patterns, direct epi f luoresence
counts, 5S SNA group specific signature characterizations* fluorescent
antibodies to datact noncultural GEMs, gene probes, and numerical taxonomy.
Using these techniques, the modification, environmental introduction,
survival, and impact of a genetically engineered organisms mill be
documented.
STATUS : On Schedule
ACCOMPLISHMENTS : The research uiork is on-going. Tha study of viable but
non-cu 1 tur abl e bacteria in the environment has been continued. Cultures
have oean sant by Dr. Martin Alexander for our laboratory to test for the
visjla but nonr ecover able phenomenon. Tha cultures hava been received. It
is anticipated that the testing regime suill yaild data for assessment of
the viable but non-cultur able phenomenon by tne end of the summer.
With regard to the problem of tracking gen-.etically altered microorganisms
relaased into the aquatic environment, aa hava focusad on probe technology.
Wa have recently reported the cloning of the chitinasa determinants of
Vibrio vulnificus in the plasmid vector p3R322, along with applications of
a probe for the chitinase genes in anvironmantal isolates (Wortman et al.,
1985, galley proofs enclosed).
The chitobiase cloning and the RNA analyses will provide us
-------
ENVIRONMENTAL R^S^ARCH LABORATORY
GULF B2EEZE , FLORIDA 32361
FY '86
DATE :
WORK PLANS
7/ 2/36
WORK PLAN CODE : Q284
Presentation "Release og of
Genetically Engineered
Microorganisms into the
Environment at Global Impacts
Applied Micro Jioloc,y VII»
Helsinki Finland
Manuscript* "Discovary cf via
but non-cul t jr aols Legianella
pneumophila by immune
epi f 1 jore senc 3 microscopy ".
of
-------
QUARTERLY REPORT
TO
THE ENVIRONMENTAL PROTECTION AGENCY
Dr. A. W. Bourquin
Microbial Ecology and Biotechnology
- Bldg. 7, Sabine Island
Gulf Breeze Environmental Research Laboratory
Gulf Breeze, Florida 32561
for
Grant No. CR812246-01-0
Provided by
Dr. Rita R. Colwell
Professor of Microbiology
University of Maryland
College Park, Maryland 20742
For the periods
December 1, 1985 through April 30, 1986
and
March 1, 1985 through May 31, 1986
-------
Appendix A
The plasmid constructs pATWSOl, pATW502 and pATW503 have been mapped
with the restriction enzymes shown in Fugure la. Please note additions to
the maps made since the last report. Specific deletion mutants have been
produced in an attempt to locate the chitobiase gene within the inserted
sequences. Those mutants are diagramed in Figure Ib. Me not know that
the inserts code for chitobiase, endochitinase and compliment a lacY mutant
of Escherichia coli, indicating that a permease may also be encoded. It
appears that the chitinase. determinants of V^. vulm'ficus are arranged in an
operon similar to that proposed for V^. harveyi (Soto-Gil and Zyskind.
Cloning of Vibrio harveyi chitinase and chitobiase genes in Escherichia
coli in John P. Zikakis (ed.) Chitin, Chitosan, and Related Enzymes.
Academic Press, 1984.), although the restriction maps of the two operons
are not similar. The deletion mapping and expression data may be explained
by the transcription of the operon onto a polycistronic message. Any
interuption of the message would, therefore, result in reduced expression
of all genes.
Two radiolabeled probes have been constructed from pATWSOl. The
first probe is the fragment from the Eco RI site i'ri pBR322 to the Sal
I site in pBR322, and includes the entire inserted sequence. The second
probe was constructed from a Kpn I/Xba I double digest of pATWBOl, and
contains no vector DNA. Although the second probe may be expected to be
the more specific of the two, it does not contain a portion of the operon
which is known to be essential for gene expression. Both probes are
currently being used to screen chromosomal and plasmid DNA from marine
and clinical isolates. Of particular interest are. those members of the
Vibrionaceae that are phenotypically negative for chitinases, and organisms
isolated from the marine environment.
-------
1. Work Status and Progress
The research work is on-going. The study of viable but non-culturable
bacteria in the environment has been continued and a manuscript describing
the method is appended. Cultures have been sent by Dr. Martin Alexander
for our laboratory to test for the viable but nonrecoverable phenomenon.
The cultures have been received. It is anticipated that the testing
regime will yield data for assessment of the viable but non-culturable
phenomenon by the end of the summer.
With regard to the problem of tracking genetically altered microorganisms
released into the aquatic environment, we have focused on probe technology.
We have recently reported the cloning of the chitinase determinants of
Vibrio vulm'ficus in the plasmid vector pBR322, along with applications of
a probe for the chitinase genes in environmental isolates (Wortman et al.,
1986, galley proofs enclosed).
The chitobiase cloning and the RNA analyses will provide us with probes
for detection of Vibrio species in the estuarine and marine environment,
an important aspect of the research in this project.
(Publications Appended)
2. Difficulties Encountered - none.
3. Preliminary Data Results and Evaluations During the Reporting Period
Galley proofs and preprints appended.
4. Remedial Actions Taken - none necessary.
5. Statement of Activity Anticipated During the Subsequent Report
Work progressing on target.
6. Changes of Key Personnel Concerned with the Project
Betty A. Ortiz-Conde, technician, was added to the project.
7. Expenditures - as budgeted.
-------
Appendix B
Three samples of rock of a sandy composition were collected in the Ross Desert of
the Antarctic and shipped to our laboratory under reduced temperature. These samples,
marked "BPL," "BPC," and "LTL" were extracted to detect and analyze nucleic acids
contained in the samples. The three samples were subjected to a variety of treatments
designed to extract any nucleic acids present from the biomass in the rocks. These
included: (1) lysozyme-SDS; (2) freeze thaw; and (3) phenol extraction. All extracts
were ethanol precipitated, suspended in a. denaturing tracking dye and electrophoresed
on thiol-soluble acrylamide (BAG: bis-acrylyl-cystamine; Hansen, 1981) to screen for
characteristic tRNA, 5S r RNA and 16S rRNA bands. The "LTL" sample yielded, under
phenol extraction, a single faint but well defined band the migration of which in 5%
BAG was characteristic of 53 rRNA. This band was carefully excised with a sterile
blade and extracted chromatographically from the solubilized gel. B^cytidine-bis-
phosphate was ligated to the purified nucleic acid using RNA ligase. The labelled
RNA was repurified and subjected to enzymatic sequence analysis, described by
MacDonell and Colwell (1984). The following sequence was obtained:
5'-nnnnUGGCGA CCAUA GCGUUUUG GACCC CACCUGA CCUCCAUUCCGAAC
UCAG AA GUG AAA CGAAAU A GC G CC AUGGUA GUGUGGGG UUU
CCCCAUGU GAGAGUA GG ACA UCGCUGGnnn-3'
This sequence confirmed that the band corresponded to a 5S rRNA, and was identifiable
as belonging a species of the genus Vibrio sensu strictu (MacDonell and Colwell, 1985)
A search of our RNA database, using a computerized sequence matching program verified
that the sequence had not been characterized previously and indicated that the LTL
RNA was shares close common ancestry with Vibrio natriegens, a marine Gram-negative
eubacterium.
An evolutionary tree indicating the phylogenetic placement of LTL amongst
characterized species of the genus Vibrio sensu strictu is attached.
Work is now in progress to perfect probe methodology, the application of which
will be to detect specific strains at concentrations as low as 1 cell/mL in environ-
mental samples, using signature sequences on 5S rRNAs as amplified genes. Probe
methodology should allow circumvention of RNA assays of large numbers of isolates
to detect a target organism in an environmental sample.
References:
Hansen, J.N. 1981. Anal. Biochem. 116:146-151.
MacDonell, M.T. and R.R. Colwell. 1984. FEES Lett. 175:183-188
MacDonell, M.T. and R.R. Colwell. 1985. Syst. Appl. Microbiol. 6:171-182
-------
a)
? y y' ? M ? ? ? ?' Y ?'
Tc
0 H* H H* K S A H H H H* X H'A
ii i iii i i i j it OATW502
fe — -
HH'KSAH HH H'X
i t i i i i it i i
b)
• H E
H'AN^-B 0 pAiwsoa
- — re
Active
Inactive
kb
Figure 1. (•) Restriction- aape of th« inaert r»yion« of pAXHSOl, {ATNS02
•ad pATNS03. Vibrio vulnificua DMA !• d«plct«d •• • bold ltn«.
Th« v«ctoc DMA, pBR322, t« d«plet«d •• • fin* lln«. IU«trlctloa
•it«* «r« d«*ign«t«d by A, Av« I; B, 89! XI; B, CcoR X; H.
Hind XXX; H'. Hp> I; K. Kpn I; S. Sph X; X. Xba I; 9. EcoR X
•It* In pBR322. Th« direction of tr«n«crlptlon Iron th«
t«tr«cyclln« promoter !• Indicated under ««ch mtp.
(b) In>«rt> cr««t«d by deletion* of plauld pAXHSOl. The
deleted pl«e«ld« «r« de>lgn«ted by SX, Sal I (pBR322) to
Xbe I deletion; AV, Ava I (pBR322) to Ava I deletion; CK,
EcoR I (pBR322) to Kpn I deletion; HIO-B and H10-C, Hind III
partial deletion*. The ability or Inability of the deletant
plaeaide to produce detectable chltoblaie activity if indi-
cated to the right of each Insert.
-------
V.
I/.
V. piote.otytiait>
V. diazotsiopkicuA
V. atQ4.no tyticuA
V. gazogenes
I/.
-------
JUDI^/AIVIC. iv.ni - jui « t »^_. . ......... - - _ . .
CLS: monthly GRP: asm2 JOB: aem-jul DIV: 6797-864
Appendix C
AND ENvmoNMtKTAL MICROBIOLOGY. July 1986, p. 000-000
0099-2240/8M)70000-OOS02.(XVO
Copyright O 1986. American Society for Microbiology
Vol. 52. No.
Chitinase Determinants of Vibrio vulnificus: Gene Cloning and
Applications of a Chitinase Probe
A. T. WORTMAN.t C. C. SOMERVILLE. AND R. R. COLWELL*
Department of Microbiology, University of Maryland, College Park. Maryland 20742
Received I November 1985/Acccptcd 31 March 1986
To Initiate study of the genetic control of chitinolytic activity in vibrios, the chltoblase gene was Isolated by
cloning chromosomal DNA prepared from Vibrio vulnificus. Chimcric plasmlds were constructed from Sau3\
I partial digests of chromosomal DNA by ((gating 5 to IS-kilobase fragments Into the Bom HI site. I.e.. in the
TC* gene, of pBR322 (Amr TcO. The resulting plasmids were transformed Into Eschtrichia co/i DH1. Chitobiase
activity of the Insert-bearing clones was detected by using a chromogeolc substrate, p-oitrophenyl-yV-acetyl-
P.D-glucoiamlnlde. and confirmed by tht appearance of a fluorescent end product from the hydrolysis of
4-mclhylumbelliferyl-p,D-^W-diacerylchlUoblosc. Endochlllnas* activity was demonstrated byjjberatlon of '•
water-soluble products produced by the degradation of (*H]chlUn. Transformation off. coli Y^JK (lacY) with
plasmlds from chitinase-positive clones restored the lactose-positive phenotype, suggesting the presence of a
permease associated with chitlnase activity. Physical mapping of plasmlds containing the chitinasc determi-
nants Indicate that transcription of these genes In £. coli may be Initiated at a V. vulnificus promoter.
Chitin, a polymer of p-1.4-bondcd JV-acctyl-D-gluco-
saminc residues found in fungal cell walls and arthropod
integuments, is among the most abundant biopolymcrs on
earth, a strong indication of its importance in nature. In
recent years significant research has been directed toward
the use of chitin and chitin derivatives in fields as diverse as
effluent water treatment, drug delivery, and wound binding
(2). Chemical dcrivitizations of chitin may be complemented
and extended by the use of chitin-dcgrading and -modifying
enzymes. The isolation and cloning of chitinasc determi-
nants from naturally occurring systems,can provide an
efficient source of such enzymes (17) as well as genetic
probes which can be used in the isolation and characteriza-
tion of related systems.
Members of the genus Vibrio, in general, produce
chitinolytic enzymes. Furthermore, there is evidence to
suggest that the association of these organisms with chitin
surfaces plays an important role in their seasonal distribution
(9-11). The interaction of vibrios with surfaces in the envi-
ronment has also proven useful in understanding the epide-
miology of disease-associated Vibrio spp.. including Vibrio
cholerae, which is the causative agent of cholera (9).
In addition to the vibrios, chitinoclastic activity is demon-
strated by a wide variety of microorganisms, including
species isolated from soil, freshwater, and the marine envi-
ronment (3, IS). The enzymes involved in chitin degradation
arc quite stable, withstanding overnight incubation in tolu-
ene, even in the absence of substrate (3). Extracts of
toluene-killed cells show persistent and measurable acetyl-
glucosamine production from chitin for more than 10 days
after treatment (3). Chitinase activity measured in one Vibrio
sp. was found to be stable within the pH range of 5 to 11.
with greatest activity measured within the pH range of 6 to 8
(II. 15).
Enzymatic degradation of chitin appears to occur in two
steps, which arc similar in both procaryotcs and cucaryotes.
An cndochitinase reduces the polymer to oligomcrs and
dinners, which arc subsequently degraded to monomers by
chitobiasc (14, 17). The enzymes appear to be coordinatcly
controlled and in different organisms can be induced by
chitosan, chitobiasc. Af-acctylglucosaminc. or glucosaminc
(1.3.15). Recently published data suggests that a third gene.
the product of which acts as a pcrmcasc, is located in a chi
opcron in Vibrio harveyi (17).
Goning of the chitobiasc and cndochitinase genes repre-
sents a first step in gaining an understanding of the function.
control, and distribution of chitinolytic enzymes of marine
and cstuarinc vibrios. Thus, the successful cloning of Vibrio
vulnificus chitobiasc is presented, and uses for the chitinasc
probe arc described.
MATERIALS AND METHODS
Strains. Escherichia coli OH1 (ATCC 33849) was used as
the cloning host, and V. vulnificus ATCC 27562 provided the
target DNA. E. coli YlGfR (lacY) (donated by M. J. Voll)
was used in complementation studies for permease activity,
and £ coli JM109 (donated by A. V. Furano) was used as the
host for deletion mapping studies.
£. coli OH1 and Y1OR were grown in LB broth (12). and
V. vulnificus was grown in broth containing 1% tryptone,
0.5% yeast extract, and 1.5% Instant Ocean (Aquarium
Systems). Agar plates were prepared by addition of 1.3%
agar (BBUMicrobiology Systems) to the broth media.
DNA Isolation. (I) Chromosomal DNA. The cell suspension
from a 1-liter overnight culture of V. vulnificus was chilled
by swirling the preparation in an ice bath for 10
• Corresponding author.
t Present address: Molecular Bioaystcma Inc., San Diego. CA
92121.
cells were collected by ccntrifugation at /
at 4*C. The supernatant was discarded, and the'pellet was
vortexed to form a slurry. Approximately 5.5 ml of a solution
of 0.02 M Tris hydrochloride (pH 8.0) and 0.2 M EDTA (TTV
tofferXwas added, followed by 5.5 ml of a 20% sodium
dodecyl sulfate solution, and the suspension was gently
mixed to complete cell lysis. Immediately after lysis, 3.5 ml
of 5 M NaQ was added and gentry mixed into the lysate.
Fifteen milliliters of cold chloroform-isoamyl alcohol (24:1)
was added, and the mixture, in a round-bottomed flask, was
gently agitated in an ice bath until an emulsion formed. The
emulsion was centrifuged at .8.000 fpHifor 2Omin?at 4*C. The
-------
WORTMAN ET AL.
Am.. ENVIRON. MICHOUIOL.
aqueous phase was placed in dialysis tubing and dialyzcd at
4*C against three changes of TE buffer (pH 8.0) over a 3-h
period. Hcat-ircatcd RNasc A (Sigma Chemical Co.) was
added to a final concentration of 50 tig/ml, and 10,000 U of
RNasc Tl (Sigma) was also added. Dialysis at 4*C against
three changes of TE buffer was carried out overnight. Thirty
microlitcrs of protcinasc K (20 mg/ml) (Sigma) was added,
and the mixture was dialyzed for 8 h against three changes of
TE buffer at room temperature. The lysate was extracted
with phenol and cthanol precipitated by the addition of 0.5
volume of 7.5 M ammonium acetate, and then 2.5 volumes of
cold 95% cthanol was layered onto the solution. The DNA
was wound onto a sterile glass rod, suspended in TE buffer,
and stored at 4*C.
(II) PUsraid DNA. £. colt DH1 (pBR322) was grown
overnight in Lfl broth containing 50 tig of ampicillin per ml
and 12.5 tig of tctracycline per ml. The pBR322 ON A was
prepared by the gentle lysis procedure of CIcwell and
Hclinski (4). Plasmid preparation from random colonies
demonstrating an ampicillin-rcsistant (Am'), tetracyline-
rcsistant (Tc*J phcnotypc was performed by the rapid boiling
method of Holmes and Quiglcy (7).
Preparation of colmerie plaiulds. Conditions for the par-
tial digestion of V. vulnificus gcnomic DNA were determined
for 5au3AJ< Bcthcsda Research Laboratories, Inc.) Enzymat-
ically cleaved DNA (400 u,g) was scdimentcd through a 5 to
40% sucrose gradient. Fractions were analyzed on • 0.8%
agarosc gel, with //indlll-digcsted lambda DNA as molecu-
lar weight standards. Fractions containing fragments of
appropriate size (5 to 15 kilobases) were dialyzed against TE
buffer, concentrated by the addition of butanol, and precip-
itated with cthanol.
SamHI-clcavcd pBR322 DNA (0.1 jig) was mixed with 0.5
tig of V. vulnificus insert DNA and 1 uJ of T4 DNA ligase
(Bcthcsda Research Laboratories) and brought to a final
volume of 12 u.1. The reaction was incubated overnight at
12'C. The ligation mixture was used to transform E, coli
DH1 (6). Transformed cells were plated on LB agar contain-
ing 50 iig of ampicillinrcolonies of ampicillin resistance
phcnotypc were transferred to LB-ampicillin master plates,
which were replicated onto LB-ampicillin containing 12.5 u,g
of tctracycline per ml.
ScmBiag for chitioolytk activity. Chitin (Bioshell Inc.)
used for the preparation of overlay plates was swelled with
phosphoric acid by the method of Monrcal and Reese (14).
Chitin was also prepared by rcacctylatkui of chitosan by the
method of Molano et al. (13).
Transformed cells demonstrating the Am' Tc* phenotype
were placed on master plates. Twenty randomly chosen
colonies were placed in 5 ml of LB broth, grown overnightat
37*C. and screened for plasmid content by a rapid boiling
method (7). V. vulnificus inserts ranged from 4 to 15
kilobases, measured by comparison with Hindlll fragments
of lambda DNA in 0.8% agarose.
The gcnomic library was replicated onto a chitin-
containing medium, and growth was monitored over 14 days.
Clones transferred to chitin overlay agar failed to produce
clear zones in phosphoric acid-swelled chitin or the more
sensitive overlay containing rcaccrylatcd chitin.
Clones grown on LB-ampicillin plates were sprayed with a
0.01 M solution of the chitobiose analog p-nitrophenylW-
acctyl-3.D-glucosaminidc in 0.1 M sodium phosphate buffer
(pH 7.5) (17). Cones producing a bright yellow color indi-
cated presumptive chitobiasc activity. Plasmid DNA iso-
lated from these clones was used to transform £ coli DH1
(6). Transformed cells were spread on LB-ampicillin plates.
Ampicillin-rcsistant colonies were again screened with p-
nitrophcnyl-jV-acctyl-p-D-glucosaminidc. Confirmation of
chitobiasc activity was done by spreading presumptive pos-
itive clones onto filter paper wetted with the umbcllifcryl
conjugate of chitobiose. 4-mcthylumbcllifcryl-3.D-A/-/V'-
diacctyl chitobiose (MUchitobiosc; Sigma), dissolved in 0.2
ml dimcthylfonmamidc, and diluted to 1.2 ml in phosphate-
buffered saline (pH 7.0), resulting in a substrate concentra-
tion of 0.83 mg/ml. A solution of 0.01 N NaOH was added to
enhance fluorescence, and the clones were observed under
UV illumination for the presence of a fluorescent degrada-
tion product.
Tritiatcd chitin was also prepared by the method of
Molano et al. (12) and used as the substrate for the chitinasc
assay (donated by R. A. Smucker, University of Maryland.
Solomons Island). Label liberated from the rcacctylatcd
chitin was measured with a Packard Tricarb 3330 liquid
scintillation counter. Samples (80 ml) of the overnight cul-
tures were sedimented at 5,000 x g for 15 min at 4*C. The
supernatant was removed, and the pellet was transferred to
a hammer mill stainless steel jar filled with liquid nitrogen.
Cells were fractured by liquid nitrogen cryoimpacting for 2
min (16). The cell powder was transferred to a tube incu-
bated at 0*C, and the powder was gently suspended in 25 ml
of buffer (0.1% MgSO,, 0.03% CaCI,, 0.85% NaCl. 0.05%
KjHPO* [pH 7.2]) to release soluble cytoplasmic compo-
nents. The membrane fraction was pelleted at 12,000 x g.
Assays were performed at 37*C for 2 h after addition of 30
uJ of pHjchitin to 100 u,l of toluene and 500 til of the
cytoplasmic or membrane fraction. The reactions were
quenched by the addition of 200 uJ of 10% trichloroacctic
acid. Controls were employed for the buffer and E. coli
harboring pBR322. Individual sample controls contained the
cell extracts quenched with 10% trichloroacctic acid before
(}H]chitin addition. Samples were then passed through
Gelman GF/C fiber glass filters (0.45-u.m pore size) to
remove undegraded chitin fibers. The filtrate was mixed with
Scintisol (Isolabs) scintillation cocktail, and the quantity of
water-soluble hydrolysis products was determined by liquid
scintillation.
Physical mapping. The physical maps of plasmids
pATWSOl, pATW502, and pATW503 were constructed from
single and double digests with various restriction
endonuclcases (Bcthcsda Research Laboratories, New En-
gland Biolabs, Boehringer Mannheim Biochemicals) by us-
ing the manufacturers' recommended conditions. Products
were separated on 1.0 to 1.2% agarose gels run at 50 to 100
V in Tris-acetate buffer (12). Gels were stained in 1 tig of
ethidium bromide per ml and photographed by transmitted
UV illumination.
Drietloa mapping. Plasmids with specific deletions were
constructed from pATW501 and screened for activity with
MUchitobiose. Deletions were made by a single digest with
Aval, double digests with Sail mdXbal. double digests with
EcoRI and Kpnl, and partial digests with Hindlll. In all
cases buffer and incubation conditions were those suggested
by the enzyme suppliers. Hindlll partial digests were done
using 2.5% (vol/vol) enzyme for incubation times from 5 to
20 min. After single restriction digestions the preparations
were treated with T4 DNA ligase (Boehringer Mannheim) in
50 mM Tris hydrochloride (pH 7.5)-5 mM MgCl-l mM
ATP-1 mM dithiothreitol-0.1 mg of bovine serum albumin
per ml. Ligation mixtures were incubated for 4 R-M- room
temperature or overnight at I5*C. Double-digested DNAs
were polished by treatment with the KJcnow fragment of
DNA polymerase I (New England Biolabs) in the presence
-------
VOL. 32, 1986
CHITINASE DETERMINANTS OF VIBRIO VULNIFICUS
TABLE I. Chitinase activity associated with cloned V, vulnificut
inictu
ChiiinaM activity (pU/cetl)
Pbtmid
pATWSOl
pATWS02
pATWS03
Extra-
cellular
NO-
ND
ND
Mcmbraac
associated
0.66
0.07
0.23
Cytoplasm
associated
0.73
1.02
0.32
Tool
1.39
1.09
OJ6
• NO. Not detected.
of adinuclcosidc triphosphatc mixture (0.1 mM each dATP,
dCTPrdGTP. dTTP) for 3 min «t 3TC. The newly formed
blunt ends were then ligatcd together as previously de-
scribed.
Deleted plasmids were transformed into £ colt JM109.
which was subsequently plated on LB-ampiciUin agar. Plas-
mids were isolated by the rapid boiling method of Holmes
and Quiglcy (7). digested with appropriate restriction
cndonuclcascs. and separated on 1% agarosc gels. Small
fragments, and those of similar molecular size, were re-
solved on 7.5% polyacrylamide gels.
Ptrmcait screening. Plasmids pBR322, pATW501.
pATW502, and pATW503 were transformed into £ coli
V^RBr Transformation mixes were plated on MacConkey
agar (Difco Laboratories) containing 50 tig of ampicillin per
ml. Untransformed cells did not grow on this medium, and
pBR322-transformcd cells produced light pink colonies.
Cells transformed with plasmids which complement the
lactose pcrmcasc mutation of £ coli YJO^proouccd brick
red colonies, indicating transport and metabolism of lactose.
RESULTS AND DISCUSSION
Three clones from a partial library of 650 insert-bearing
transformants were found to be positive for chitobiase
activity after screening with p-nitrophenyl-A/-acetyl-p,D-
glucosaminide and the umbelliferyl conjugate of chitobiose.
All clones demonstrating the chitobiase phenotype were also
ampicillin resistant, suggesting that the DNA encoding the
chitinasc activity was located on the chimeric plasmids. The
number of positive clones found in a gene library of this size
may indicate that the chitinasc determinants are present in
more than one copy on the V. vulnificus chromosome. An
investigation of this possibility is in progress.
Cones grown on plates overlaid with colloidal chitin did
not produce a clear zone, suggesting that the chitinase
enzymes were not exported by £ coli DHL This observa-
tion was corroborated by experiments with I'HJchittn. Su-
pernatant fluids from cultures harboring pATWSOl,
pATW502. and pATW503 were devoid of both endochitinase
and chitobiase activity. Chitinase activity was detected in
the cytoplasmic and membrane fractions of all three clones
by the ( HJchitin assay. Activities associated with cytoplas-
mic fractions were greater than those found in membrane
fractions. Activities associated with pATWSOl and
pATW502 were greater than those associated with
pATW503 (Table I). The assay for chitobiase activity seems
to detect the enzyme in cells lyscd during spreading onto the
filter paper or cell debris transferred from the agar plate.
The chimeric plasmidr'pATWJOl. pATWS02. and
pATW503, obtained from the chitobiasc-positive clones,
were mapped with restriction cndonucleases (Fig. 1). The
inserts were found to be 5.1.5.7, and 5.7 kilobases in length.
respectively. All three contained unique restriction sites for
Kpn\, Sph\, and Xbal. Multiple Hpai and HindHl sites were
also present. The inserts contained no BuinHI, Su/l. EcoRI.
Psl(, or Xmal restriction sites. The inserts were found to
have identical maps within the 5.1 kilobases of common
DNA. The orientation of the insert DNA in plasmid
pATW503 is opposite to those in plasmids pATWSOl and
pATW502. The restriction pattern of these inserts is not
similar to the chitinasc opcron isolated from Vibrio harvcyi
(17).
Transformation of plasmids oATWSOl. pATWS02. and „
pATWS03 into £ coli YJOS^fiRluccd dark red colonies on
MacConkey agar plus ampicillin. indicating that the cloned
V. vulnificus insert complemented the lactose pcrmcasc
mutation in the host strain.
Regulation of the chitinase genes in V. vulnificus is not yet
known. However, the chitinasc genes arc expressed by £
coli DH1 in their present configurations. Even though initial
screening and confirmatory tests were not performed in the
presence of a known chitinase inducer, the clones produced
strong, positive results. This may be due to the high gene
dosage imposed on the cell by the use of pBR322, which has
a relaxed mode of replication. The fact that inserts in both
orientations (Fig. 1) are expressed indicates the recognition
of a V. vulnificus promoter by the host RNA polymerase,
rather than transcription from a strong vector-carried pro-"
moter region. The efficiency of transcription cannot be
implied from the present work. Figure Ib illustrates delet-
ants formed by digestion of pATWSOl with various restric-
tion enzymes. Only one of the deletants constructed thus far
demonstrates chitobiase activity by hydrolysis of the
MUchitpbiose. The positions of these deletions indicate that
expression of the chitobiase gene depends upon uninter-
rupted transcription of a relatively large (4.4-kilobase) V.
vulnificus fragment. Since pATWSOl has also been shown to
specify an endochitinase and to compliment an lacY mutant.
a)
1 f?
• N' M M' • S » I
MM MX M4
M' K S * M MM
W
IX
»v
e«
MlO-i
MO-C
*«cr««
IHttnt
FIG. I. (i) Restriction maps of the insert region* of pATWSOl.
PATW502. and pATWSOJ. V. vulnificut DNA is depicted as a bold
line. The vector DNA. pBR322. is depicted as a fine line. Restriction
sites: A. Aval. B. Bgfll; E. EcoRI; H. Mndlll; H1. Hpa\; K. Kpn\\
S. Sp*I; X. Xbal. 0. fcoRI site in pBR322. The direction of
transcription from the tctracycline promoter is indicated under each
map. (b) Inserts created by deletions of plasmid pATWSOl. Deleted
plasmids: SX.5a/1 (pBR322)-te-Xbiil deletion; AH. Aval (pBR322)>
WhAval deletion; EK. EcoRI (pBR322)-to-A^iI deletion; H10-B and
H10-C. /rtndlll partial deletions. The ability or inability of the
dcletant plasmids to produce delectable chitobiase activity is indi-
cated to the right of each insert.
-------
CLS: monthly VJKI-:
-<.... „. _.
WORTMAN ET AL.
APPL. ENVIRON. MICKOBIOL.
it is likely that the chitinise dctenninants in V, vulnificta are
organized in an opcroo such as that previously proposed for
V. harveyi (17).
V. vulnificta exports chitinase enzymes into the surround*
ing medium and produces large, clear zones in colloidal
chitin overlays. Gear zones were not produced by £. colt
colonies carrying the cloned genes, indicating that in £. coti
the enzymes remain cell associated, a hypothesis supported
by the lack of cndochitinase and chitobiasc activity in the
culture supcmates. The detection of the hydrolysis of p*
nitrophcnylW-acctyl-B.D-glucosaminide and MUchitobiose
probably reflects enzymes released by cell lysis.
Further subcloning is in progress to define more precisely
the chitinase determinants which can be used as a genetic
probe to assay for the presence of identical or closely related
gene sequences in Vibrio and related species. The probes are
being employed to measure evolution of the chitinase gene
complex in procaryotes and cucaryotes as well as to deter-
mine the distribution of the chitinase gene among marine
vibrios from the deep sea, coastal waters, estuaries, and
brackish water regions.
ACKNOWLEDGMENTS
We thank R. A. Smucker for his assistance in performing assay*
with I'H|chitin.
This research was supported in pan by Office of Naval Research
Contract NOOOI4-8I-K-0638, National Science Foundation Grant
BSR-84-01397. and Environmental Protection Agency Contract
CR8I2246-01-0.
LITERATURE CITED
1. BCBMO, C. B.. and M. A. Hood. 1980. Effects of cultural
conditions on the production of chitinase by a strain of Bacillus
megatariiun. Dev. Indus!. Microbiol. 21:357-363.
2. Brine, C. J. 1984. Chitin: accomplishment* and perspectives, p.
xviii-xxiv. lit i. f. Zikakis (ed.). Chitin, chitosan and related
enzymes. Academic Presa. Inc., New York.
3. Clark*. P. ML, a*d M. V. Traeejr. 1956. The occurrence of
chitinase in some bacteria. J. Gen. Microbiol. 14:188-194.
4. CIcwcU. O. 0., a*d D. R. HeliisU. 1969. Supercoiled circular
DNA-protein complex in EscHerichia coti; purification and
induced conversion to open circular form. Proc. Nail. Acad.
Sci. USA 62:1159-1166.
5. Cortrtll, R. R. 1983. Biotechnology in the marine sciences.
Science 222:19-24.
6. HanshsB. D. 1983. Studies on transformation of Eicherichia coti
with plasmids. J. Mol. Biol. 166:557-580.
7. Holaw, D. S., and M. Qulglcjr. 1981. A rapid boiling method for
the preparation of bacterial plasmids. Anal. Biochem. 114:
193.
8. Horwte. M.. J. RtU. ud D. OgrydzUk. 1984. Genetic improve-
mem of chitinase production by S. marccscens, p. »•* m. In
J. P. Zikakis (ed.). Chitin, chitosan and related enzymes.
Academic Press, Inc., New York.
9. Huq, A.. E. 8. Small, P. A. West. M. I. Huq. R. Rahman, and
R. R. ColwtD. 1983. Ecological relationships between Vibrio
cnoUrat and planktonic crustacean copepods. Appl. Environ.
Microbiol. 45:275-283.
10. Hu4|, A^ P. A. Wnt. E, B. Small, M. I. Hw|. aod R. R. CohrcU.
1984. Influence of water temperature, salinity and pH on sur-
vival and growth of toxigenic Vibrio cholera* serovar Ol
associated with live copepods in laboratory microcosms. Appl.
Environ. Microbiol. 48:420-424.
11. KsMka, T., sad R. R. Corwttt. 1973. Ecology of Vibrio
parakaemofyticia and related organisms in Chesapeake Bay. J.
Bacteriol. 113:24-32.
12. Manlad*. T.. E. f. FriUch, and J. Sambrooh. 1982. Molecular
cloning: a laboratory manual. Cold Spring Harbor Laboratory.
Cold Spring Harbor. N.Y.
13. Molano. J., A. Duns, and E. Cablb. 1977. A rapid and sensitive
assay for chitinase using tritiated chitin. Anal. Biochem.
83:648-656.
14. Moroni, J., aod E. T. RMM. 1969. The chitinase of Strratia
marcescent. Can. J. Microbiol. 15:689-696.
15. Obtakan, A.. M. MlUutoml, and Y. Ucblda. 1979. Purification
and some properties of chitinase from Vibrio sp. J. Ferment.
Technol. 57:169-177.
16. Smuclur, R. A^ and R. M. Pfliter. 1975. Liquid nitrogen
ctyoimpacting: a new concept for cell disruption. Appl. Micro-
biol. 30:445-449.
17. Soto^U. R. W., tmd J. W. ZysUad. 1984. Cloning of Vibrio
Harveyi chitinase genes in Esckerichia coli, p. 169-177. In 1. P.
Zikakis (ed.). Chitin. chitosan and related enzymes. Academic
Press, Inc.. New York.
-------
Appendix D
FLUORESCENT ANTIBODY STAINING METHOD FOR ENUMERATION OF
VIABLE ENVIRONMENTAL VIBRIO CHOLERAE 01
P. R. Brayton and R. R. Colwell
Department of Microbiology
University of Maryland
College Park, MD 20742
Submitted to: Journal of Microbiological Methods
Date: May, 1986
*
Corresponding author
-------
ABSTRACT
A membrane filtration method has been developed which is useful for
enumeration of viable Vibrio cholerae 01 in environmental water samples by
inatunofluorescent staining. The samples are incubated with yeast extract
and nalidixic acid. Substrate responsive cells, i.e. viable cells, elon-
gate and after staining with specific antiserum and fluorescein conjugate,
viable ^. cholerae cells appear as long, peripheral fluorescent green
banded bacilli when viewed under the microscope. Using an ocular reticule,
the number of viable cells per ml can be calculated. The procedure has
been adapted for use with other bacterial species if specific antisera is
employed.
-------
INTRODUCTION
Enumeration of indicator organisms or, preferably, direct detection of
pathogen* in the aquatic environment is .essential if water quality is to be
estimated. Public health safety, of course, is determined from the results
of such tests. The choice of method for assessment depends on specificity,
reproducibllity, and statistical validity, in conjunction with time and
cost efficiency and ease of operation. Immunofluorescent-epifluoreBcent
detection methods fulfill these criteria and are being used to detect a
variety of organisms (4, 8, 11, 12, 14).
The viable, but non-culturable stage of bacteria has been established
(1, 8-10). In addition, results of studies have shown that nutrient
starved and injured cells are incapable of growing on standard culture
media and, therefore, indicator organisms in that state will not respond to
standard methods for assessing microbial populations. The virulence of
viable but non recoverable cells may persist, demonstrated by animal
studies (1, 13).
Fluorescent antibody (FA) procedures can overcome the problem of
organisms remaining viable but yielding "no growth" in standard tests,
since the presence, absence, or size of specific cell populations can be
easily detected microscopically. In the conventional FA procedures, all
organisms possessing a species-specific antigen can be observed. However,
it is not possible to determine which cells are viable and which are dead.
The method described here is advantageous because it allows detection of
viable cells. By combining the indirect fluorescent antibody procedure of
Xu £t a_l. for direct detection of bacteria in natural water samples (15),
with the direct microscopic viable count by Kogure et al. (7), enumeration
-------
of viable, specific populations of bacteria can be achieved. We described
here a procedure we have used to detect viable ^. cholerae serovar 01. The
method, however, is adaptable to any organism, provided specific antisera
are available.
Procedure
1. Collect water samples in sterile containers following standard proce-
dures for sample collection and employing aseptic techniques. Add
filter-sterilized and autoclaved solution of yeast extract to obtain a
final concentration of 0.025%. It is recommended that the concentra-
tion of yeast extract be prepared so that 1.0 ml of 0.25% yeast
extract solution can be added to a 10 ml sample.
2. Incubate for 20 min at a temperature appropriate to the environment
from which the sample was collected.
3. Prepare an 0.2% solution of nalidixic acid (Sigma Chemical Co.,
St. Louis, MO) in 0.05 N NaOH. Filter sterilize. Add 0.1 to 10 ml
of sample.
4. Incubate at the appropriate environmental temperature for 12 h.
5. Filter the water sample through an 0.2p x 25mm polycarbonate membrane
filter (Nuclepore Co., Pleasanton, CA), mounted on a 25mm glass fil-
tration unit (Millipore Corp., Bedford, MA). Filters are prestalned
overnight in 0.2% irgalan black (Ciba-Geigy Corp., Greensboro, NC)
dissolved in 2% acetic acid and rinsed several times in distilled
water before use. Place filters in a 30 x 10mm petri dish.
6. Fix cells to filters by placing petri dishes on the surface of a test
tube rack partially submerged in a 55°C water bath. Incubate for
20 mln.
-------
7. Prepare a 1:20 dilution of rhodamine isothiocyanate-bovine serum
albumin (RITC, Dlfco, Detroit, MI). Place one drop on the surface of
the membrane, and place a coverslip' on top of the membrane to spread
the reagent across the filter.
8. Incubate 35-37°C for 30 min. in a dark moist chamber.
9. Rinse filters three times with phosphate buffered saline (PBS, per
liter: NaCl, 8.5g; Na2HPO4, 9v1 g; KHjPC^, 1.5g; pU - 7.3) and allow
the filters to soak for 10 min. in the petri dish. Remove PBS and air
dry the filters. Care must be taken to ensure that the top of the
filter always remains upright throughout the procedure.
10. Add one drop of V. cholerae 01 antiserum and place a coverslip on top
of the drop.
11. Repeat steps 8 and 9.
12. Add one drop of fluorescein isothiocyanate anti-rabbit goat serum
(FIIC, BBL, Cockeysville, MD). Place coverslip on top.
13. Repeat steps 8 and 9.
14. Mount each filter on a glass slide with a coverslip using FA mounting
fluid, pH •> 9 (Oifco).
15. Examine with a microscope equipped with a neofluor objective, IVFL
epifluoresence condenser, 100 W halogen lamp, BP 450-490 band pass
filter, FT 510 beam splitter, and LP 520 barrier filter, or equiva-
lent. Slides should be examined as soon after preparation as possible
to avoid fading of the intensity of the fluorescence. Slides should
be stored in a dark box until used. The V. cholerae 01 cells that are
-------
counted are those that appear as long and/or fat rods possessing a
peripheral green band just below the cell wall (Fig. 1). Calculate
total viable cells/ml of sample, according to the size of the ocular
reticule eaployed in the microscope (6).
Discussion
The mechanism of nalidixic acid centers on its ability to inhibit DMA
*•
gyrase. At the concentration employed in this study, protein and RNA syn-
thesis continue, but cell division is interrupted. As a consequence, the
cells exposed to the nalidixic acid and yeast extract mixture elongate
(3, 7).
The length of the yeast extract-nalidixic acid incubation is at the
discretion of the investigator. Bacterial cells that are starved or
injured may need a longer time to respond to added substrate. It is sug-
gested that 6, 12, and 24 h time intervals be used initially, in order to
establish wnich incubation interval produces the largest number of elon-
gated cells per sample. Experience has shown that adjacent sampling sites
in the field require the same length of time for incubation, providing a
guide for future FA-DVC work in the same geographical area.
For convenience, formaldehyde may be added to samples after incuba-
tion, stopping the reaction (0.2 ml of filtered 37% formaldehyde per
10 ml of sample). The samples can then be stored at 4°C. During
processing, dried filters may be frozen after the filtration step, or,
after the RJTC and/or antiserum application. For best results, the
filters should be viewed the day the FITC is applied.
-------
It must be emphasized that the reliability of the FA-DVC procedure
depend* on the specificity of the antiaerum used. The antiaerua must be
carefully examined for possible cross-reactivity with other bacterial
species, and should be absorbed with those strains to eliminate binding
activity. Experimental control slides must be prepared to test each new
batch of antiserum and FIIC to determine which concentration of each
reagent will produce the maximum intensity of fluorescent cells.
The filter method permits filtration of increasing volumes of water
when the bacterial count is low. The maximum volume filtered will be
dependent only on the degree of turbidity of the water. If the turbidity
of the water sample is high, the sample can be prefiltered through a
1.2y filter (Gelman Sciences- Inc., Ann Arbor, MI) to remove large particu-
late matter. The yeast extract-nalidixic acid incubation can then follow
immediately. FITC has a tendency to absorb non-specifically to organic
particles and detritus. RITC counterstaining can limit this problem
because the counterstain occupies active sites on the particle surfaces.
The red background provides good contrast for fluorescent cells.
Polycarbonate filters are essential for the success of the FA-DVC
procedure (5). The flat surface and uniform pore size of the polycar-
bonate filter prevents bacteria from being imbedded inside the membrane
and, instead, retains the sample on the surface. Irgalan black dye
eliminates autofluorescence of the filter.
The membrane filter (MF) method has gained acceptance and recognition
as an effective, reproducible, and rapid procedure (2). It is convenient,
in that samples can be collected, incubated, fixed and stored for later
staining.
-------
The cultural protocol for enumeration of V. cholerae 01 involves
sample filtration, transfer of the filter to thiosulfate-citrate-bile
salts-sucrose agar (TCBS), incubation for 24 h, followed by biochemical
testing of all sucrose positive colonies with subsequent serological
confirmation. For most probable number (MPN) determinations, samples
should be enriched, employing alkaline peptone broth and incubation for 6h.
Then, a loopful from each tube is streaked onto TCBS agar. The same bio-
chemical and serological. protocols as above should be followed. Inaccurate
counts can result if ^. cholerae is overgrown by other organisms during the
enrichment procedure and, therefore, fails to appear on the plating media.
Final identifications and counts for both procedures can extend for several
weeks. In comparison, the immunofluorescent technique is very time and
cost effective. A total FA count of ^. cholerae 01 using the MF method
(yeast extract-nalidixic acid step is eliminated to count live and dead
cells) can be prepared in 3 h. The viable count, or FA-DVC, can be com-
pleted the same day, or by the next day, depending on the length of the
incubation period. The time of the staining procedure can be further
reduced if an antiserum-FA conjugate is available. This direct staining
process would then involve 2, rather than 3, reagent applications.
The advantage of the FA-OVC far exceeds the conventional plating
methods for viable bacteria. Plate counts considerably underestimate the
viable population in the aquatic environment. No culture media is capable
of supporting growth for all organisms. It is probable that all viable
bacteria are incapable of enlarging in the DVC procedure with yeast extract
as a substrate, and therefore this too, underestimates the true viable
count. However, the FA-DVC can more closely portray the viable environ-
mental population than traditional culture methods.
-------
ACKNOWLEDGEMENTS
This work was supported by grants from the Agency for International
Development (Grant No. DPE-5542-G-55-4060-00, the World Health
•Organization (Grant No. C6/181/70), and Environmental Protection Agency
Contract CR 812246-01-0.
-------
REFERENCES
1. Colvell, R.R., P.R. Brayton, D.J. Grimes, D.B. Roszak, S.A. Huq and
L.N. Palmer. 1985. Viable but non-culturable Vibrio cholerae and
related pathogens in the environment: implications for release of
genetically engineered microorganisms. Bio/Technology _3:817-820.
2. Franson, Mary U. (ed.). 1985. Standard methods for the examination
of water and wastewater. Sixteenth Edition. American Public Health
Association, Washington, D.C.
3. Goss, William A., W. H. Oeitz and I.M. Cook. 1965.- Mechanism of
action of nalidixic acid on Escherichia coll. II. Inhibition of
deoxyribonucleic acid synthesis. J. Bacteriol. 89:1068-1074.
4. Gray, L.D. and A.S. Kreger. 1985. Identification of Vibrio
vulnificus by indirect immunofluorescence. Diagn. Microbiol. Infect.
Dis. 2:461-468.
5. Hobble, J.E., R.J. Daley and S. Jasper. 1977. Use of nuclepore
filters for counting bacteria by fluorescence microscopy. Appl.
Environ. Microbiol. jj^:1225-1228. .
6. Jones, J.G. 1979. A guide to methods for estimating microbial
numbers and biomass in fresh water. Freshwater Biological Assoc.
Scientific Publication No. 39.
7. Kogure, K., U. Simidu and N. laga. 1979. A tentative direct micro-
scopic method for counting living marine bacteria. Can. J. Microbiol.
25:415-420.
-------
8. Pearson, A., R. Colwell, J. Byrd, J. Dennis, J. Grimes, M. Hood, D.
Uussong, C. Hutchinson, J. Lee, M. O'Brien, J. Stott and J. Walker.
1986. Discovery of viable but non-culturable Legionella pneumophila
by immune epifluorescence microscopy: evidence of amplification and
sources in five case studies. Bio/Technology (submitted).
9. Rollins, D.M. and R.R. Colwell. 1985. Viable but non-culturable
stage of Canpylobacter jejuni and its role in survival in the natural
aquatic environment. Appl. Environ. Microbiol. (in press.).
10. Roszak, D.B., D.J. Grimes and R.R. Colwell. 1984. Viable but non-
recoverable stage of Salmonella enteritidis in aquatic systems.
Can. J. Microbiol. 30;334-338.
11. Sauch, J.R. 1985. Use of immunofluorescence and phase contrast
microscopy for detection and identification of Giardia cysts in water
samples. Appl. Environ. Microbiol. 50:1434-1438.
12. Schmidt, E.L., R.O. Bankole and B.B. Bohlool. 1968. Fluorescent
antibody approach to study of Rhizobia in soil. J. Bacteriol. 95:
192-198.
13. Singh, A., R. Yeager and G.A. McFeters. 1986. Revival, growth and
pathogenicity of Escherichia coli after copper and chlorine-induced
injury. Abstract. Annual Meeting of the American Society of
Microbiology.
14. Strayer, R.F. and J.M. Tiedje. 1978. Application of the fluorescent-
antibody technique to the study of a methanogenic bacterium in lake
sediments. Appl. Environ. Microbiol. 35:192-198.
-------
10
15. Xu, H.-S., N.C. Roberts, L.B. Adams, P.A. West, R.J. Siebeling,
A. Huq, M.I. Huq, R. Rahman and R.R. Colwell. 1984. An indirect
fluorescent antibody staining procedure for detection of Vibrio
cholerae serovar 01 cells in aquatic environmental samples.
J. Microbiological Meth. 2:221-231.
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Appendix E
IN
PRESS
APPLICATIOH OF IOW EXCHANGE HPLC Dl THE PURIFICATION
OF 5S rRNAa SUITABLE FM SEQUENCE ANALYSIS
by
M.T. MacDonell*
Center of Marine Biotechnology
Uniyersity of Maryland
Adelphi, MD 20783
S.G. Morris
Beckfflan Instruments Inc.
Coluabia, MD 21045
B.A. Ortiz-Conde, C.J. Pillidge, and R.R. Colvell
Dept. of Microbiology
University of Maryland
College Park, MD 20742
Submitted to: J. Chromatography
Date: March. 1986
Running title: 5S RNA Purification
Corresponding author
-------
SOMAST
Ion exchange high performance liquid chromatography (HPLC) was
applied to the separation of transfer ribonucleic acids (tRNA) and SS
ribosomal ribonucleic acid (5S rRNA) from cell lysates. A simple
method involving a step elution of RNAs froa DEAE-cellulose followed by
separation of RNA species by HPLC was developed. This technique yields
5S rRNAs of a purity sufficient for ^sequence analysis in a fraction of
the time necessary for equivalent gel methods.
DfTBODOCriON
A simple, dependable size exclusion or ion exchange method for the
liquid chromatographic separation of tRNAs and 5S rRNA is not
available. Indeed, the method of choice for purification of small RNA
species consists of electrophoretic separation on denaturing
polyacrylamide gels. Methods for purifying small oligo-ribonucleotides
using either conventional (1,2) or thiol-soluble (3) polyacrylamide
gels are well developed. Although these methods are dependable and
result in minimal loss during recovery of samples from the gel, they
nevertheless fall short of liquid chromatographic methods in terms of
speed, labor, reduced risk of error, and ease of sample recovery.
Even though tRNA and SS rRNA molecules are significantly different
in length, chromatographic separation of the two molecules is not
necessarily easy. Transfer RNAs comprise a heterogenous collection of
specific amino acid acceptors, the average length of which is ca. 75
bases; 5S rRNA is virtually homogenous, comprising 116 to 122 bases in
prokaryotes, depending upon species. Despite a significant difference
in length, tRNAs and 5S rRNAs share remarkable similarities in
-------
secondary and tertiary structure (Figure 1), possibly attributable to
having derived from a common origin (4).
In this paper we describe a rapid and reliable method for
purifying of 55 rRNA from biological samples, with sufficient
homogeneity of the preparations for sequence analysis.
EXPERIMENTAL
Samples
An aqueous nucleic acid solution, rich in tRNA, messenger RNAs
(mRNA), 5S rRNA, and 16S rRNA, was prepared from bacterial cell paste
(approximately 1 gm wet weight) as follows. Cells were lysed using a
freeze-thaw technique (5) and phenol extracted (6) with a solution
composed of 892 (w/v) phenol, 0.1Z (w/v) 8-hydroxyquinoline in 50 mM
Tris borate EDTA (TBE), pH 8.3. After centrifugation for 10 min, at
12,000 x G, the aqueous (upper) phase was collected and precipitated in
2 parts cold absolute ethanol. The ethanolic precipitates were chilled
on crushed ice for 10 minutes (7) and collected by centrifugation (10
min at 12,000 x G). This "total nucleic acid" fraction was then dried
using a vacuum line.
Enrichment for RNAs by Ion Exchange Chromatography
The dried nucleic acid pellet was resuspended in 10 ml of 50 mM
TBE (pH 8.3) and adsorbed onto DEAE-cellulose (Cellex D, Bio Rad,
Richmond, CA), poured to a bed height of 4 to 5 cm in a 150 X 9 ma
disposable glass column (Chromoflex, Kontes Scientific Glassware,
Vineland, NJ). The column was washed with 2 volumes of 50 mM TBE, pH
8.3, followed by a 2 column volume rinse with 0.2 M NaCl in 50 mM TBE.
-------
The "small RNA" fraction was eluted with 0.5 M NaCl, and 7 M urea in SO
nM TBE. The RNA was precipitated by addition of 2 volumes of cold
absolute ethanol and chilling on crushed ice for 10 minutes. The
precipitated RNA fraction was collected and dried as described above.
Separation
The precipitated "small RNA" fraction was dissolved in 200 - 500
microliters of sterile*distilled water and injected, in 200 microliter
aliquots, onto a Spherogel DEAE TSK-IEX 5PW Ion exchange column (7.5 x
75 mm) (Beckman Instruments, Berkeley, CA) in the presence of 200 mM
KC1, 5 M urea in 20 mM NaP04, pH 6.9. and fractionated using a 2-step
linear gradient. A Beckman model 334 HPLC with a model 165 detector
was used throughout. The gradient program used on the HPLC controller
is given in Table 1.
Polyacrylaw.de Gel Electropboresis
Dried RNA samples were suspended in a tracking dye loading buffer
consisting of .052 (w/v) xylene cyanol, 0.05% bromophenol blue, 10 M
urea, 20 mM TBE (pH 8.3) and electrophoresed for 90 minutes at 40 V/cm
and stained with ethidiuo bromide. Presumptive identifications of tRNA
and 5S rRNA were Bade on a basis of banding pattern and location with
respect to tracking dyes.
RESULTS AND DISCUSSION
To test for the separation of tRNA and 5S rRNA, a mixture of
purified tRNA and unlabelled 5S rRNA was loaded onto the HPLC column
and a two-step linear gradient was run (see Experimental section).
-------
Results of this separation are shown in figure 2a. The chromatography
32
was then duplicated using P-5S rRNA (figure 2b). It was observed
that 5S rRNA was successfully separated from tRNAs in biological
samples by a two-step process involving enrichment for small
oligoribonucleotides, using conventional DEAE-cellulose ion exchange
column chromatography, followed by separation by ion exchange HPLC. In
this procedure, small fragments, monomers and very small oligomers
( <40 bases) are eluted from the DEAE-cellulose with 0.2 M NaCl in 50
mM TBE, after which the fraction containing tRNA, 5S rRNA, mRNA and 16S
rRNA was eluted with 0.5 M NaCl, 7 M urea in 50 mM TBE. Very large
oligonucleotides, plasmids and chromosomal DNA remain on the DEAE-
cellulose under these conditions. We have concluded that further
separation of tRNA and 55 rRNA is not feasible at this step since these
RNAs co-elute from DEAE-cellulose regardless of solvent conditions.
The "small RNA" fraction, i.e., tRNA through 16S rRNA, fraction was
precipitated in 2 volumes cold absolute ethanol, collected and dried as
above. The dried pellet was suspended in 100 ul of TBE and injected
onto the HPLC column. The chromatogram (figure 3a) shows several peaks
in the vicinity of the expected 5S rRNA peak. Of considerable interest
were those RNAs which eluted fro. (i) 322 to 38Z B, (ii) 381 to 39Z B,
and (iii) 41Z to 43Z B.
Samples were collected corresponding to peaks numbered 1, 2 and 3
in the chromatograa (Figure 3) and the nucleic acids precipitated. An
aliquot corresponding to peak 2 was reinjected onto the HPLC column
(Figure 3b) to evaluate chromatographic purity. In addition, aliquots
corresponding to peaks 1 through 3 were collected and electrophoresed ~
on 5Z polyacrylaaide to presumptively identify the RNA species, based
-------
on characteristic mobilities. Bands corresponding to peaks 1 and 3
•igrated as tRNAs, while peak 2 migrated as 5S rRNA (figure 4).
To identify peak 2 as 58 rRNA, nucleic acid was collected, end-
32
labeled with (gamma) P ATP, using the method of Richardson (8), and
sequenced enzymatically (11). The results of the sequence analysis
indicated that the 5S rRNA band was homogenous and clearly identifiable
as the sequence of 5S rRNA.
V
CONCLUSIONS
Despite the tendency of tRNAs and 5S rRNAs to co-elute during
chromatographic separations of crude RNA solutions prepared from
cell lysates, adequate separation is possible by ion exchange HPLC to
allow sequence analysis of 5S rRNA collected directly from the eluate.
This represents a considerable savings in time, labor, and materials,
and prevents loss of sample, compared with purification using
polyacrylamide gels.
ACKNOWLEDGEMENTS
The authors wish to acknowledge D.B. Roszak for helpful discussion.
Support for this research was provided by NSF Grant BSR 84-01397,
Office of Naval Research contract NOOO-14-81-K0638, and The University
of Maryland Center of Marine Biotechnology, and Environmental Protection
Agency Contract CR812246-01-0.
-------
KEFcREIfCu
1 J.N. D'Alessio, RNA Sequencing, In D. Rickvood and B.D. Haaes
(eds.)t Gel Electrophoresi3 of Nucleic Acids. IRL Press, Oxford,
(1982) 173.
2 B.R. Jordan, Studies on 5S RNA conformation by partial ribonuclease
hydrolysis, J. Mol. Biol., 55 (1971) 423.
3 J.N. Hansen, Use of solubilizab,le acrylamide disulfide gels for
isolation of DKA fragments suitable for sequence analysis, Anal.
Biochem., 116 (1981) 146.
4 D.V. Mullins, Jr., J.C. Lacy, Jr., and R.A. Hearn, 5S rRNA and
tRNA: Evidence for a common evolutionary origin, Nat. New Biol.,
242 (1973) 80.
5 L. Zablen, L. Bonen, R. Meyer, and C.R. Woese, The phylogenetic
status of Pasteurella pestis, J. Mol. Evol., 4 (1975) 347.
6 J. Marmur, A procedure for the isolation of deoxyribonucleic acid
from micro-organisns, J. Mol. Biol., 3 (1961) 208.
7 J.A. Zeugin and J.L. Hartley, Ethanol precipitation of DNA, Focus
7/4 (1985) 1.
8 C.C. Richardson, Phosphorylation of a nucleic acid by an enzyme
from a T4 bacteriophage-infected JE_. coli.. Proc. Nat. Acad. Sci.
USA, 54 (1965) 158.
9 S.R. Holbrook, J.L. Sussman, R.V. Warrant, and S.-H. Kin, Crystal
structure of yeast phenylalanine transfer RNA. II. Structural
features and implications, J. Mol. Biol., 123 (1978) 631.
10 T. Pieler and V.A. Erdoann, Three-diaensional structural model of
eubacterlal 5S RNA that has functional implication, Proc. Nat.
Acad. Sci. USA, 79 (1982) 4599.
-------
11 H.T. MacDonell and R.R. Colwell, Nucleotide base sequence of
Vibrionaceae 5S rRNA, FEBS Lett. 175 (1984) 183.
-------
TABLE 1. HPLC Controller Program Listing
Time3
0:00
0:00
0:00
4:00
14:00
Function
Percent B
Flovrate
Chartspeed
Percent B
Percent B
Value Duration
20Z
1 ml/min
0.1 cm/min
30Z 10 min
602 - 60 min
aExecution time from start of program.
Length of time allowed for the completion of a program instruction.
Example: in the last line, percent B would increase from 30Z (previous instruction)
to 60% over a period of 60 minutes.
-------
FIGURE LBGEMDS
Figure 1. Predicted tertiary structures of (a) tRNA (adapted from
[9]) and (b) 58 rRNA (adapted from [10]).
Figure 2. HPLC Chromatograas of an adaizture of purified yeast
phenylalanine tRNA and purified Escherichia coli 5S rRNA.
Column - Beckjnan Spherogel TSK-IEX DEAE-5PW, mobile phase
200 to 600 oN KC1 in 5 M urea, 20 mM Sodium phosphate
buffer, pH 6.9, in a two-step linear gradient. Flowrate » 1
ml/min, chart speed - 1 mm/min, range - 0.2. (a) optical
density profile, (b) repeat of (2a) in which 5S rRNA was
identified in the elution order by spiking the admixture
with E. coli 32P-5S rRNA.
Figure 3. HPLC chromatograms of the "small RNA" fraction (see text for
discussion), (a) Chromatogram of the crude RNA fraction
prepared from the bacterial cell lysate. Eluates
corresponding to peaks 1-3, selected on a basis of their
elution order and positions relative to the buffer gradient,
were collected and ethanol precipitated. Aliquots of RNAs
from each peak were electrophoresed on polyacrylaaide for
verification (see Figure 4). An aliquot of peak 2 was
reinjected onto the HPLC column, (b) Chromatogram of the
RNA collected from peak 2. Chromatography conditions were
as described in Figure 2.
-------
Figure A. Ethldium bromide-stained SZ polyacrylaaide gel after
electrophoretic separation of ethanol precipitates
corresponding to peaks 1-3 (see Figure 3). Lanes 1 and 3
correspond to peaks 1 and 3 in the chromatograa. The
pattern and location of bands in lanes 1 and 3 are identical
to those expected of tRNAs. The prominent band in lane 2
(corresponding to peak 2) is consistent with the expected
«»
position of. 55 rRNA (see text for discussion). Locations of
tracking dyes bromophenol blue (bpb) and xylene cyanol (xc)
are indicated.
-------
140X
80 X
-------
Range 0.2 —
10 20 30 40 90 00
,- 16
I- 12C
80
40C
MIN
-------
a.
b.
Range 1 O -
GRADIENT
%B
60
SO
4O
tRNA
20 30 40
MIN
-------
-------
OU COD: I
OBJECTIVE
TITLE
CODE
ENVIRONMENTAL RESEARCH LA30RATORY
3ULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q285
• L134 Chemical Testing t Assassmant
TITLE : G Develop and Validate Improved Risk Assess Mthds
?PA CODE I TITLE : 02 Environmental Aspects of Biotechnology
PROJECT CODE £ TITLE : 24 rcol Consequences of Gen Eng Microorgs
PLAN TYPE
INVESTIGATOR :
PHONE
FT:
: COOP
3 CusKey
635-9011
PRINCIPAL INVESTIGATOR: R Miller
WORK PLAN TITLE : Genetic Transfer in Aquatic Environmants.
WORK PLAN OBJECTIVE : To determine the ability of genetic determinants to ba
trsnsferrad between organises in an aquatic environment. The three known
macnanisms of DMA transfer oetwaen organisms; transformation* transduction,
and conjugation mill be tested in this regard.
'•JCRK PLAN APPROACH : Pseudomonas aeruginosa mill be usad in transduction and
conjugation experiments. The tamperate bacteriophage, F116, which has been
shoujn to promote DNA transfer in natural env/ironments mill also be used in
the transduction studies. -Both plasmids FP2 ^nd R63.45 which promote
conju-al transfer of DNA between organisms mill be used in conjugation
studies. The use of R65.45 is especially appropriate for this work because
it has baan shoan to transfar to a wide range of gram negative organisms.
Psaudomonas stutzari 'jill be usad for transformation studies because it is
naturally transformed (as opposad to P. geruginosa u/hich must be made
competent for trans^ormation ," artifically). Laboratory defined media as
uiall as sterile laka ojater samples uiill be us?d to determine the maximal
rat^s of genetic transfer. Paramstars to be approached in th<3se initial
studies include: minimum densities of donor and recipient populations!
multiplicity jf inf-3ctiont temperature, pHf surface substrate effects and
c oncentration s of dissolved organic matter. These ujill ba studied with
botn ;Dure cultures an-3 mixed populations.
Field in ves ti ja t ions .uill be conducted in conjunction with the laboratory
o xp>ir im ?n t s. Initial studies mill determine the phy sico-chamical and
microbiological nature of botn a ocllutsd and clean habitat selected for
study. The prasenca of homologous ONA (to that being tasted in "released"
organises) in t h e s a environments jjill bg determined using colony
hybrinzstion techniques. In lat>?r exp3rim?nts» jene transfer u/ill be
tested using f 1 ouu-t nrough chambers.
STATUS
On Schedule
ACCOMPLISHMENTS : Laboratory studies of the naturally
which mas previously shoujn to transduce plasmid ON A
was shO'jun to be a generalized transducing pha^e and
w a i 3 h t than previously described tr^nsducinc; phages
intraspacific transfer of plasmid DNA via transduct
coirplatad in this quarter. Transduction of plasmid
recipient strain u/ss tested using phage lysat?s and
recipients* Analysis of research results indicated
was greatest jhan the transducing phage was lysogen
or tha racipiant. Tha 3hort-lived viability of pha
lysatas was probably the reason that low levels of
saen in this tast system. T h a s a data indicate that
in aithar donor or tha recipeint calls can transfer
aquatic ecosystems. Tha addition of natural bacter
isolated bacteriophage
ware completed. This
had a larger molecular
. Field studies on
ion were started and
DNA to a model
lysogenic donors or
that plasmid transfer
ic in either the donor
ge added as cell-free
plasmid tran-s4er wara
lysogenic phage present
plasmid DNA in natural
ial isolates to tha test
-------
ENVIRONMENTAL RESEARCH LA3Q3ATGRY
3ULF BREEZE , FLORIDA 32361
FY '86 WORK PLANS
DATE : 7/ 2/36
•VORK PLAN CODE
Q285
systems caused a loss of viability of both Honor and recipeint strains,
Field studies on conjugal plasmid transfer were i
MILESTONES. (DATES) : TAR3ET
Optimization studies on 01/86
transduction of plasmid DMA
Conventional laboratory 01/85
experiments on conjugation
systens and genetic markers to
b 3 u s 3 d
Collection of field survey data 01/86
Dat3rnination of natural 04/86
populations from f i •» 1 d sites
Analysis of field data on 04/85
intraspecific transJuction
Winter experiments to test 04/35
seasonal variation -it field
sites
REVISED REVISED ACTUAL
o i / im
01
04/
04/3
Analysis of field data on
interspecific transduction
Conventional laooratory
exp^rimants on tr,e
transformation of plasm id D'JA in
P. stutieri
Start conjugation studies at
Doth test sites
07/85 10/SS
07/36
10/35
07/35 10/8-
Start transformation studies at 07/35
both test sites
Laboratory studies on 10/86
conjugation optimization
Laboratory studies on 10/86
transformation oatinization
Journal article: Intraspecific 07/35
transduction of plasmid 0 N A in
situ
Journal article: Trsnsformat ion
10/36
-------
of P. stutzeri
plasmid DNA
ENVIRONMENTAL RESEARCH LA303ATQ3Y
GULF BREEZE , FLORIDA 32561
FY '36 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : QZSS
ujith homologous
Final report on the effacts of
physico-chemical and biological
factors on genetic exchange in
aquatic environments ( 7 2 7 1A )
12/87
-------
PROGRESS REPORT 1 APRIL, 1986 TO 15 JUNE, 1986
ASSISTANCE ID NO CR812494-01
"GENETIC TRANSFER IN AQUATIC ENVIRONMENTS"
Robert V. Miller, P. I.
During this period we have concentrated our efforts on
various factors involved in the transfer of plasmid DNA by
transduction. We have carried out experiments both in the
laboratory and at field sites. Several different models of the
introduction of plasmid DNA into the environment have been
investigated in tfrese initial field trials. While the analysis
of our field data is not complete (the experiment was concluded
14 June, 1986) several interesting findings are emerging from
both the laboratory and environmental experiments.
Laboratory Studies
As outlined in our previous report, we have been using two
transducing phages in our studies. While F116 is well
characterized, f^DSl was isolated because it was suspected of
transducing plasmid DNA. As we wished to use
-------
sequences. This will be estimated by in situ colony
hybridization.
3. Effect of Mg++ on transduction.
We had observed that transduction frequencies were
several fold higher when phage* adsorption was carried out in
TNM buffer than when it was carried out in water from the
test sites. As TNM buffer contains 10 mM Mg"*"*", we asked if
varying the concentration of Mg in lake water samples
would effect the transduction frequency by 4DS1. The
presence of Mg increased the frequency of transduction
(ie. transductional events/10 input phage) by approximately
3 fold. By ir-tself, Mg does not appear to be a highly
significant variable.
4. Models of -transduction in defined media.
We have hypothesized that the natural reservoirs of
temperate transducing phage are likely to be lysogenic
bacteria. We therefore set up two model systems in the
laboratory to test the potential for plasmid transduction in
systems containing lysogenic bacteria as the source of
transducing phage. These experiments were carried out in
large volume cultures.in a defined minimal medium. Two
systems were investigated. In the first, the plasmid
containing donor strain was lysogenic for the phage and the
recipient strain contained neither the phage nor the
plasmid. In the second, the plasmid-containing strain was
non-lysogenic and the recipient strain was a «$DS1 lysogen.
While lysogeny makes a strain immune to superinfection, it
does not inhibit adsorption of phage and the injection of
DNA. Lysogenic recipients show higher frequencies of
transduction because- transductants are protected from lysis.
We found transfer of the plasmid to the recipient strain in
both test systems. The second system is particularly
significant as it suggests that resident populations have
the potential to act as the source of the transducing phage.
Field Studies
During the past two weeks we have been carrying out field
studies at Fort Loudon Lake in Eastern Tennessee. Several
different model environments were tested. These included systems
in which transducing lysates and non-lysogenic recipients were
introduced into the test environment, systems in which the
plasmid donor was also lysogenic for the transducing phage, and
systems in which the plasmid donor was not lysogenic but the
recipient strain was a lysogen. F116L and
-------
1. The direct introduction of phage particles into the
model system was not as effective as the introduction of a
lysogen. Viability of phage lysates was short lived while the
induction of lysogens supported a high phage titer
throughout the experiment. This was the case even though
there was no substantial increase in viable cell numbers in
the test chambers. •-.
2. The resident bacterial community reduced the ability of
both the plasmid-containing donor strain and the introduced
recipient strain to survive in the test environment.
3. Transducjtion was observed in test chambers which
contained a non-lysogenic plasmid donor and a lysogenic
recipient strain.
These preliminary results support our hypothesis that the
most effective environmental reservoir for transducing phage is
the presence of lysogenic bacteria. In addition, it would appear
from this preliminary analysis that such lysogens can serve not
only as a source of transducing virus but also as recipients for
transduction of plasmid DNA introduced into the environment in
non-lysogenic bacterial strains.
-------
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY "86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN cooe : 0292
DU CODE £ TITLE : L104 Chemical Tasting and Assessment
OBJECTIVE CODE & TITLE : 3 Develop & Validate Improved Risk Assess Meth
?PA CODE & TITLE • 02 Environmental Aspects of Biotechnology
PROJECT CODE & TITLE I 24 ceo Consequents of Gen Eng Microorgs
JORK PLAN TYPE : COO?
INVESTIGATOR : A Bourquin PRINCIPAL INVESTIGATOR: R. Olsen
PhONc NO : FTS 636-9011
WORK PLAN TITLE : Development, Characterization and Utilization of Bacteria
Benchmark Piasmids
WORK PLAN OBJECTIVE : Determine the likelihood of» and the molecular basis for
changes (.nutations) in GEM's that might occur subsequent to the development
and ralease of a GEM for usa to facilitate studies for risk-assessment,
WORK PLAN APPROACH : For our worK towards tha stated objectivias, we mill
utilize broad host range bacterial cloning vectors previously developed in
our laboratory (5) and other vectors of extanded host range currently under
development in our laboratory as oart of an E^A collaborative agreement
(EPA grant no. 312679). Thasa vectors have the advantage of being
transferrable to a full range of psaudomonads and related Gram-negative
bacteria by either transformation or mobilization. The behavior of the
cloned fragments can therafora be determined in combination with the unique
metabolic traits of a wide range of bacterial species. The sources of ONA
for these initial experiment's" will be plasmid pJP4 (3) and the cloned
fragment reportad by Amy et al. (4). The racombinant plasmids uie derive
will be physically nappedi using common restriction endonucleases
appropriate to locate and define CPO activity as determined by assay of
cell preparations which contain the clones and their subclones. Once the
foregoing has Daen accomplished» tha relevant clones will be exposed to the
2»4-D analogue, phenoxyacetic acid (PAA). Previous work by Pemberton et
al. suggests that tne 2,4-C CPO gana of pJP4 nay be duplicated and mutate,
resulting in activity towards PAA. We have confirmed this by in vivo
experimants in strain PJM134 bearing the archetypal plasmid pJP4
(unpublisned observation). This mutant strain, designated AE0101, aiill
also be used to clone the irutant CPO activity for comparision uiith parental
analogous clones and clones which hava bean mutatad towards CPO activity
subsequant to cloning.
Such conpan'sons will bs done to alucidata at the molecular level
evolutionary divergance with regard to substrata specificity or the
evolution of broader activity towards structural analogues of the parent
compound, 2,4-D.
Parental and mutated donas will be analyzed for tha regulation of CPO
activity. Previous reports suggest that such activity -nay be constitutive
in one instance towards the herbicide 2,4,5-trichlorophenoxyacetic acid
(2,^,5-T) (6). Howaver, tha plasmid showing such activity was the result
of extensive and indeterminant molecular reorganization concurrent with the
progress of a "olasmid-assisted molecular breeding" experiment (7). Ona
cannot oe sure, then, that tha enzymic activity studied was, in point of
fact, constitutive in the parental progenitor. On tha other hand, .nutation
towards constitutivity nay annanca the utility of an avolved pathway. We
hopa during tne course of cur studies to distinguish between these
possioilitias with regard to the CPO activity originating in plasmid pJP4
or the cloned fragment reported by Amy at al. (4).
-------
ENVIRONMENTAL RESEARCH LA3Q3ATC'*Y
GULF BREEZE , FLORIDA 32561
FY '8.5 .JORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q292
A topical summary of the experiments to ba done is as follows:
1. Clone the chlorophenol oxygenase (CPO) of the c hlorophenoxyacetata
upper pathway CD.
2. Mutate the substrate specificity of CPG
3. Qetarmine the regulation of CPO. -
4. Match tre cloned CPO activity uiitt\othar phenol degradative activities
and determine the potential for mutation towards the mineralization of
phenolic structural analogues.
5. Apply dataction and enumeration protocol as they obtain from other
E PA-sponsorsd tuork to the parant and evolved GEM's.
STATUS : Final Report: Development of bf.ctarial models for ris* assessment:
Use of benchmark plasmids end DNA saqu2nc?s.
ACCOMPLI SH IE NT S : °ursuant to ou- original rss-^arch plun, benchmark bacterial
strains hava bean ascartain ad and cnaractarizad, In addition* benchmar*
plasmid donor strains containing a ujide spectrum of transposons encoding
antibiotic resistance have be *n developed. W? have initiated distribution
of these strains to EPA-funded projects.
Wa nave b?sn consulting with tha Ti?dje Laboratory* Michigan 5tata
University and assistin-j them in tha incorporation of the benchmark
trans f err able plasmids in th3ir E°4 supaortsd ras-??rch and other projects
in this laboratory,
Non t r ans f srraol ? versions of. several of th? banch.nark plasmids have baen
constructed ?.id evaluated. these p!?snids contain antibiotic resistance
m-arxers for c ar 'ssnici 11 in » te tr acycline and/cr trimethoprim.
We have dav = lDp3d a bacterial 'transformation system for the bacterium •
P seuoomon as cepacia. Tnis 5yst-?m h?s oaen 'jpti'nized for several P. cspacJti
strains. To our knouiledgej this 1-3 the first example of such a system
u/hich noui p?r:nits uxpanmsnts. in molecular ^?netics direction (i.e.*
s-al f-cl on in 5) on this ecologically significant bacterial strain,
(Manuscript m praparation for submission to t h 3 journal of Bacteriology*
July, 195 6)
We have dsvelopad plesmid cloning vectors designated pR32317, pR02320,
p.%C23st c /to-au to toxici ty) for
use tuith benchmark plasnids. This strain is -sli.nin 2t 3d from microbial
populations cultured or plstad under certain nutrient conditions* allowing
groMitn of o*.h.?r bacterial strains. This cont ^ ins'ola strain is not I
auxotrophic end therefore can bj includad md s-jstained under environme ntM.
conditions >a h e r a mineral -3 aits and a cs^bor so urea =»ra the nutrients.
MILESTONES (DATES) : TARGET RJVISfD REVISED REVISED ACTU
Construct* test first series of 12/35
1
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '36 WORK PLANS
D4T5 : 7/ 2/36
WORK PLAN CODE : Q292
benchmark plasmids CTra + u/ith
transposons Tnl, TnSOlt Tn904,
Tnl722)
Derive .uork plan and set-up 12/85
post-doctoral felloju lab at
Corvallis
Derive mork plan and set-up 02/36
post-doctoral felloui lab at Gulf
Breeze
Derive and authenticate plesmid Q4/8S
p J P 4 gene banks at (Ann Arbor
and Corvallis labs).
Construct g^nomic gene oanks of 05/86
biomass converting
streptortycetes Cat Ann Arbor an j
Gulf freeze labs).
Derive and test Tra- benchmark 36/85
plasmid sat for E?A
investigators (at Ann Arbor
lab).
Evaluata ?XiDr9S5ion of 07/85
biomas5-convsrting ^ene bark
clongs Cat Ann Aroor ?ind Gulf
Breeze Laos)
Identification of Qjna oank 10/35
clones associated uiith the
degradation of lignin Cat Ann
Arbor and Gulf ireeze labs).
Derive and test Tra- benchmark 12/86
plssmid 33t ujj.tr> transposition
functions del9t?d for EPA
investigators Cat Ann Arbor and
Gulf 3reeze labs.)
Complv?te enzymological 3nd 02/37
regulation studies on pJP4 c,ene
bank recombinant plasttiids Cst
Ann Arbor and Corvallis labs)
Physical and functional mapping 02/87
of gene bank clones Cat Ann
Arbor and Gulf freeze labs).
Prepare and inventory benchmark 06/37
plasmid specific probes iuith
knouin sequences for distribution
-------
ENVIRONMENTAL RESEARCH LABORATORY
;ULF
BREEZE
FY '86
CATS :
to EPA investigators
Arbor lab).
Cat Ann
, FLORIDA 32551
WORK PLANS
7/ 2/36
WORK PLAN CODE : Q292
Prepare recominant plasmids for 06/37
use by EPA investigators in
microcosom studies Cat julf
Bre3Z3» Corvallis and Ann Arbor
labs).
Characterization of the 06/87
enzymological basis for biomass
conversion of metabolizable
compounds Cat Ann Arbor and Gulf
Breeze labs).
Final report on pJP4-related
work at Corvallis and Ann Arbor
labs
11/87
Final report
activities
on Gulf 3reeze lab
01/88
Collaborate, consult uith Gulf 07/83
Breeze - biomass project and '
Corvallis - c'nloro-hydrocarbon
degradation projects; prepare
publications and reports.
Expression of strepto'nycete DNA 03/85
cloned into Pseudomonas
asruginosa ?AQ1. CProc. A.S.M.)
Journal Article! °rotocat3chuic 06/86
acid dioxyg3n353 from
?seudomonas capaciai Cloning the
structural genes using an
IncW-derived vector, pRC2317.
C xxx xo )
ASM Abstract: Cloning and
expression of halo—aromatic
genss in Pseudomonas cspacia
D3C1.
09/86
-------
Quarterly Report (06/20/86)
Ronald H. Olsen* Ph.D
University of Michigan* Ann Arbor MI
This report is constructed in three sections: (i), work only performed at
the Ann Arbor laboratory; (ii), work performed at the Ann Arbor laboratory and
at the ERL, Gulf Breeze Florida; (iii),' work performed at the Ann Arbor
laboratory and at the ERL, Corvail is Oregon in collaboration with Dr. Ramon
Seidler.
I. Work only at the Ann Arbor Laboratory.
PRINCIPAL INVESTIGATOR:
Ronald H. 01sen
WORK PLAN TITLE:
Development! characterization and utilization of bacterial benchmark
plasmids for environmental risk assessment and biotechnology applications.
WORK PLAN OBJECTIVE:
Construct a "standard set" of, plasmids to be used in ecological studies to
serve as a base line - reference for the evaluation of behavior of other
unrelated and perhaps uncharacterized plasmids indigenous to systems proposed
for the individual projects.
WORK PLAN APPROACH:
Plasmid R388 will serve as a starting point for the subsequent development
of derivative plasmids with special properties including disabled transfer, the
addition of unique antibiotic resistances (transposable or not) and other DNA
sequences to be determined by the needs of the EPA environmental research
laboratories. Standard sets of donors and recipients will be characterized to
be used in laboratory studies using conventional genetic transfer techniques
and for used in microcosm/mesocosom models in ecological studies.
STATUS:
Ahead of schedule.
ACCOMPLISHMENTS:
1. We have been consulting with the Tiedje laboratory) Michigan State
University and assisting them in the incorporation of the benchmark
transferable plasmids in their EPA supported research and other projects in
this laboratory.
2. Non transferable versions of several of the benchmark plasmids have
been constructed and evaluated. These plasmids contain antibiotic resistance
markers for carbenicillin, tetracycline and/or trimethoprim.
3. We have developed a bacterial transformation system for the bacterium
Pseudomonas cepacia. This system has been optimized for several P. cepacia
-------
Quarterly Report to ERL, 06/50/86 (continued)
strains. To our knowledge» this is the first example of such a system which
now permits experiments in molecular genetics direction (i.e., self-cloning) on
this ecologically significant bacterial strain. (Manuscript in prepartion for
submission to the Journal of Bacteriology, July, 1986.)
4. We have developed plasmid cloning vectors designated pR02317,
pRQS320, pRQ2321 from the parental benchmark plasmid, R388. The utility of
these cloning vectors has been verified in bacterial strains P. putida, P.
aeruoinosa, P. ceoacia. Escherichia coli and Alkalioenes eutroohus.
5. Using the above benchmark-derived cloning vectors, we have cloned and
determined the expression of the ring fission enzyme, protocatchuate
dioxygenase, from P. cepacia in disparate Gram negative bacterial strains.
6. Studies are under way to determine gene expression in disparate hosts
for the enzyme, catechol dioxygenase, cloned from P. aeruginosa.
7. Preliminary work is completed, and utility established for a
bacterial donor strain which is self destructive (selective cyto-autotoxicity)
for use with benchmark plasmids. This strain is eliminated from microbial
populations cultured or plated under certain nutrient conditions allowing
growth of other bacterial strains. This containable strain is not auxotrophic
and therefore can be included and sustained under environmental conditions
where mineral salts and a carbon source are the nutrients.
MILESTONES (DATES):
Final report on benchmark plasmids and biotechnology applications due
09/31/86.
II. Work done at ERL, Gulf Breeze and Ann Arbor laboratories.
PRINCIPAL INVESTIGATOR:
Ronald H. Olsen
CO-PRINCIPAL INVESTIGATOR:
Susan McCarthy (Post-doctoral fellow at ERL, Gulf Breeze, Florida
WORK PLAN TITLE:
Development of risk-assessment protocol for evaluating heterogenetic DNA
(Gram positive) detection in Pseudomonas; cloning and characterization of
lignase-related metabolism from Streptomyces viridosoorus to Pseudomonas
bacteria.
WORK PLAN OBJECTIVE:
Construct and characterize a model GEM - gene sequence(s) for future use
in the evaluation of detection protocol related to risk-assessment.
-------
Quarterly Report to ERLi 06/50/86 (continued)
WORK PLAN APPROACH:
A series of cloning vectors has been developed for the EPA as part of the
benchmark plasmid series (Ann Arbor). Me Mill use these vectors for the
cloning of lignin metabolism-related genes from Streptomvces viridosoorus into
other Gram negative bacteria. Preliminary work has established the efficacy of
this using DNA cloned from S. viridosoorus which encodes the degradation of
ferulic and para-coumeric acidst lignin-related monmers. We are duplicating
this result* starty with 5. viridosoorus DNA since this strain has been
reported to degrad polymeric lignin. Thus, such clones derived from the strain
should contain a broader range of degradative activities on lignin substrates.
STATUS:
Behind schedule (reflecting technical difficulties at the Gulf Breeze
ERL.) This deficiency has been corrected.
ACCOHPLISHHENTSs
1. Plasmid vector and S. viridosporus ONA has been prepared at the Gulf
Breeze laboratory.
2. Or. McCarthy spent a week at the Ann Arbor laboratory and
successfully started the construction of a S.. viridosoorus gene bank library.
HILESTONES (DATES)1:
Final report on benchmark plasmids and biotechnology applications due
09/31/86.
III. Work at ERL, Corvallis Oregon and Ann Arbor laboratories.
PRINCIPAL INVESTIGATOR:
Ronald H. Olsen
CO-PRINCIPAL INVESTIGATORS:
Alan Marker* Research Associate (ERL, Corvalis Oregon)
Ramon Seidler (Director, Biotechnology and terristrial microbiology,
Corvallis Oregon)
UORK PLAN TITLES
Studies related to development of risk-assessment protocol for an
evaluation of the mutability of a genetically engineered microorganism (GEM):
cloning and characterization of the 2,4-D chlorophenoxyacetate oxygenase (CPO).
UORK PLAN OBJECTIVE:
Determine the likelihood of, and the molecular basis for changes
(mutations) in GEM's that might occur subsequent to the development and release
of a GEM. We Mill derive plasmids to determine the influence of these, changes
on detection for risk-assessment studies.
-------
Quarterly Report to ERL» 06/50/B6 (continued)
WORK PLAN APPROACH:
Ma utilize broad host range bacterial cloning vectors previously developed
in our laboratory and cloning vectors developed recently as part of the
benchmark plasmid plasmid derivations described elsewhere herein to clone,
characterize and mutate genetic determinants intrinsic to the 2,^-dichloro-
phenoxyacetic acid degradative plasmid pJP<». The choice of the pJP<» metabolic
plasmid was mitigated by its utility and use by many laboratories as a starting
point for genetic alterations useful for the treatment of toxic chlorinated
aromatic compounds in the environment. We are particularly concerned with the
first step in the pJP4-encoded pathway, the side-chain cleaving enzyme (2,<»-
dichlorophenoxyacetate oxygenase). The literature indicates this step may
mutate to accomodate related substrates. We are studying the mutability of
this step to determine the influence of this on detection. We are preparing
gene banks of pJP
-------
Quarterly Report to ERL> 06/50/86 (continued)
in our laboratory.) (Ann Arbor lab.)
8. Enzyme assays have been developed for pJP^-encoded enzymes expressed
in Alkaligenes or P. ceoacia DB01. (ERL-Corvallis lab.)
9. Growth and cell harvest protocol have been developed and optimized
for the production and assay of pJP<»-encodedv enzymes. (ERL-Corvallis lab.)
10. We have transferred the cloned Hindi 11 fragment from recombinant
plasmid pSA122 (Amy et al., Corvallis) to vector pROS3Sl and therefore gene-
expression studies can now be done in PseudtKnonaa, and AJkalioenes bacterial
strains now that we have developed of transformation systems for these
bacteria. (Ann Arbor lab.)
MILESTONES (DATES): •-»*-——.. —,
Final report on benchmark piasmids and biotechnology applications due
09/31/86. - - --
-------
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , <=LORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q282
DU CODE & TITLr : L104 Chemical Tasting £ Assessment
OBJECTIVE CODE £ TITLE : G Develop & Validate Inproved Risk Assessment
Mthds
"PA CODE E, TITLE : 02 Environmental Aspects of Biotechnology
PROJECT CODE & TITLE : 2<* ccol Consequences of G?n Eng Microorgs
WORK PLAN TYPE : COOP
INVESTIGATOR : T Barkay PRINCIPAL INVESTIGATOR: 3 Saylar
PHONE MO : 636-9011
WORK PLAN TITLE : Genetic Approachas for Dat ar.iiining Parsistance and Effects
of Introduced Species.
WORK PLAN OBJECTIVE : Effects of genetically engineered organisms in estuarine
coastal and open ocsan waters is of fundamental interest and an
undarstanding of the p-otential affects must be achieved if adequate
controls sre to be provided. Before the broader goal of understanding
effects can be accomplished, however, there must first be developed the
means by which the survival and effects of novel organisms can be measured
accurataly and precisely. The overall goal of this project is to evaluate
the use of QNA reassociation kinetics or total genetic complexity as a
measure of the survival and effects of GEMs on nicrobial communities*
The specific objectives of this project are tot 1) develop a method for
measuring the complexity of the population genone Can organismal
rapresentation of the total genetic complexity of a microbial community)
using DMA raassociation kinetics, 2) measure genetic complexity of the
population genome for an aquatic microbial community, and 3) evaluate the
survival and effects of DNA elements introduced into the population of an
aquatic microbial community through augment-it ton of DMA reassocia tion
kinetics Jith DNAZCiJA hybridization probe analysis.
WORK PLAN APPROACH : The complexity of the population genome, measured by
r aassoc ia t ion kinetics (Cot values), mill b.? us-ad to define the genetic
diversity of natural aquatic bacterial communities. 3y analogy to
eukaryotic genomes, the population genome is defined as the complement of
DNA in which each sequence is present in tha sa>ne relative frequency as it
is in the population, and in which the least frequent sequence is present
onca. Changes in the ecosystem as a result of the application of G'-Ms
could ba followed by their effect on tha genetic complexity of the
microbial community. TJJO independent factors may effact the observed
r aas soci a t ion curves: A) The fraq-jency of a specific species in the
community and 3) The frequency of specific DNA sequences in each species or
common to some species. AS a result, interpretation of the observations
necessitates that additional specific DNA s
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
Ff '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : 0282
2. Methods for cell lysis and purification of community genomes have been
developed and found appropriate for obtaining DNA for raassociation
kinetics. These methods are currently applied to analyse the community
complexity of samples obtained from the field.
3. A detailed study of the sorption Qf DNA to sediment constituents has _
been completed. From the results* it Appears that the presence of clays !•
environmental samples seriously affects the efficiency of DMA recovery. •
Alkaline extraction is appropriate for recovery of the sorbed ONA. Organic
matter had little effect on the recovery of DNA. Houisvar, humic substanc
interfered with reassociation studies. This problem jias solved by apply!
column chromatography prior to GC analysis
4. A community genome comprised of equal mass of ONA originating in 9
representative organisms has been obtained.
4. w
I
MILESTONES (DATES) : TARGET REVISED REVISED REVISED ACTU<
Final Report on Genetic 12/87
Complexity of Aquatic Microbial
Communities as a Measure of
Ecosystem Effects Induced by the
Application of Gems. (7270A)""
-------
Progress Report - 7-1-85 to 6-30-86
Cooperative Agreement; CR812488-01-0
Project Title; Genetic Approaches for Determining the Persistence and Effects
of Introduced Species
Principal Investigators: Gary S. Sayler, Professor
William S. Riggsby, Professor
Institution: The University of Tennessee
Department of Microbiology and The Graduate Program of Ecology
Project Objective^
(I) Develop methods for measuring the genetic complexity of a raicrobial
community using DNA reassociation kinetic analysis (Cot analysis).
(2) Measure the genetic complexity of an aquatic microbial community and,
(3) Evaluate the survival and effects of DNA elements introduced into the
community using reassociation kinetics and DNA:DNA hybridization probe
analysis.
Research Progress
(1) Cell concentration and-harvesting: For population reconstruction
experiments individual bacterial species were harvested from culture via
conventional centrifugation prior to cell lysis and DNA extraction. For
populations recovered from natural field samples, it was calculated that up to
1000 liters of reservoir water would need to be processed to recover sufficient
cell mass for DNA extraction and reassociation kinetic analysis. Hollow fiber
ultrafiltration (Microgone) was initially examined for concentrating cells from
large volumes of dilute reservoir water. This method proved inefficient due to
slow processing rates and clogging of hollow fibers by suspended clay particles.
A Millipore Pellicon, tangential flow, cell harvesting system was eventually
chosen for concentrating cells from reservoir water. The final system uses 10
sq. ft. of 100,000 MW ultrafiltration membrane in a coarse screen channel,
operated at a tangential flow across the membrane of approximately 3 gal
min~l. A 5 urn, nominal cutoff, polypropylene prefilter was selected reduced
contamination by suspended particulates and larger eucaryotic organisms. The
system operates in a recirculation mode through a retentate tank. Average
processing rates are approximately 1500 ml rain"* with a total processing
time for a 1000 liter sample of approximately 10 hrs. Samples up to 980 liters
have been concentrated to 1.5 liters at which time the sample is iced and
returned to the laboratory to harvest concentrated cells via centrifugation.
(2) Cell lysis extraction and purification of DNA; Initial procedures for
standardizing the lysis of a heterogenous microbial population was established
for 10 microbial species of varying degrees of ease of lysis with Brevibacterium
spp. the most difficult to lyse. Based on microscopic observations and plating
of the lysate, lysis was achieved for all species following four passages
through a French pressure cell. DNA was also sheared to an appropriate size for
reassociation.kinetic studies, ie. 500 bp. For natural field samples, cell
concentrates could not be lysed by this procedure on a routine basis. Hydraulic
abrasion from clay associated with cell pellets severely damaged valves and
-------
piston assemblies of the Preach press. For this reason a lysis procedure using
a "bead beater" cell horaogenizer was developed.
(3) Sorption and extraction from suspended sediments; In preliminary
experiments, significant losses of DNA during extraction were associated with
ONA sorption to suspended sediments hypothesized to be clay dominated. Detailed
studies were undertaken to determine DNA sorption and optimized extraction of
sorbed DNA. These studies used characterize^ soils as model sediments and
conditions for optimal DNA extraction were employed in the DNA extraction and
purification protocol described later.
Five soils and one acid-washed sand were used in these studies. The
properties of these soils are listed in Table 1. All soils were ground to pass
through an 80-mesh sieve (nominal diameter, 177 microns).
Commercially obtained herring sperm DNA was used in all of these
experiments. For the high molecular weight DNA studies, the DNA was dissolved
in SSC (0.15 M NaCl, 0.015 M trisodium citrate, pH 7.0) after several passes
through a 21 gauge needle. DNA for the low molecular weight DNA sorption
isotherms was sheared by four passes through a French Pressure Cell Press
(Aminco, Inc.) at 20,000 psi. The fragment size of this DNA was found to
average 500 base pairs by agarose gel electrophoresis using lambda phage DNA
restricted by Hind III as a size marker.
The extent of DNA sorption to these sediment/soils was determined by a
batch slurry method similar to that described by Green et al. (1980). Two grams
of soil were weighed into siliconized glass tubed, wet up to 1% SSC, and
autoclaved for 30 minutes to inactivate heat labile nucleases. Four railliliters
of a range of DNA concentrations between 0 and 100 ug/ral were added to the
soils, mixed well, and shaken on a tumbling shaker overnight. These tubes were
centrifuged, and the supernatant analyzed for DNA concentration. The amount of
DNA los from solution was assumed to be sorbed by the soil. The sorption data
was analyzed by the Freundlich equation,
S =• KCn,
where S is the sorbed concentration (ug/g), C is the solution concentration
(ug/ml), and k and n are constants, or by the Langmuir equation,
(Smax KG)
S - 1 + KG ,
where Smax is the maximum adsorption capacity (ug/g). All experiments were run
in triplicate.
DNA concentrations were analyzed in one of two ways. Large molecular
weight DNA concentrations were determined by a fluorometric method similar to
that described by Paul and Myers (1982). Two mis of supernatant were mixed with
one ml of 1.5 x 10~5 M Hoescht 33258, allowed to sit in the dark for 10
minutes, and the concentrations analyzed on a Perkin-Elraer Model MPF-44A
fluorescent spectrophotometer. Standard curves were made using a range of DNA
concentrations in the appropriate soil extract. The DNA concentrations for the
low molecular weight studies were determined by using radiolabeled DNA. DNA
solutions were spiked to 20,000 cpm/ral with ^?-labeled sheared herring
-------
TABLE 1, SELECTED PROPERTIES OF SOIL
SOIL
wW I L*
MAURY
IBERIA
MEMPHIS
HARTSELL
EUSTIS
ACID-WASHED
SAND
•
TEXTURE
1 ^/\ 1 U |\^ •
SILT LOAM
SILT CLAY
SILT
LOAM
FINE SAND
SAND
MINERALS
Ill l*h»l UlbW
VERMILCULITE, MONTMQRILLONITE
KAOLIN ITE, ILLITE, QUARTZ
MONTMORILLONITE, KAOLINITE
QUARTZ
MONTMORILLONITE, ILLITE
KAOLINITE, QUARTZ
VERMICULITEA ILLITE
KAOLINITE, QUARTZ
' QUARTZ
QUARTZ
CLAY
Vui 1
24
49,3
10,0
14.0
3,2
0
ORGANIC
CARBON i
vrMUJUn • '
0.90
2,16
0,76
1.98
0,6
0
CEC
[Mpo/inn,
U IcU/ ^UUI
17,50
40,80
13.78
4.85
5.2
—
-------
sperm DNA before addition to soil, and the equilibrium concentrations were
related to cpra remaining in solution. The cpra was determined by addition of 0.5
ml supernatant of 10 ml commercial scintillation cocktail, and counted in a
liquid scintillation counter.
The tracer for the small molecular weight study was labeled with
orthophosphate ^2p nidc translation, and was ,.shown to be the same size as
the non-labeled DNA by agarose gel electrophoresis. The nick translation
procedure followed was that supplied in a commercial nick translation kit
(BRL).
DNA extraction studies were performed using Iberia soil and
sheared herring sperm DNA at a concentration of 20,000 cpm/ml mixed with 100
ug/ml sheared herring sperm DNA. The DNA was loaded into the soil by adding
four mis of the above solution to two grams of soil and shaking on a tumbling
shaker overnight. The extent of loading was determined by centrifuging the soil
and sampling the supernatant, as described above. The supernatant was removed,
and replaced with an equivalent volume of pH 10 NaC buffer (0.025 M NaHCC>3 *
0.025 M ^2003), mixed well and shaken overnight, the tubes centrifuged, and
the supernatant sampled, counted, and the extraction repeated. The final pH of
the solution was 8.0.
(4) Summary outline for DNA extraction, purification and reassociation kinetics
I. Isolation and purification of DNA from natural concentrates and
clay/sediment sorbed DNA'
A. Pretreatment
1. Centrifuge concentrate at 6000 rpra (5846 x g) for 20 minutes.
Discard supernatant.
2. Wash pellet first with 100 ml 0.1 NaP buffer (0.05 M
Na2HPC>4, 0.05 M Nal^PC^), pH 7, recentrifuge and
discard supernatant, and then wash with 100 ml 0.15 M NaCl, 1
raM EDTA, pH 8.0, to remove some humic materials.
B. Lysis
1. Resuspend pellet in 25 ml 0.15 M NaCl, 1 raM EDTA, pH 8.0, and
add 100 mg lysozyme. Incubate at 37°C for 1 hour.
2. Add 1 ml 25% SDS (sodium dodecyl sulfate) to above and incubate
at 65*C for an hour.
3. Complete lysis in bead beater.
C. Extraction of DNA from sediments
1. Centrifuge lysate and resuspend in 20 ml 0.12 M NaP buffer
(0.06 M Na2HP04, 0.06 M NaH2P04), pH 8.0. Shake well,
centrifuge, and save the supernatant. Repeat and combine the
supernatants.
2. Bring the concentration of the combined supernatants up to 8 M
urea and 0.7 M KCl for further removal of humic substances.
Store on ice overnight.
3. Centrifuge, keep supernatant, and reextract the pellet with 20
ml 8 M urea and 0.7 M KCl. Let sit at 4°C for an hour,
centrifuge, and combine the supernatants.
-------
D. Purification of DNA
1. Apply sample in 10 ml aliquots to DEAE-Sepharose column (inner
diameter 3.6 cm, bed volume 60 mis) and elute with 0.7 M KCl,
10 raM Tris-HCl buffer, pH 7.3. DNA elutes between 40 and 50
mis.
2. Combine fractions containing DNA, mix with 20 mis
hydroxylapatite for one hot^r, and pour into glass column with
siliconized glass beads as base. Elute starting buffer under
air pressure, wash with 30 mis 8 M urea, .24 M NaP, or until
absorbance at 260 and 240 is zero. Remove urea with 150 mis
0.014 M NaP, and elute DNA with 0.4 M NaP, pH 6.8.
II. Protocol for DNA ^association kinetics
A. Tracer DNA is prepared by removing 1 ug of sheared and purified DNA
in 0.12 M NaP buffer (0.06 M Na2HP04, 0.06 M NaH2P04), pH
6.8, and labelling with ->2p by nick translation in accordance
with a commercially available nick translation kit (BRL).
Unincorporated nucleotides are removed by passage over Sephadex
6-50, with 0.12 M NaP, pH 6.8, as the eluent. Specific activity of.
the nick translated DNA is determined by scintillation counting of .
a 5 ul aliquot.
B. Snap-back DNA is removed from the tracer DNA by denaturing the DNA
in a boiling water bath for 10 minutes, and placing immediately on
a 3 ml hydroxyapatite (HA) column kept at 65*C. Single-stranded
DNA is eluted with 8 to 10 1 ml fractions of 0.12 M NaP, and
double-stranded DNA is eluted with the same number of 0.40 M NaP
fractions. This procedure is then repeated with az 20 ul aliquot
taken from the single-stranded fractions. Twenty ul aliquots of
these fractions are analyzed by scintillation counting to determine
the amount of snap-back DNA remaining.
C. Tracer DNA is then added back to 2 ml of driver (unlabeled) DNA to
a final specific activity of about 2000 cpm/ug. Divalent cations
and mononucleotides are removed from this mixture by passage over a
two-layer column consisting of Sephadex 6-50 and Chelex, with 0.12
M NaP, pH 6.8, as the eluent. Fractions containing DNA are then
combined and concentrated by using Centricon tubes to between
200-350 ug/ml.
D. The exact concentration and purity of the DNA is assessed by
scanning ultraviolet spectrophotometer, and the specific activity
is determined by scintillation counting. Twenty microliter
aliquots of this mix are loaded into siliconized 50 ul capillary
tubes, and the tubes sealed by flame at both ends.
E. Tubes are placed in a boiling water bath for 10 minutes, and
quickly transferred for incubation in a 65°C water bath. Tubes are
incubated to various Cot values, removed, frozen in a dry
ice-ethanol bath, and stored at -20°C.
F. Tubes are opened and contents emptied into 1 ml aliquots of 50
ug/ml herring sperm DNA in 0.12 M NaP, pH 6.8, and refrozen until
further analysis.
-------
G. Samples are thawed and placed on 3 ml HA column at 65°C, with
single-stranded DNA eluted by 0.12 M NaP, double-stranded DNA
eluted with 0.40 M NaP. Fractions are sampled and the amount of
DNA in each fraction determined by scintillation counting.
H. Data are related to Z DNA reassociated with Cot, and analyzed by a
least squares FORTRAN based statistical program developed for Cot
analysis. v
Research Results
(I) Sorption and extraction of DNA from sediment; DNA sorption to five
soils and an acid-washed sand were analyzed using standard batch slurry methods
and by fitting the data to either the Freundlich or the Langrauir equation.
Sorption isotherms for a range of soil types revealed that the presence of
montmorillonite chlay dominates the sorption of DNA at low to neutral pH's.
This is not surprising since Greaves and Wilson (1969) have reported that 1 ml
of pure montmorillonite may sorb more than 1 rag of DNA at 5. The intercalation
of DNA in montmorillonite seems to be dependent upon the ionic state of the DNA.
At low pH's (below 7 in these soils), DNA is neutral and moves into the anionic
interior of the clay lattice, where it is sorbed. At higher pH's, DNA is in the
ionic form and is excluded from intercalation.
As can be seen from Table 2, soils which contain a significant amount of
montmorillonite sorb all of the DNA added to the system, up to at least 200
ug/g. The single exception is the Memphis soil (Figure 1), which sorbs a
maximum of 15.4 ug/g and is described by the saturating Langrauir equation. The
pH of this soil is relatively high (7.2), and it is likely that not all of the
DNA was neutral. That DNA which was neutral was probably intercalated into the
clay, while the remaining ionized DNA was left in solution. When the pH of the
solution was adjusted to 10, no DNA was sorbed, and when the pH was adjusted to
5.9, all of the DNA was sorbed. This indicates that the surface pH of some
natural soils and sediments may be near the pKa of DNA, and that significant
amounts of DNA could be present in the aqueous phase even in the presence of
montmorillonite.
The adsorption of DNA to sands was studied .at two different shear sizes and
two different pH's. Sands generally showed a Freundlich type isotherm at lower
pH's within the range of concentrations studied. For both sheared and unsheared
DNA, the Eustis soil and acid-washed sand were described satisfactorily by the
Freundlich equation. In both sands, unsheared DNA was sorbed to a greater
extent than the sheared DNA, as well as showing a more linear isotherm in the
range of concentrations studied. Eustis fine sand (Figure 2) yielded a k
(sorption coefficient) of 2.36 and an n of 0.95 for the unsheared case, and a k
of 1.51 and an n of 0.86 for the sheared case.
The sorption isotherm for acid-washed sand (Figure 3) yielded a k of 2.82
and an n of 0.62 for unsheared DNA, and a k of 3.15 and an n of 0.42 for the
sheared case. This is somewhat different from the Eustis soil in that the k's
for the unsheared versus the sheared case are very close. The unsheared
isotherm resembles that of the sheared at low concentrations, but more sheared
DNA is sorbed at equilibrium concentrations greater than 20 ug/ral. It should be
noted that the Eustis isotherms are much more linear than those for acid-washed
sand, indicating a higher sorptive capacity.
-------
TABLE 2, SUMMARY OF DNA SORPTION CHARACTERISTICS
M
PlAURY
T
IBERIA
M •
MEMPHIS
u
HARTSELL
EUSTIS
LARGE**
rIOLt WT,
SMALL**
MOL. WT.
Ac ID- WASHED
SAND
MOL. WT
»
SMALL**
MOL, WT,
FREUNDLICH LANGMUIR
p" K N S^^
c nc •••
O.ID ~ — - —
6r ••• ',
,j ~
7 9 in? i5/i9
1 ,L J.,U_. 1.3. 4/
C Q ••• _— _ -
J,O — — — —
in n ,— -
1U U — — _____
c n n . '.__
0,y U — --- — —
•
60 9 7K n QS -_
,0 Z.JD U.3.3 _— —
6,8 1,51 0,86
6,8 2,82 0.62
in a 99 zz KK
±\J ^
-------
20
18
16
14
12
UG/
g 10
8
6
4
2
0
LANGMUIR
K » 1.02
SMAX 315.42
r2»0.95
0 I 23 45 678 9 10
Ce
12 13 14 15 16 17 18 19
Figure 1. Memphis soil DNA sorption isotherm at pH 7.2. This soil showed a
saturation Langmuir type isotherm even in the presence of a significant amount
of montmorillonite.
-------
50
80
60
UG/G
40
201
FREUNOLICH
K- 2.36
N* 0.95
r2-0.87
B
0.95
10 20 30 40 50
ce(/xg/ml)
10 20 30 40 50
Ce (/xg/ml)
ISO
160
140
120
100
80
60
40
20
Figure 2. (A) High raol. wt. DNA. (B) Low raol. wt. DNA. Eustis sand DNA
sorption isotherms for two different molecular weights of DNA at pH 6.8. Both
curves were analyzed by the Freundlich equation.
-------
160
140 -
B
0
10
FREUNDLICH
K- 2.82
N- 0.62
r2-0.897
FREUNDLICH
K » 3.15'
N » 0.42
r2-0.987
20 30 40
Ce (jig/ml)
50
10 20 30 40
Ce(/zg/ml)
50
160
140
120
100
80
60
40
20
Figure 3. (A) High raol. wt. DNA. (B) Low mol. wt. DNA. Acid-washed sand DNA
sorption isotherms of two different molecular weights of DNA at pH 6.8. Both
curves were analyzed by the Freundlich equation.
-------
At higher, ionizing, pH's, unsheared DNA yields a saturation type isotherm
on acid-washed sand that is best described by the Langrauir equation (Figure 4).
The maximum aount of DNA sorbed (Sraax) was found to be 55.66 ug DNA/g. This
type of curve is indicative of an ion exchange mechanism that sorbs all
available DNA until the maximum is reached. Any DNA free in solution after this
sorption maximum has been reached will not be sorbed. It is interesting to note
that at pH 10, the amount of DNA sorbed by the sand is much lower than at pH
6.8. Apparently, there are more sites available for the sorptive mechanisms at
work in the lower pH's than there are at higher pH's.
The contribution of organic carbon to DNA sorption seems to be minimal at
the normal pH of these soils. Hartsell soil, which contains no montmorillonite
and 1.98% organic compound (Table 1), sorbed no DNA. Organic carbon may,
however, play a more important role in DNA sorption at higher pH's. Iberia
soil, which contains a significant amount of raontmorillonite and 2.16% organic
carbon, sorbs all DNA up to 200 ug/g at pH 8.0. At this pH, we would expect no
DNA to be sorbed by the montmorillonite, leaving organic carbon as the only
likely sorbant. Other reports (Torsvik, 1980) have noted the association of DNA
with huraic acids at this pH. It may be that in soils and sediments with high
organic carbon contents and pH's above 7.0, organic carbon may be an important .
sorbant of DNA.
Iberia soil was chosen as the model soil on which to perform DNA extraction
studies. It has the highest levels of montmorillonite and organic carbon of the
five soils, and it was believed that Iberia would be the most difficult from
which to extract DNA. There is also preliminary evidence which indicates that
the predominant clay mineral in Fort Loudon sediments, and hence in the water
column, is a smectite (such as raontraorillonite). In an exploratory experiment,
it was found that one gram of Fort Loudon sediments adsorbed 195 ug of DNA out
of 200 ug added. For extraction experiments, DNA was loaded into the sediment
taken from Fort Loudon reservoir, and sequentially extracted with 0.12 M NaP
buffer, pH 8.0. Using a solution to sediment ratio of 2, over 95% of the
adsorbed DNA could be removed after four extractions, with an average extraction
coefficient of 0.50.
In the extraction experiment with Iberia soil, sequential extraction were
made with pH 10 NaC buffer. It was found that five pH 10 extractions with 3 mis
of buffer per extractant were needed to desorb 99.99% of the DNA intercalated
into 2 grams of soil (starting concentration of 200 ug/g). The number of
extractions could doubtlessly be reduced if a larger volume per extraction were
used.
(2) DNA reassociation kinetics of reconstructed population: In an effort
to simulate the reassociation kinetics of a mixed community, an equal mass of
chromosomal DNA from nine different organisms were mixed together and the
kinetics of reassociation measured. These nine organisms were:
1) Proteus vulgaris
2) Flavobacterium aquatile
3) Bacillus cereus
4) Rhizobium japonicum
5) Streptococcus faecalis
6) Bacillus subtilis
7) Escherichia coli
8) Brevibacterium amoniagenes
9) Micrococcus lutea
-------
100
80
S
ug/g
60
40
20
LANGMUIR
K*4.22
SMAX = 55'66
* 0.96
1 - 1
20
40 60 80
C (jug/ml)
100
Figure 4. Langrauir sorption isotherm for high molecular weight DNA on
acid-washed sand at pH 10. Curve indicates saturation of available sites at a
much lower point than at pH 6.8.
-------
If these organisms are assumed to have approximately the same genoraic
sizes, somewhere in the range of 2.0-2.5 x 10' daltons, we would expect the
time for 50% of the DNA from this reconstructed community to reassociate (the
Cot 1/2) to be approximately nine times the Cot 1/2 for a single species. The
Cot 1/2 value for ]!. coli is approximately 8, although this number is dependent
upon reassociation conditions and strains. If all nine strains used have the
same genoraic size as _E_. coli, and no interspecies homology or repeated
sequences, we would expect a community Cot 1/2 of 9 x 8 =• 72.
The reassociation data (Figure 5) from the reconstructed community was
analyzed by a least squares curve fitting program specifically designed for
reassociation kinetics, and the Cot 1/2 was determined to be 54.2, with an RMS
error of 5.6Z. The lower than predicted Cot 1/2 value is not a complete
surprise, however, due to the extensive DNA homology between £. coli and Proteus
vulgarist and between Bacillus subtilis and Bacillus cereus. The more homology
between different community members, the faster the reassociation rate.
Repetitive genes for nitrogen fixation in _R. japonicum probably also contributed
to the faster rate.
(3) Extraction and purification of DNA from field samples. The efficiency
of the concentration of the lake water and the recovery of cells entering into
the filtration process were checked by plate counts on YEP6 agar. Since it is
conceivable that some cells may pass through the filtration process 1000 times,
there was some concern that a substantial number of cells may be lysed during
the concentration. This possibility was checked by the introduction of a known
quantity of easily traceable organisms into the prefiltrate and measuring
concentrations before and after the concentration process. The marker of choice
was the NAH plasmid, harbored by Pseudomonas putida. The final concentration
after filtration of 941 1 was 4 x 10' cfu/ral in 2 1, or a total of 8 x
10^0 cells (both the introduced £. putida and the natural community).
Efficiency of cell recovery was only 1% due to losses by prefiltration and
shearing of cells by repetitive passages through the filtration pump.
Assuming an average genome'size of 2.5 x 10^ daltons, we would expect a
total of 0.332 rag of DNA to be contained in the final 8 x 1010 cells. It
should be noted that this number only accounts for those cells that can be
cultured on YEPG agar. After extraction and purification of DNA from the final
concentrate, approximately 0.4 mg of high quality DNA was recovered (Figure 6).
At the time of this report, DNA reassociation kinetic determinations for
purified DNA from two separate field populations are being completed. The
tracking of specific DNA (NAH 7 plasraid DNA) by dot blot filter hybridization of
aliquots from hybridization mixtures is also being completed. These results
will be in hand by July 1 and will be reported at the upcoming meeting a
GB/ERL.
Conclusions
1) Filtration techniques are inefficient for concentration and harvesting cells
from large volumes of dilute environmental sample.
2) Mixed culture cell lysis DNA extraction and purification is efficient and
provides sufficient DNA suitable for the analysis of DNA reassociation —
kinetics.
-------
1 0 0
%
;ss
50
0,0
o
3,0
LOG COT
Figure 5. Cot curve for DNA reassociation of a nine raerabered mixed microbial
population.
-------
Scant 82.01
0.500
Display Node
[Single]
Function
[Peak Pick]
x
408, 5 3,003
32R.R 0.HH4
315.5 0.805,
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iLLb. U £!. "il
fa .r"'7
'
0.000
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*
4...
°'
00
4
vT
/
/
J
yh
i
\
\
\
L
3.0
,
\
32
i
3.0
i
ww
i
3.0
44
_
3J
Ciiq
._FKJ-j
y'"-
il xl!~1H
sj, f!jy
ra -iiiii
U. -jtltl
n^R
R Iflfl
R.HMR
600
_x Hcb bUUKLL
b00.0 0.0015 Uis/UU Scan
-f»^i.-. .-.r—
IKHIL
Figure 6. Absorbance spectrum of purified DNA recovered from natural field
populations.
-------
3) The presence of clay3 in environmental samples seriously effects the
efficiency of DNA recovery due to sorption. However alkaline extraction is
appropriate for recovery of DNA from clays. Organic matter has little effect
on DNA recovery.
4) Huraic materials co-extract with DNA and effect the quality of DNA for
reassociation studies. Appropriate column chromatography can be used for
purification of DNA prior to Cot analysis.
-------
-------
DU CODE & TITLE
OBJECTIVE CODE L
PPA CODE & TITLE
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q281
L104 Chamical Testing I Assessment
TITLE : G Develop & Validate Inproved
Mthds
! 02 Environmental Aspects of 3iotechnology
Risk Assessment
PROJECT CODE £ TITLE : 24 £col Consequences of Gen Eng Microorgs
WORK PLAN TYPE I C30P
INVESTIGATOR : T Sarkay
PHON5 NO : 686-9011
PRINCIPAL INVESTIGATOR: o stahi
WORK PLAN TITLE
Populations
The Use of rRNA Sequances to Characterize Natural Microbial
WORK PLAN OBJECTIVE : I) Use com rumen as a model system to develop and
evaluate techniques for employing the 5S rRNAs and 16s rDNAs/rRNAs as
determinants of natural microbial diversity and abundance. Steps include:
1) Develop the use of 53 rRNA oligonucleotide fingerprinting for A) genera]
evaluations of microbial ecosystem perturbation and 8) tracing the fate of
individual microorganisms in complex microbial community settings! and 2)
Determine the general applicability of sequencing 16s rRNA genes cloned
from environmental samples for tha characterization of natural microbial
communities. This includes evaluating different methods for extracting an<
cloning 16S rRNA from a heterogeneous population of microorganisms and
defining unique regions of sequence within the 16S rRNAs useful for specie?
identification. II) Determine 16S rRNA nucleotide sequences from
characterized rumen microorganisms.
WORK PLAN APPROACH : The nucleotide sequences of rRNA molecules will be used
to characterize natural microbial communities. Two approaches mill be
employed: A) Fingerprints of total 5s rRNA directly isolated from the
environment will be obtained. Comparison between patterns of stressed vs
unstressed systems will indicate changes in the structure and composition
of the community. 3) 16s rRNA genes originating in DNA directly isolated
from the environment will be cloned in bactariophage vectors and
characterized by DNA sequencing. These genes will be then utilized as
probes to measure the relative abundance of specific sequences in DNA
isolated from natural samples. The relative abundance of each species will
ba defined as the ratio between hybridization signals of the specific probe
and those of a :nixed kingdo-n probe (i.e.* representing 16s rRNA of
eukaryotes archeobacteria and eubacteria). Alternatively! abundance will
ba measured by DNA:RNA hybridization to improve the sensitivity of the
method. Tha feasibility of these methods will be tested by following the
response of the rumen flora to the application of GHMs and an ionophore
antibiotic. Further* the applicability of tha developed methods to aquatic
systems mill be verified by conducting a similar study with anaerobic
s ediments.
STATUS : On Schedule
ACCOMPLISHMENTS : 1) Sequencing of 16s rRNA of 17 rumen strains has been
completed. For taxonomical identification as well as ecological abundance
studies, 652 Cor about 1000 nucleotides) of the rRNA sequence is
sufficient. Example data have been collected to allow taxono.nic analysis
of 3acteriodis succinoganes.
2) Preliminary work indicated tnat 5s rRNA fingerprinting of rumen material
is subjected to interferences by nuclaic acids of plant origin. The
-------
r i
I -« L.
GULF BREEZ: , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q281
nucleic acid fractions extracted from natural waters and sediments by Gary|
Sayler (University of Tennessee* project CR812483-01-1) will be used to
test the utility of 5s rRNA fingerprinting for tha characterization of
natural microbial communities.
3) Procedures and protocols for cloning of 16s rRNA from whole rumen
content have been designed and improved.
4) Threa ribosomal SNA probes hava bean prepared. An E. coli probe is
currently used to follow the fate of E". coli strains introduced to the
rumen. Rasults of hybridization with whole rumen nucleic acid fractions
are compared uiith those obtained by direct plating on selective media. A
species-specific B. succinogenes probe and a strain-specific probe (for 8
succinogenes 385) have been prepared and testad. These probes and
additional ones which are currently under construction will be used to
follow population shifts in the rumen environment as a result of monensml
feeding.
5) Dr. Richard Devereux has arrivad in Gulf Breeze and initiated a study on
sulfate-reducing populations in marine sediments. Culturing of
sulfate-reducing bacteria for the sequencing of these 16s rRNA is undarwal
Salt marsh sediments have baen altered and extraction of DNA is currently
attampted.
I
on
I
MILESTONES (DATES) :
Pariod 2 - Continued cultivation
and 16s rRNA sequence
determinations of four or five
listed rumen bacteria. Pilot
rumen sampling and R N A
extractions. Fingerprinting of
total rumen 5s rRNAs.
Period 3 - Continued cultivation
and 16S rRNA sequence
determinations of four or fiva
listed rumen bacteria. Pilot
DNA extractions and evaluation
of blunt-end ligation cloning
protocols. A post doctoral
associate (Dr. Richard Devereux)
arrives at the University of
Illinois to learn the basic
experimental methodology. A
detailed experimental design for
the application of these
techniques to the study of
anaerobic s?dim3nt microcosms
developed in collaboration uiith
the Gulf Breeze scientists.
T4R3ET
03/86
REVISED REVISED REVISED
ACTUAL
03/^6
f
36/86
OS/
Pariod <* - Continued cultivation
09/35
-------
ENVIRONMENTAL RESEARCH LA3CRATORY
GULF BREEZE » FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q281
and 16S r3NA sequence
determinations of four or five
listed rumen bacteria* Design
and synthesis of selected
species-specific oligonucleotide
probes. Cloning of DNA isolated
from total rumen contents.
Identification of 16S rONA
containing clones and initial
sequencing screans for
redundancy.
Period 5 - Continued cultivation 12/86
and 16S rRNA sequence
determinations of four, or fivs
listed rumen bactarial isolates.
Sequence determinations of 165
rDNA clones derived from ru.nen
contents. Acquisition of the
fistulated staer. °ilot rRMA
and rONA targeted hybridizations
with organism specific
oligonucleotide(s).
Period 6 - Sequence 03/87
determinations of four or five
16S rRNAs and tuio 5S rRNAs from
the listed rumen bacteria.
Sequence determination of 16S
rONA clones derived from ru^en
contents. Tracing the
elimination of 5 . coli from the
rumen by 5S rRNA fingerprinting,
rRNA targeted hybridization and
viable counts.
Period 7 - Sequ?nce 06/87
determination of 16S rDNA clones
derived from ruman contents.
Initiate monensin rumen
perturbation experiment.
Period 8 - Sequence 09/87
determinations of 16S rDNA
clones derived from rumen
contents. Complete monensin
perturbation experiments.
Final Report on the Detection of 11/37
GEM's and Their Abundance in
Complex Environments by RNA
Fingerprinting and Sequencing.
C7434A)
-------
Quarterly Progress Report June 1986
The Use of Ribosomal RNA Sequences to Characterize Natural Microblal Populations
David A. Stahl, Ph.D., University of Illinois
Berdina Flesher, M.S., Research Technician,..University of Illinois
Richard Devereux, Ph.D., Postdoctoral Fellow\;
The work has closely followed that outlined in the original grant proposal.
Sequencing of 16S ribosomal RNAs isolated from representative rumen
microorganisms is well along. Reference organisms now in hand and the
percentage of the 16S sequence completed are listed below. Complete sequences
for all strains are not required for the ecology studies. About 1000
nucleotides of sequence (65%) is adequate for an accurate determination of
phylogenetic relatedness and to select appropriate target sites for
oligonucleotide hybridization probes. Thus, for the purposes of this project a
65% completed sequence is considered "complete".
Cell Paste in Hand Approximate percentage
of 16S rRNA sequence completed
1. Bacteroides succinogenes S85 90%
2. B. succinogenes HM2 65%
3. B. succinogenes NR9 65%
4. B. succinogenes A3C 65%
5. ^. succinogenes DR7 65%
6. Butyrivibrio fibrisolvens Dl 65%
7. £. fibrisolvens 49 65%
8. B. fibrisolvens A38 65%
9. Selenomonas ruminantium HD4 65%
10. S. ruminantium GA192 65%
11. Ruminococcus albus 7 65%
12. R. albus 8 65%
13. R. albus KB1
14. Ruminococcus flavefaciens C-94 65%
15. R. flavefaciens FD-1 65%
16. Ruminococcus bromii JB-2A 65%
17. R. bromii 6833 65%
18. R. bromii 56B-47 65%
19. Oxalobacter formigenes OXB
20. £. formigenes SOX4
21. £. formigenes BA-2
22. Anaeroplasma intermedius 7LA —
23. Anaeroplasma. abactoclasticum 6-1 —
24. Asterolplasma anaerobium 161
-------
25. A. anaerobium B-5
26. Lachnospira multiparus 40 65%
27. L_. multiparus D32 65%
28. Megasphaera elsdenii B159 65%
RNA fingerprinting. Preliminary fingerprinting suggests that the rumen is not
a. suitable model system for this approach to a diversity assessment. Without
fractionation of plant and microbial contents, RNA degradation products derived
from plant material severely contaminate fractionated 5S rRNAs. Because many
rumen microorganisms (the cellulolytics in particular) are tightly associated
with the plant material, fractionation would introduce a serious bias. An
alternative that may be explored if time permits is the feeding of a total grain
diet. However, before doing so, an alternative environment will first be
explored in collaboration with Dr. Gary Sayler's group. Dr. Sayler will provide
total nucleic acid (RNA and DNA) derived from water column or sediment samples.
These samples will be used, for Cot analysis by Dr. Sayler's group and for 5S
rRNA fingerprinting by us. Thus, both assessments of diversity will be applied
to the same population.
Cloning of 16S rDNA from total rumen contents. Several DNA extraction regimens
of total rumen contents have been explored. The approach that appears to offer
the most representative isolation is an extended protease digestion with
concurrent dialysis. The DNA isolated by this approach is high molecular weight
(>50 Kb) and restrictable. A preliminary partial Sau 3A digestion and shotgun
cloning using the EMBL4 phage lambda cloning vector yielded about 2 x 10^
putative recombinants. Cloning is now being repeated to increase the yield
prior to screening the library for 16S rDNA recombinants.
Ribosomal RNA targeted hybridization probes. Three synthetic oligonucleotide
probes have been synthesized for preliminary hybridization studies. One is
specific for Escherichia coli and will be used in the study of the elimination
of E_. coli from the rumen.
Because of inhibition by volatile fatty acids, few E_. coli are normally
present in the rumen (102-lo3 per ml). Their artificial introduction therefore
offers a convenient model for tracing the elimination of a microorganism from a
complex microbial population. Since few of the normal rumen flora are capable
of aerobic growth, and most facultative members are inhibited by bile salts,
direct plating offers a convenient assessment of culturable numbers. Culturable
numbers are now being compared to those numbers derived from rRNA targeted
hybridization of total rumen nucleic acid.
Two synthetic DNA oligonucleotides probes have been designed to address
population variation among various strains of Bacteroides succinogenes. Five
strains of B^. succinogenes have so far been characterized by comparative rRNA
sequencing (above). Although phylogenetically coherent, the evolutionary
distance separating certain strains suggests the need for taxonomic revision,
with a new species designation replacing at least one phylogenetic cluster
defined (so far) by strains NR9 and DR7. Although these "strains" are
genetically distinct, they are not well circumscribed by the so far defined
phenotypic characters (vitamin requirements and substrates) and pure culture
techniques do not provide a convenient or reliable approach to their
identification and enumeration. The use 16S rRNA targeted hybridization should
offer a rapid assessment of presence and population variation among the various
-------
"strains". From the now available 16S rRNA sequence information we have
synthesized a strain specific and a species specific oligonucleotide probe. One
is specific for the type strain (B^. succinogenes S85), the other encompasses all
strains of B. succinogenes so far characterized by 168 rRNA sequencing. The
specificity and generality of these probes is supported by preliminary
hybridization studies. Additional probes for Selenomonas ruminantium and
Lachnosplra mutiparus are being fabricated .for the raonensin population shift
experiment (below).
Monensin population shift experiment. The polyether antibiotic monensin is
routinely added to cattle feed to increase feed efficiency. In part, increased
animal productivity has been attributed to a change in the composition of the
rumen mlcrobial population. The 16S rRNA targeted oligonucleotide probes
(above) will be compared to traditional pure culture enumerations of certain
rumen microorganisms (B. succinogenes, ruminococci, S. ruminantium and L.
multiparus) before and following a monensin induced population shift. This work
is in progress in collaboration with Dr. Larry Montgomery of the Department of
Animal Sciences at the University of Illinois.
Anaerobic sediments Dr. Richard Devereux, having spent several months in Urbana
becoming familiar with basic methodology, has initiated work with anaerobic
sediment microbial populations at the Gulf Breeze laboratory. The project will
follow the basic experimental approach as outlined for the rumen investigation,
emphasizing the sulfate reducing bacteria. Initial work will involve the
compilation of a representative data base of 16S rRNA sequences from a wide
variety of sulfate reducing bacteria. Dr. Friedrich Widdel at the University of
Illinois has provided technical advice, pure cultures and lyophilized cells of
the following sulfate reducing bacteria: Desulfotomaculum ruminis, I), orientis,
Desulfomonas pigra, Desulfovibrio vulgaris "hildenborrough", £. vulgaris
"marburgh", J). vibrio "baarsii", Desulfobacter vacuolatum, I), hydrogenophilus,
D^. curvatus, D_. latus, Desulfobacter spp. Sac10 and 4acll, and Desulfobulbus sp.
3prlO. Continued cultivation and sequencing of sulfate reducing bacteria and
pilot studies on extraction of DNA and RNA from sediment samples are now in
progress.
Significance. The comparative sequencing of representatives of the rumen
mlcrobial flora has emphasized the limitations of the usual determinative
techniques in assessing natural microbial composition and diversity. Not only
are many organisms not amenable to culturing but also, a poorly described
species (e.g. JB. succinogenes or Butyrivibrio fibrisolvens) may lump together
genus level (or higher taxon) diversity. The ribosomal RNA sequencing offers a
good assessment of genetic homogeneity and provides a sensitive (ca 10^
hybridization targets per cell) measure of environmental presence and abundance.
Given a phylogenetically coherent collection (e.g. B^. succinogenes) a single
hybridization probe can be designed to evaluate the entire assemblage in the
environment. For certain phylogenetically coherent groups of microorganisms it
should be possible to fabricate hybridization probes that assess function, e.g.
sulfate reduction and methanogenesis. The experiments now ongoing, monitoring
elimination of E. coli from the rumen ecosystem and the monensin induced
population shifF, will evaluate the sensitivity and fidelity of these techniques
in a complex natural setting.
-------
H
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE t FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q280
3U CODE & TITLE : L104 Chemical Testing 6 Assessment
OBJECTIVE CODE & TITLE : G Develop and Validate Improved Risk Assess Mthds
PPA CODE & TITLE : 02 Environmental Aspects of Biotechnology
PROJECT CODE & TITLE : 24 Environmental Assessment Biotechnology Applications
WORK PLAN TYPE : Inhouse/Contract
INVESTIGATOR : T BarKay
PHONE NO : FTS 636-9011
WORK PLAN TITLE : Fate and Effects of a Mercury-Reducing Bioengineered
Organism in Estuarine Ecosystems.
WORK PLAN OBJECTIVE : To establish a model system to study the processes
governing the fate of a bioengineered organism in aquatic environments. An
organism carrying the genes uihich mediate the detoxification of Hg2+ by
reduction to the volatile elemental form (Hg) will be applied to microcosms
dosed u/ith Hg. The establishment of the applied organism in the indigenous
aquatic microbial community and its effects on the structure and function
of the aquatic ecosystem and the fate of the added Hg will be investigated.
Defining the inter-relationships which exist between the applied organisms
and these ecological phenomena will reveal some of the processes and
hazards involved in the release of bioengineered organisms in th» aquatic
environment.
RATIONALE: Until sufficient data suggest genetically altered microbes can
be field tested* microcosms* as natural simulations* must be used to study
the processas which affect tha fate of these organisms in aquatic
environments. Microcosms have been used successfully in the study of the
biodegradation of toxic chemicals. Models derived from microcosm data can
reliably describe similar processes as they occur in the field. Bacterial
Hg resistance is selected as a model study system because: CD Mercury has
delatarious effects on biological systems and several instances of
environmental pollution problems created by this element have been
documented in the scientific literature; (II) the biogeochemical cycling of
Hg in the biosphere has been elucidated; and (III) the biochemical and
genetic systems which mediate the r»sistanc>? and detoxification of Hg are
well understood. Thus» the response of aquatic ecosystams to Hg is of a
practical significance* the interactions of environmental variables with Hg
have been dafinad and the detailed knowledgs of the bacterial response to
Hg provides essential msans for the detection of specific organisms and
ganes in environmental samples.
WORK PLAN APPROACH : Simulated estuarine environments will be seeded with a
Pseudomona strain containing Hg-resistanca plasmids. The host strain will
be marked with two mutations (antibiotic resistance and auxotrophy) to
enaole its detection by using simple selective laboratory growth media.
The following parameters will ba measured:
1. The survival of the novel organsims mill be followed by common
microbiological and molecular procedures.
2. The response of indigenous microbial communities to the additions of the
novel organisms and toxic Hg2+ will be determined by estimating community
structure (by diversity analysis) and following essantial functions (a.g.»
element cycles).
3. Tha spread of the Hg-resistance gene from the novel organism to
indigenous bacteria will be followed by common genetic molecular analysis.
4. The fate of the Hg in the microcosm systems will be followed by
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE f FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q280
analysing Hg in uiatert sediment and air. An attempt to separate organic
from inorganic forms of Hg mill be carried out by differential extractions.
5. Environmental factors knoun to affect tha speciation of Hg u/ill be
measured* including organic matter content* particulate matter* pH»
temperature and Eh.
Controls mill consist of sterile syst'ems* sterile systems to which the
Hg-resistant Sioengineered organism is1 added and "non-sterile" syst9ms
tuhich do not contain the bioengineered organism.
By simultanous analysis of all of these parameters, relationships betaiean
the survival of bioengineared organisms* gene transfer, and the effect of
stressors and physico-chemical parameters of the ecosystem -iiill emerge.
STATUS : On Schedule
ACCOMPLISHMENTS : Tha development of simulated aquatic systems has been
progressing on schedule. The system is now applied to freshwater* marine
and salt marsn systems to study Hg2 + adaptation of the indigenous microbial
communities. Construction of Hg2 + resistant GEMs has suffered some
setbacks. Strategies to overcome these difficulties have been designed and
experiments are progressing satisfactorily.
MILESTONES (DATES) : TARGET
Preliminary results: Survival of 12/35
the mercury-resistant organims
in aquatic microcosn.
Construction of GEM's 12/85
Establishment of simulated' 12/85
aquatic systems to test GEM's
Accomplishment of studies on the 03/85
survival and activity of GEM's
in aquatic systems.
Accomplishment of studies on the
effect of genetic exchange on
ecosystem functions.
ASM presentation: Mechanisms and
processes of adaptation of
natural aquatic bacterial
communities to Hg stress
Bacterial g°na transfer in soil 03/86 08/86
and aquatic environments (69448)
ISOME presentation: Ganstic 08/86
exchange as an adaptive process
to environmental stress
REVISED REVISED REVISED
ACTUAL
12/35
03/86
08/86
03/86
09/86
09/86 12/86
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATS : 7/ 2/86
WORK
PLAN CODE
Q280
analysing Hg in mater* sediment and air. An attempt to separate organic
from inorganic forms of Hg mill be carried out by differential extractions.
5. environmental factors known to affect tha speciation of Hg mill be
measured* including organic matter content* particulate matter* pH»
temperature and Eh.
Controls mill consist of sterile systems* sterile systems to uihich the
Hg-resistant bioengineered organism is? added and "non-sterile" systems
which do not contain the bioengineered organism.
By simultanous analysis of all of these parameters, relationships betmean
the survival of bioengineared organisms, gene transfer, and the effect of
stressors and physico-chemical parameters of the ecosystem uiill emerge.
STATUS : On Schedule
ACCOMPLISHMENTS : Tha development of simulated aquatic systems has been
progressing on schedule. The system is noiu applied to freshwater* marine
and salt marsh systems to study Hg2 + adaptation of the indigenous microbial
communities. Construction of Hg2 + resistant GEMs has suffered some
setbacks. Strategies to overcome these difficulties have been designed and
experiments are progressing satisfactorily.
MILESTONES COATES) : TARGET
Preliminary results: Survival of 12/35
the mercury-resistant organims
in aquatic microcos-n.
Construction of GEM's 12/85
Establishment of simulated ' 12/85
aquatic systems to test GEM's
Accomplishment of studies on the 03/86
survival and activity of GEM's
in aquatic systems.
Accomplishment of studies on the 09/86
effect of genetic exchange on
ecosystem functions*
ASM presentation: Mechanisms and 03/86
processes of adaptation of
natural aquatic bacterial
communities to Hg stress
Bacterial gsna transfer in soil 03/86
and aquatic environments (69448)
ISOME presentation: Ganetic 08/85
exchange as an adaptive process
to environmental stress
REVISED REVISED REVISED
ACTUAL
12/35
08/86
03/86
09/86
12/86
08/85
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ENVIRONMENTAL RESEARCH LA30RATORY
JULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q280
Internal report on regulatory 09/86
research for biotechnology,
identification and enumeration
techniques? survivability and
genetic exchange in genetically
altered microorganisms. (6944A)
Journal Article: Adaptation to 10/86
HG2 in =stuarina Microbial
Communities. (6945B)
Qualitative t quantitative 09/83
determination of genetic
exchange and its effect on
microbially mediated activities
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE t FLORIDA 32561
FY '86 WORK PLANS
OATS : 7/ 2/86
WORK PLAN CODE : 0239
OU CODE & TITLE : L104 Chemical Testing I Assessment
OBJECTIVE CODE £ TITLE : G Develop and Validate Improved Risk Assess Mthds
PPA CODE £ TITLE : 02 Environmental Aspects of Biotechnology
PROJECT CODE £ TITLE : 24 Ecol Consequences of Gen Eng Microorgs
WORK PLAN TYPE : COOP
INVESTIGATOR : D Chattarjee
PHONE NO : 636-9011
WORK PLAN TITLE : An Approach to Assess the Fate and Effects of Genetically
Engineered Microorganisms in Simulated Natural Environments.
WORK PLAN OBJECTIVE : The consequences of the release of genetically
engineered microorganisms in the open environment are of great concern. A
reliabla method to monitor the released orggnism(s)» survivability of such
strain(s)» movement of genes present in such strainCs) as mall as the
stability of the genetic structures should be assessed before introduction
of novel organisms into the environment. Plasmid-bearing strains capable
of degrading 3-chlorobenzoic acid and 2f4»5-trichlorophenoxyacetic acid
mill be used in model ecosystems to answer these questions* Gene movement
mill be followed in the presence and absence of plasmids known to help
mobilize chromosomal and extrachromosomal genes. The fate of an *ukaryotic
gene segment present as a recombinant mill also be tested. This research
project mill alloaj the assessment of potential risks associated »ith the
release of novel organisms in the environment.
WORK PLAN APPROACH : The fata of microorganisms containing novel genetic
sequences deliberately released to the environment is the subject of much
speculation. Factors such as competition uiith the native microflora*
predation* ability to adapt to varying nutritional and environmental
conditions all affect uihethar the novel microorganism will become a
permanent member of the microbial community. Of perhaps greater concern is
the fata of the novel genetic sequences contained in these organisms.
Foreign genes are routinely incorporated into bacterial plasmids due to
ease of manipulation. Most of th» recombinant plasmids are made in
non-conjugative vectors purposely in order to minimize the spread of such
recombinants. However several mechanisms are knoiun in mhich
non-conjugative plasmids can oe mobilized into other bacteria. Various
drug resistance plasmids including the naturally occurring RP4 plasmid can
enhance mobilization of plasmid encoded genes C3arth, 1979). Several
additional mechanises are knoum by uihich th* chromosomal genes may be moved
from ona organism to another. The naturally occurring mobilizing plasmid
R68.45, for example, can graatly increase the rats of conjugal transfer of
chromosomal genes ta other gram nagative bacteria (Hollou;ay» I979)t
Transduction and transformation can also lead to axchange of genetic
information. This proposal suggests axperiments to determine to uihat
extent thssa processes are significant uiith regard to novel gene sequences
in the environment. Tha axperiments involva several bacterial strains
tuhich have bean u;ell characterized• yet which contain interesting genes for
the biodegradation of chlorinated hydrocarbons.
Since 3-chlorobenzoate (3cba) and 2»4»5-trichlorophenoxyacetic acid
(2»4f5-T) dagradation are unique in bacteria developed in our laboratory
(Chatterjee et.al 1981a, Chatterjee et.al 1981b), these model systems have
direct application to proposed deliberate ralease situations. Some of the
strains carry dagradative genes exclusively on a recombinant plasnaidt othar
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c ill V
ULF
BREEZE
FY '86
DATE :
z. o c A ,-, u n i- « a u ,->' H i <_• •% i
, FLORIDA 32561
WORK PLANS
7/ 2/86
WORK PLAN CODE : Q289
strains carry such genes on both plasmids and the chromosome. The
chromosomes or plasmids of these strains are often marked with antibiotic
resistance markers and one plasmid will contain both prokaryotic
biodegradative genes and a aukaryotic gane sequence as a model of a
r.ecombinant plasmid system. Using a variety of molecular biological
tecnniques, I propose to monitor the stability of the native and novel gene_
sequences in model ecosystems as well a.s the extent of genetic exchange
between novel organisms and naturally occurring representatives in the
presence and absence of RP4 and R68«45.
STATUS : On Schedule
ACCOMPLISHMENTS : The stability of the new recombinant DNA construct has been,
studied both in laboratory media and in simple natural systems. We found
under selective and nonselective conditions the plasmid is stable up to
more than 100 generations. Isolation of plasmid DNA from randomly picked
colonies showed presenca of intact plasmid as evidenced by restriction
digestion patterns. The stability of the plasmid and the
plasmid-containing strain ware testad in starile freshwater sediment .and
sterile sound water (salt-uiatar) and found to be stable and to grow in
sterilized sediment* more than 2 logs of the added micororganism (10/6gm
sediment). The number remained constant for 4-5 days and then declined.
After 10 days the cell number fall 2 logs and after 2 weeks the cell numb
was 10-4 per ml of sediment,. During thesa axperiments approximately 100
colonies u/ere picked and spotted onto 3 cba (the plasmid phehotype) and
carbenicillin plate (tha resistance marker). All antibiotic resistance
strains wera 3 cba positive, suggesting that the plasmid was stable under
such conditions.
In sterilized sound water* tha plasmid-bearing strain was unable to grow.
However* all of the antibiotic resistance colonies tested were found to b
3 cba positive which suggested that the strains did not loose the plasmid
but rather diad under tast conditions. The cell count was measured by
antibiotic resistance* was undetectable aftar 15 days.
Presently* colony hybridization with b-globin gena sequence is underway t
confirm tha presence of such sequence. Also* 3 cba will be spiked into
sadimant and sound -aater to datarmine whether the calls will be able to
grow and be datacted.
MILESTONES (DATES) : TARGET REVISED REVISED REVISED ACTUAL
Initial baseline studies on fata 09/85 06/86
of microorganisms carrying novel
sequences in microcosms
Construction of mar
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q289
the recipients
Journal Article: Genetic 06/86 09/86
Stability of Altered
Microorganisms in Natural
Environment
Journal Article: Genetic 06/86 12/86
Analysis of 2t4»5-T
Biodegradation
Qualitative and Quantitative 09/88
Determination of Genetic
Exchange and Its Effect on
Microoially Mediated Activitias
in Aquatic Environments. C6945A)
Internal Report on Regulatory 09/86
Research for Biotechnology:
Identification and Enumeration
Techniques? Survivability and
Genetic Exchange in Genetically
Altered Microorganisms. (6944A)
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ENVIRONMENTAL RESEARCH LA30RATORY
GULF BREEZE i FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q286
OU CODE £> TITLE : L104 Chemical Testing 6 Assessment
OBJECTIVE CODE L. TITLE : G Develop and Validate Improved Risk Assess Mthds
PPA CODE & TITLE ' 02 Environmental Aspects of Biotechnology
PROJECT CODE & TITLE : 2* Ecol Consequences of Gen Eng Microorgs
WORK PLAN TYPE : Inhouse/Contract
INVESTIGATOR : S Cuskey
PHONE NO : FTS 686-9011
WORK PLAN TITLE : The Effect of Conditional Lethal Genatic Determinants on
Cell Survival in Selected Environments.
WORK PLAN OBJECTIVE : To determine the feasibility of creating strains Cor
plasmids) whereby daath of the cell is probable under conditions determined
by the researcher. Both chromosomal and plasraid-encoded conditional lethal
determinants mill be created and tested under laboratory conditions*
Promising systems will be tested in a microcosm model environment to
determine 1) ability of altered organisms to compete against native
microbiota, 2) extent of lethality under selected conditions and 3) extent
of genetic transfer prior to the onset of lethal conditions.
WORK PLAN APPROACH : Concerns for the fate of environmentally released,
genetically altered DNA into the environment may be assuaged if it can be
shown that organisms carrying such DNA have a short lifa-span. Additional
concerns have suggested that mutations which would insure a rapid clearance
of released microorganisms put thasa strains at a competitive disadvantage
to the astablishad microbiota, precluding any hoped-for, positive effactCs)
of release. Dn'S way to satisfy both concarns would be to employ
conditional lethal genetic determinants in strains of interest.
Chromosomally encoded conditional lethal mutations have been known for
decades. These mutations show a wild-type phenotypa under permissive
conditions. Upon switching to nonpermissive conditions* the mutation is
expressed and the cell dies.
STATUS : On Schedule
ACCOMPLISHMENTS : 3acterial strains with cold-sensitive mutations in a gene
mediating resistance to ultraviolet irradiation wera testad under
laboratory conditions. A dacreasad viability was seen, but a high rate
reversion to prototrophy was also seen. This conditional lethal genetic
daterminant was, therefore, rejected as a practical method for biological
control of released GEM's due to instability and lack of mobility to other
strains. Bacteria containing a conditional lethal plasmid based on mercury
toxicity were tested under laboratory conditions. A reduced viability was
seen in these cells. A low incidence of plasmid loss or deletion was
noted, indicating that with this systam, tha conditional lethal genes may
be more useful if present on the chromosome. Construction of a conditional
lathal plasmid based on the 3-chlorobenzoat9—dependent expression of tha
EcoRI methylase gane and the constitutive expression of the endonucleasa
gene progressed. A 1.6 kb DNA fragment was isolated with the
3-chlorobenzoate regulatory gane and promotar. This was placed in front of
the promotei—less EcoRI methylase structural gene to determine if
3-chlorob»nzo3te-dependent expression of the methylase occurred. Growth
ratas of cells containing a plasmid encoding constitutive expression of the
methylase gena were not significantly different from cells containing the
vector plasmid alone.
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY "86
DATE :
WORK PLANS
7/ 2/36
WORK PLAN CODE
Q286
In collaboration iuith DPS. M. Nelson and ?. Pritchard at Gulf Breeze-ERL,
large plasmid was discovered and isolated in the trichloroethylene
degrading strain, G4« Tests are underway to determine if the plasmid
encodes the ability to degrade triehlorethy lene.
A presentation entitled "The Effect Of Conditional Lethal Genetic
Determinants Tn C. M. Cusk^y,
F. Genthner and A. W. 3ourquin. To be presented
Symposium on psaudomonads in the Environment and Medicine, Geneva*
Switzerland, 9-36.
Phenylpropanoid metabolism in Pseudomonas putida
S. M. Cuskey, J. R. Lute and R. H. 31sen. To be
on ""licrobial Metabolism and the Carbon Cycle, St.
Degradation of Trichloroethylane by a Bacterial
and Streptomyces setonii.
presented at a symposium
Paul Mn., 7-86.
Isolate. M. Nelson, S. M.
Cuskey» S. Q. Montgomery and P. H. Pritchard. To be presented at a
symposium on bacterial degradation of toxic uiastes, Aberdeen, Md., 11-85.
MILESTONES
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ENVIRONMENTAL RESEARCH LABORATORY
3ULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q286
Identification and Enumeration
Techniques. Survivability and
Genetic Exchange in Genetically
Altered Microorganisms. C6944A)
Qualitative and Quantitative 09/83
Detarmination of Genetic
Exchange and its Effect on
Microoially Mediated Activitias
in Aquatic Environments (74744)
Report on the Effect of Novel 11/83
Genetic Arrangements on Genetic
Exchange and Cell Survival in
Aquatic Environments C7653A)
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K
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ENVIRONMENTAL RESEARCH LABORATORY
3ULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q291
OU CODE & TITLE : L104 Chemical Tasting L Ass3ssment
OBJECTIVE CODE & TITLE : G Develop and Validate Improved Risk Assess Mthds
PPA CODE t TITLE ' 02 Environmental Aspacts of Biotechnology
PROJECT CODE £ TITLE : 2<» Ecol Consequences of G>sn Eng Microorgs
WORK PLAN TYPE : Inhouse/CDOP ORGANIZATION : Univ. of 111.
INVESTIGATOR : T. 3arkay PRINCIPAL INVESTIGATOR: R. Devereux
PHONE NO : FTS 636-9011
WORK PLAN TITLE : Use of rRNA Sequences to Characterize Natural Microbial
Populations
WORK PLAN OBJECTIVE : To develop tools necessary for detection of specific
microorganisms in mixed sediment communities. Implamentation of these
tools will permit an evaluation of the dissemination of GEMs and their
effect on microbial populations.
WORK PLAN APPROACH : Characterization of roicrobial populations often relies
first and foramost upon the prerequisite growth of colonies of bacteria on
agar plates. Since only a small precentage of sediment bacteria are. likely
to. grow into colonies when platedt the prerequisite plating effectively
precludes characterization of a significant portion of the microbial
community. Tnus, populations of bacteria capable of forming colonies on
agar plates are not likely to represent an accurate sample of the natural
populatio ns.
Characterization of low-complexity microbial populations by 5S rRNA
sequence analysis has baen performed with a hot spring community and
hydrothermal vent-associated symbionts. Due to the high-complexity of
marine sediments* cloning and sequencing of 1SS ri?NA genes mill be followed
for its characterization. Although the cnaractarization of mixed microbi^
communities through analysis of 16S rRNA sequences has not yet been done?
the strategy is conceptually and technically feasible. We will use 16S
rRNA sequence analysis therefore to charactsrize marine sediment microbial
communities with 3 focus on an ecologically significant physiological
groups of bacteria: tne sulfate-raducers.
A) Data 3asa - A reference collection of 165 rRNA sequences from
sulfate-raducin 3 bacteria will be compiled. In conjunction with Dr.
Frisdrich Widdel, University of Illinois* fifteen strains of
sulfate-reducers including species of Dasulfovibrio» Qesulfotomaculum*
Desulfobact=?r and Oesulfobulbus will be characterized by 16S rRNA
sequencing. Nuclaotide sequencing of their 15S r?NAs will be obtained,
using the chain terminating-reverse transcriptase procedure with 16S rRNA
as the template.
3) Purification of ONI from environmental samples - Protocols for
extraction of DMA from soils have been described which yield ONA of
relatively high purity. Such DMA however has an average size of about 1 Kb
and is not suitable for the type of cloning and analysis proposed hare.
For our experiments we will raquire DNA of about 30 Kb of sufficient purity
to alloai endonuclease restriction, ligation to vector DNA and packaging in
phage particles.
C) Cloning and packaging - The purified DMA will be partially digested with
the endonuclease Sau3A which cuts both methylated and unmethylated ONA.
Cleavaga of DNA with a restriction enzyme with a four-base pair recognition
sequence, such as Sau3A, produces a nearly random distribution of
fragments. Restriction fragments of 15-20 Kb will be ligated into the
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY *86 WORK PLANS
DATE : 7/ 2/36
WORK
PLAN CODE : Q291
BamHI site of the lambda cloning vector EMBL4. This vector can accomodate
large DNA inserts and carries genetic markers for the selection of
recombinant phage. Packaging of tha ligated DMA into infectious phage
particles will be accomplished, using packaging extracts prepared from E.
coli strain SMR 10. SMR 10 carries a lambda prophage uiitn a deletion in
the packing origin, rendering its chromosom? unpackagaable. This packagin
system therefore is of higher efficiency with l?ss background than the tmo
strain packaging systems.
D) Screening recombinant phage - Recombinant phage 'jjill be plated and the
plaques transferred to nitrocellulose filters. Phage carrying 16S rRNA
mill be identified by hybridization tuith 16S rRNA specific probas. Probes
mill oe either 165 rRNA prepared by using the chain terminating
reverse-transcriptase procedure tuith 165 rRNA purified from the
environmental sample as the template or synthetic DNAs designed from tha
compilation of sulfate-reducer 165 rRNA sequences.
STATUS : On Schedule
ACCOMPLISH^
technique
been requ
bacteria
inoculati
leading i
informati
Pond sa.np
technique
the sadim
amount of
approach
NTS : Initial studies .jere conductad at Univ. of 111 to develop
s and protocols while equipment and supplies for the research
isitioned and amplacsd. 1) Six strains of marine sulfata—reducing
have been maintained in culture and are pr3sently scaled up for
on into 10L carboys. 2) Letters have been sent to investigators
n the field of DNA purification from soils or sediments requestin
on and advice. 3) Ssdiment ;uas collected from the Range Point
ling site and processed in a pilot nucleic acid purification
. An ethano1-precipitated nucleic acid fraction was obtained fro
ents. This nucleic acid fraction appears to contain a large
humic substances. Experiments ar? undaruray to determine the bes
to purify the nucleic acids aiuay fro/n the humus.
I
MILESTONES (DATES) :
Sulfate-raduc;r 16S rRNA
sequences 1) 6 organisms
Sulfate-raducer 165 rRNA
sequences 2) additional
organisms
Sulfate-reducer 165 rRNA
saquencas
Characterization of Natural
Populations 1) DNA purification
tahcnique established
Characterization of Natural
Populations 2) ONA clonad,
filter hybridizations
TARGET REVIS3D REVISED REVISED ACTUAj
03/86
11/86
02/87
08/36
09/86
Charactarization of Natural
02/87
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q291
Populations 3) Subcloning
initial sequence c
haracterization
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L
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ENVIRONMENTAL RESEARCH LABORATORY
SULF BREEZE * FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE t Q287
DU CODE & TITLE : L104 Chemical Testing 6 Assessment
OBJECTIVE COOE & TITLE : 5 Develop and Validate Improved Risk Assess Mthds
PPA CODE & TITLE : 02 Environmental Aspects of biotechnology
PROJECT CODE & TITLE : 24 Ecol Consequences of Can Eng Microorgs
WORK PLAN TYPE : Inhouse/Contract
INVESTIGATOR : c Genthner ^
PHONE NO : FTS 686-9011
WORK PLAN TITLE : Capacity of Bacteria Isolated from Natural Aquatic Sourcas
to Exchange Genetic Material.
WORK PLAN OBJECTIVE : 1) To monitor the survival of appropriate donor and
recipient strains and the appearanca of exconjugants in sterilef freshwatar
sediment. 2) To assess the maintenance of transferred genetic traits in
broth and sediment. 3J) To evaluate the productivity of several selective
and differential plating media to be used for enumerating stressed
fluorescent pseudomonads in the aquatic environment.
WORK PLAN APPROACH : The frequency of conjugal transfer in sterile freshwater
sediment will be determined aiith those isolates identified as
recipient-active. To examine the effect of surfaces on conjugal transfert
frequency mill be measured in sediments amended•with various clays and in
mixtures of sand, silt, clays, detritus. Using these mixtures, various
paramat3rs (e.g., siza, surface, and charge of particles) can be
manipulated to define those conditions which affect conjugal transfer.
Surface slicks and other locations mill be examined for genetic transfer.
In these locations, plasmid transfer uiill be facilitated by using
indigenous isoganic strains. If significant transfer is noted, further
studies will be initiated. High concentrations of nutrients and bacteria
in such aquatic niches may favor colonization and gane transfer by released
ganetically engineered microorganisms.
5TATUS : On Schedule
ACCOMPLISHMENTS : Final data is being compiled for a journal article on the
transfer of plasmids from Pseudomonas aaruainosa to bacteria indigenous to
freshwater sedinent. To test a plate mating technique for predicting
horizonal spread of plasmid-encoded genas, tha 42 racipient-minus isolates
ware reaxaminad for their capacity to act as 9P4 recipients, using the more
sensitive, 'out labor intensive, filter-mating procedure. Only one isolate
(WTL 9401, genus unknown) demonstrated RP4 transfer. Of the 25
recipient-active isolates, 9 were racipients for the nonconjugative
plasmid» R1162, by RP4 mobilization. Transfer frequencies were compared by
using 3 procedures? broth, plata and filtar-mat ings. A solid surface for
transfer was better than liquid medium for ail recipients. In general,
filtai—mating was the most sensitive technique. This study confirmed that
transfer efficiency dapends upon environment.
The effect of surfaces on the frequency of conjugal transfer was examined
using Ps. aeruginosa as the R?4 donor and C. violacaum as recipient. Broth
matings, 30 deg C without shaking, uiere performed using 2 mis of donon and
recipient calls in 125 ml flasks. Sediment CO, 2.5, 5.0, 10.0 and 20
mg/ml) was suspended in plate count broth and used as the test variable.
Results demonstrated that the presence of sediment increased the transfer
frequency almost two fold. Similar results wera observed using the
benchmark plasmid R383.
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK
PLAN CODE : Q287
The potential for chromosomal mobilization was an additional goal of this
UIOTK plan. Difficulties mere encountered in constructing a donor harborinf|
chromosomal genes (TOL-plasmid) for the catabolism of toluene. An attempt
will be made to devisa an alternate model system. Chromosomal genes for
the catabolism of benzoate mill be mobilized into suitable recipients usinfc
plasmid R68.45. If dif f iculities are encountered, this goal mill not ba
continued as it is not considered important to the overall objective of thf*
project.
Genetic maintainance studies mere completed with C. violaceum (RP4, R1162)
After approximately 100 generations in Plat? Count 3roth (generation timet
45 min.), 66? of the cells had lost RP4 and 28% had lost R1162. In
constrast, only 3? had lost RP4 and all the cells had retained R1162 after
approximately 100 generations in sediment (generation time, 94 min). To
address the question of whether this effect tuas due to the growth medium or
the growth rate, plasmjLd maintenance studies were conducted in a dilute
medium nhich allowed a doubling time comparable to that in sediment. In
this medium plasmid loss was not observed. Thus, plasmid maintainance
appeared to be controlled by the grouith rate.
A study designed to evaluata plating media used for tracking the benchmark
strains in the aquatic environment is nearly complete. The data shows thag
either the differential Pseudomonas P or Pseudomonas F ftgar with the
addition of 0.5 mg/ml NX is the medium of choice for tracking, enumerating
and recovering thesa organisms. The addition of catalase did not
significantly improve the productivity of these media. However, the
productivity of other media when used to emunerate injured pseudomonads,
particularly SI, was improved with the addition of catalase. One of the
remaining experiments will be to examine the influence of fungal inhibitor
on the productivity of these media.
On April 17, I served as a judge in the Microbiology division of the
Florida State Science and Engineering Fair. In May I wrote a section of al
chaper on "Sacteria and the Env ironroent" for the book A Revolution in
Biotechnology. My section aias on microbial mining and metal recovery.
Monsanto data for the release of ?. syringaa harboring the insect toxin
gene ujas also reviewed in May. On June 11, I met with Dr. Greg Stewart
the University of South Florida - St. Petersburg to discuss a possible
cooperative agreement. On June 13» I mat with Dr. A. M. Guarino, US
FQA-Qauphin Island, AL, and discussed bacteria harboring multiple
resistance plasmids associated with catfish ponds.
MILESTONES OATES) : TARGET RSVIS^D REVISED REVISED
Begin constructing 11/85 06/86
p ar achlor obiphenyl degrader
of
Test for conjugal transfer in
freshwater sediment, slicks, and
fish intestines
11/83
04/86
04/<
Begin conjugal transfer studies 11/85
using sediments amended with
clays and mixtures of silt,
clays, sand, and detritus
Paper Presentation (SETAC): 11/35
Capacity of bacteria isolated
from natural aquatic sources to
participate in genetic exchange.
11/35
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN cooe : 0237
Journal Article: Transfer of 03/86 09/86
plasmids from Ps. aaruginosa to
bacteria indigenous to
freshwater sediment (6945C)
Internal Report on Regulatory 09/86
Research for Biotechnology?
Identification and Enumeration
Techniques* Survivability and
Genetic Exchange in Genetically
Altered Microorganisms. C6944A)
Report on Environmental Factors 09/88
Which Limit and Contro_l Survival
and Grouith of New Genotype.
(6945A)
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M
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q290
DU CODE £ TITLE : L104 Chemical Tasting £ Assessment
OBJECTIVE CODE L TITLE : 5 Develop and Validate Imporved Risk Assess Mthds
PPA CCOE 6 TITLE : 02 Environmental Aspects of Biotechnology
PROJECT CODE t TITLE : 24 ccol Consequences of Gen Eng Microorgs
WORK PLAN TYPE : Inhouse/COOP
INVESTIGATOR : S. McCarthy PRINCIPAL INVESTIGATOR: R. Olsen
PHONE NO : FTS 636-9011
WORK PLAN TITLE : Development of risk-assessment protocol for evaluating
heterogenetic DNA (Gram positive) detection in Pseudomonasi cloning and
characterization of lignase-relatad metabolism from Streptomyces
viridosporus to Pseudomonas bacteria,
WORK PLAN OBJECTIVE : Construct and characterize a model GEM - gene
sequence(s) for future, use in the evaluation of detection protocol related
to risk assessment.
WORK PLAN APPROACH : 1. Vectors developed for EPA
2. Gene bank construction as done previously for other streptomycetes and
other Gram negative bacteria.
3. Selection of relevant clones (i.e., FER/COU).
4. Search for other activities a. Indulin AT b. Model compounds
A series of cloning vectors has been developed for the EPA as part of the
bench mark plasmid series. These vectors are multicopy? IncW replicators
to uihich various selective antibiotic resistance markers mith distinctive
sites for restriction endoncleases have been added. Vector pR02317 is
comprised of the tetracycline resistance determinant from p3R322 and its
restriction sites. The carbenicillin resistance of pR023l7 is in part
derived from p8R322 and in part from the transposon, Tnl. A similar
vectorf pR02321 has also bean constructed. Its trimethoprim gene was
derived from plasmid R322 and substitutes for the carbenicillin determinant
of pR02317. These vectors are broad host range and therefore have
potential utility for broad;application among Gram negative bacterial
species. Preliminary experiments indicate that their utility is equivalent
to that reported previously for the broad host range Pseudomonas cloning
vectort p*Q1614. We will use these vectors for the cloning of lignin
metabolism-related genes from S. viridosporus into P. aeruginosa.
We have developed protocol for the groiuth and DNA harvest from S.
viridosporus. This DNA mill be partially cleaved with restriction
endonuclease, Pstlf and ligated to one of the vectors described above under
conditions promoting the maximum recovery of recombinant Plasmids. This
ligated DNA mill then be transformed into P. aeruginosa strain PAOlc uiith
selection for tetracycline resistance since this antibiotic resistance gene
mill be unaffected by the procedure. Tetracycline resistant colonies mill
be streaked onto homologous medium and medium which contains carbenicillin.
StreaKS sensitive to carbenicillin mill be designated and collected from
the permissive plate Ctatracycline) onto a plate of tetracycline medium
(100 carbenicillin sensitive clones per plate). Following overnight
incubation* the plates mill be mashed with buffer and used as an inoculum
for the growth of cells from oihich plasmid ONA mill be harvested using
appropriate techniques. DNA suspensions derived from a mixed culture of
100 independently isolated colonies mill constitute a gene bank for
subsequent testing.
The next task mill focus on obtaining a sample of S. viridosporus DNA that
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ENVIRONMENTAL RESEARCH LA30RATORY
GULF BREEZE , FLORIDA 32561
FY *86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q290
is unambiguously in P. aeruginosa. For this work, we mill intially search
gene banks for clones which have acquired the ability to utilize ferulic
acid as a sole source of ^carbon for growth. Our previous work has shown
that S. setonii DNA associated juith farulic acid metabolism is expressed ir
P. aeruginosa. In view of this observation* we expect to isolate the
analgous clone cerived from S. virido.sporus. Pending this and the possibB*
expression of other distinctive metabolic steps* we uill test the ability™
of gene bank transf ormant s to acquire the metabolic ability to groin on
polymerized lignin (e.g.* Indulin AT) and* perhaps* model compounds. If
the progress of the work is as anticipated, we should make available for
further study an example of a biomass - degrading recombinant DNA clone
(GEM) for further evaluation and testing by the EPA ERL.
Topical summary of experiments to b a done.
1. Prepare DNA required for the cloning sxoeriments Ci*e«* vector pR023lT
plasmid DNA and S. viridosporus chromosomal DNA.
2. Streptoniycete DNA will be shot-gun cloned into pR02317 following
partial digestion with restriction endonuclease* PstI* and ligation with
transformation into P. aeruginosa strain PAOlc which has been rendered
phenotypically non-restricting for hetero-ONA. Gene bank ONA containing
100 indapendently isolated recombinant plasmids will be prepared as
reported by Olsen, et a'l (8).
3. Gena banks will be searched by their transformation into P. aeruginoqp
for transformants that have acquired the ability to groa on ferulic acid
shown previously by Cusky and Qlsan.
4. Positive clones from the above search will be analysed for the physica
and functional characteristics of the cloned DNA. Such results will be
compared with similar clones reported by Cuskay and Olsen for the cloning
of DNA from S. setonii.
5. Following the successful conclusion of the above preliminary
exparimants* the gene banks will now be searched analagously for the
isolation of recombinant plasnids allowing growth on lignin model compound
(3.3.* veratryglycerol-beta-guaiacyi ether) or the commercially available
low molecular weight lignin substrate, Indulin AT.
STATUS : Final Report: Development of oactar'ial models for risk assessment:
Use of banch nark plasmids and DNA sequances Due 07/83
ACCOMPLISHMENTS : Streptomyces viridosporus chromosomal ONA and vector pR017™f
plasmid DNA hava been purified for jse in cloning experiments.
Several transformatants have been obtained via shotgun-cloning of S.
viridosporus DNA into p3Q1727 with subsaquent transformation of P.
aeruginosa PAOlc cells. To accomplish this, S. viridosporus DNA mas
partially digested with restriction endonuciease PstI and shotgun-ligated
into Pstl-cleaved pRO!727 vector DNA. Ligated DNA ujas used in the
transformation of P. aeruginosa PAOlc, followed by selection of
transformants on tetracycline-containing (50 nig/ml) medium. Tetracycline
resistant colonies were rsstr^aked to tatracycline medium and
carbenicillin-containing (600 mg/fll) medium. Tetracycline resistant,
carbenici11 in sensitive clones were examined for utilization of ferulata,
collected from permissive (tetracycline) plates* and frozen for future u^a
in the construction of a gene bank.
The week of April 23 - May 2 was spent in the laboratory of Dr. Ronald H.
Olsan at the University of Michigan. During this periodt transformations
of P. aaruginpsa PAOlc cells were carried out using S. viridosporus DNA
which had baen shotgun-cloned into vector pR02317.
-------
ENVIRONMENTAL RESEARCH LABORATORY
SULF 8RE6Z5 , FLORIDA 32561
FY '86 WORK PLANS
DATE t 7/ 2/86
WORK PLAN CODE : Q290
MILESTONES (DATES) :
Identification of gene bank
clones associated with the
degradation of lignin (at Ann
Arbor and Gulf Breeze labs).
Derive and test Tra- bench mark
plasmid set with transposition
functions deleted for EPA
investigators (at Ann -Arbor and
Gulf Breeze labs).
Complete enzymological and
regulation studies on pJP4 gene
bank recombinant plasmids (at
Ann Arbor and Corvallis labs).
TARGET REVISED REVISED REVISED ACTUAL
10/86
12/86
02/87
Physical and functional
of gene bank donas (At
Arbor and Gulf Breeze labs).
mapping
Ann
02/37
Prepare and inventory bench mark
plasmid specific probes with
known sequences for distribution
to EPA investigators (at Ann
Arbor lab).
06/87
Prepare recominant plasmids for 06/87
use by ?PA investigators in
microcosom studies (at Gulf
Breeze and Ann Arbor labs).
Characterization of the 06/37
enzymological basis for biomass
conversion of metabolizable
compounds (at Ann Arbor and Gulf
Breeze labs).
Final report on pJP4-related 11/87
work at Corvallis and Ann Arbor
labs
Final report on Gulf Breeze lab 01/88
activities
Collaboratet consult with Gulf 07/83
Breeze - biomass project and
Corvallis - chloro-hydrocarbon
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q290
degradation projects; prepare
publications and reports.
Expression of streptomycete DMA 03/86
cloned into Pseudomonas
aeruginosa PA01. CProc, A.S.M.)
Jounal Article: Protocatechuic 06/86
acid dioxygenase from
Pseudomonas cepacia: Cloning the
structural genes using an
I ncW-deri v/ed vector? pR02317.
ASM Abstract: Cloning and 09/86
expression of halo-aromatic
genes in Pseudomonas cepacia
D801.
-------
-------
ENVIRONMENTAL RESEARCH LA30RATQRY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q288
DU CODE & TITLE : L104 Chemical Testing & Assessment
OBJECTIVE CODE & TITLE : G Develop and Validate Improved Risk Assess Mthds
PPA CODE & TITLE : 02 Environmental Aspects of Biotechnology
PROJECT CODE & TITLE : 24 Ecol Consequences of Gen Eng Microorgs
WORK PLAN TYPE : Inhouse/COOP
INVESTIGATOR : A Bourquin PRINCIPAL INVESTIGATOR: R Walter
PHONE NO : FTS 686-9011
WORK PLAN TITLE : Construction of Bacillus subtilis Strains Containing a
Marker Sequence Allowing Quantitative Enumeration in Environmental Culture:
WORK PLAN OBJECTIVE : The possibility of releasing bioangineered
microorganisms either intentionally or by accident prompts us to understanc
how these microbes uiill survive in their native habitat. As important as
understanding the hosts ability to survive is the question of whether the
added Cbioengineered) ONA sequences placed in them would be transferred to
other organisms and so persist in the environment. To approach these
questions I propose to construct a short (1 to 2 kbps) DNA sequence (market
sequence) which confers chloramphenicol resistance on B. subtilis strains
harboring it and acts as a specific marker in DNA hybridization
experiments. This marker sequence would be cloned in B. subtilis at
various places including: 1) several chromosomal locations* 2) phag«
genomes (transducing and nontransducing), 3) plasmid genomes* and 4) the
transpoison Tn917. The marked strains thus constructed would b» followed
separately (or in combinations) in artificial microcosms to detemine both
the survival of the host and the ability of the marker sequence to be
transferred to other species or genera.
WORK PLAN APPROACH : The emergence of bioengineered microorganisms for use in
agricultural* industrial, and research applications prompts us to begin to
ask questions concerning the stability of such organisms in native haoitat;
(1*2). It seems prudent to understand how a novel DNA segment will affect
both the ability of the engineered microbe to maintain itself in its niche
and the effact* if any* on tha surrounding environment imposed by it.
Microoranisms have several means of transferring DNA sequences between
members of a species including conjugation* transformation* transduction,
and transposition (via cointegrata formation uuith conjugative plasmids).
Transfer of genetic material between and across ganatic barriers has been
demonstrated in enteric and other microbes in vivo (3»4»5»6*7). Once a
novel DMA sequence has entered a neuj genus* no matter how improbable this
event may be* it seems likely that it may spread among the members of the
new host (and closely related organisms) with extreme rapidity using the
above mentioned modes of genetic axchange.
A question which bears investigation concerns the comparative survival of
bioengineered organisms in which the introduced (foreign) DNA segment is
located at different locations in separate members of otherwise isogenic
strains. For instance one may ask* if a cloned DNA segment is in the host
chromosome, is it mora stable (ie. less stressing to the hosts survival
and/or less able to be transferred to other niche innabitants) than if the
sama segment is placed on a nonconjugative plasmid? Is a DNA sequence
cloned in a phage genome as readily transmitted to other species as the
same sequence cloned in a transpioson or conjugative plasmid? Does the
site of cloning play a role in tha stability of the sequence or mill a host
"scramble" the sequence among phage* or plasmids picked up from other
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE t FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK
PLAN CODE : Q288
members of the native habitat?
in <; in u *; i o wi v 11 *? IIOI.A.W 11 a hs .L i. a i i
In order to approach thesa questions T will construct a DNA sequence whichB
can be easily followed by both biological (selective plating) and physical*
(DNA hybridization) methods. Using standard recombinant DNA techniques I
will then place this DNA segment in: 1) the host genome at various
locations» 2) conjugative and nonconjugative plas.nids* 3) transducing and
nontransducin3 phage genomes* and 4) a transposon. A set of strains in
which each member contains the same foreign ONAt but at a different site»
would allow one to compare the fate of both the host organism and the novej
DNA sequence in an artificial microcosm through time. Release of one or
combination of these marked strains uiith appropiate monitoring of their
survival may allow us to understand how an organism which is to be releaseB
in the environment should be constructed in order to attain the desired
benefits without compromising the native habitat.
STATUS : Cn Schedule
ACCOMPLISHMENTS : The DNA cassette contains two unique sequences (plant DNA
from plasmid pnz» and animal DNA from plasmid RVH832) flanking a biological
marker has been constructed and its structure confirmed by southern
hybridization. Ananlsis using the cassette for bacterilogical
determination and enumeration has been started and is undermay.
Construction of Bacillus subtilis strains carrying the cassette at various
locations is being attempted.
MILESTONES (DATES) : TARGET
Construction of strains carrying 10/85
the marker sequence at different
sites in t h a same genome.
REVISED
06/86
REVISED
03/86
REVISED ACTUi
Initial baseline studies on fata
of host in mictocosin and
stability of narkar sequence.
01/8S . 03/36 09/36
Placement of 'narkar sequence in 31/85
extra-genomic entities.
Analysis of fate of marker in 03/85
extra-genomic entities after
release in microcosms.
03/36 09/36
11/36
Report on Environmental factors
which limit and control survival
and growth of n a w genotype.
(6954A)
09/37
Journal Article: On the Survival
of Genetically Engineered
Bacillus in Microcosms.
06/86 08/86
Transfer of Ganetic Material in
07/86 10/85
-------
ENVIRONMENTAL RESEARCH LABORATORY
3ULF BREEZE * FLORIDA 32561
Fr '86 WORK PLANS
DATE : 7/ 2/86
WORK
Microcosms by Bacillus subtilis-
Qualitative and Quantitative 09/88
Determination of Genetic
Exchange and its Effect on
Microbially Mediated Activities
in Aquatic Environments (7474A)
Internal Report on Regulatory 11/88
Research for Biotechnology I
Research for Biotechnology!
Identification and Enumeration
Techniques* Survivability and
Genetic Exchange in Genetically
Altered Microorganisms C694<»A).
Report on the Effect of Novel 11/88
Genetic Arrangements on Genetic
Exchange and Cell Survival in
Aquatic Environments C7653A)
PLAN CODE
Q288
-------
-------
ENVIRONMENTAL RESEARCH LABORATORY
3ULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE
DU CODE & TITLE : L104 Chemical Testing L Assessment
OBJECTIVE CODE £ TITLE : M
0191
PPA CODE & TITLE
Validated Exposure Assess Mthds Related to Eco
Risk
03 Evaluation of Exposure Assessment Methods
PROJECT CODE L TITLE : 07 Microcosms L Field Applicability
WORK PLAN TYPE
INVESTIGATOR :
PHONE NO : F7S
: Inhouse/Contract
P Pritchard
636-9011
WORK PLAN TITLE : Laboratory Microcosms as Verification Tools for Studying
Fate and Transport Processes in Natural Estuarine Systems
WORK PLAN OBJECTIVE : Use of Eco-core and other microcosms to verify the
accuracy of exposure assessment models and lao test methods which assess
biode gr ad at ion potenti.al of estuarine microoes associated uiith salt mater*
sediment, and coastal or estuarine plant/root surfaces.
WORK PLAN APPROACH : With the larga number of toxic chemicals to be registered
by SPA» mathematical modeling mjst be used to provide assessments of risk.
Validation of these modals and tha methods for deriving input data for tha
models will be nore expedient and cost saving u»ith microcosm studies than
validation tuith field studies.
If microcosms are simulations of natural sites they can be used to assess
fate of toxic chemicals in aquatic environments in lieu of field studies.
To make predictions about the field from microcosm studies* three
verification steps are required? a) microcosm simulation* b) systems
analysis of lab derived process data and c) conceptualization of
mathematical model framework. Microcosm simulation
-------
ENVIRONMENTAL RESEARCH LA30SATORY
GULF BREEZE t FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK
PLAN CODE : Q191
Report on Extrapolation of 09/86
Laboratory 3i.odegradati.on Data
to Microcosms and Field Studies
C6319A)
Biodegradation of Chlorophenol 04/86
in a Salt Marsh Environment
C6319E)
Report on Viability of 04/86
Biodegradation (heterotrophic)
Acitivity Levels in an Estuarine
Environment (6319G)
Biodegradation of 4-Chlorophenol 11/85
in Estuarine Environments
(6319H)
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE i FLORIDA 32361
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : 0195
DU CODc & TITLr : LIO^ Chemical Tasting £ Assessment
OBJECTIVE CODE £ TITLE : M Validated Exposure Assessment Methods Related to
Eco Risk
PPA CODE & TITLE I 03 Evaluation of Exposure Assessment Methods
PROJECT CODE £ TITLE : 07 Microcosms and Field Applicability
WORK PLAN TYPE : Inhouse/Con trac t 4.
INVESTIGATOR : P Pritchard
PHONE NO : FTS 636-9011
WORK PLAN TITLE : Role of Cometabo1 ism in the 3iadagradation of Mixtures of
Alkyl Substituted Benzenes by Microbial Comnunities in Aquatic
E n v i r o n m e n t s .
WO.RK PLAN OBJECTIVE : To compare the relative rates of biodegradation of
specific alkyl benzanes singly and in complex mixtures. To datermine the
effect of the biodegradation of one alkyl benzene on the degradation of
another. To assess through pure and mixed culture studies» the mechanisms
by jihich natural communities degrade mixtures of aromatic hydrocarbons.
WORK PLAN APPROACH : Our general experimental plan mill be to folloui the
relative rates at which individual components of a defined mixture of alky]
benzenes disappear in sterile and continuous flow test systems Csee below)
using saaaiater from polluted and pristine estuarine sites. Individual
alkyl bsnzenes aill ba run through a separate continuous flow system to
enrich for specific dagrader organisms. The pure cultures will be
characterized for their ability to degrade alkyl benzenes isomers and other
substances. Attempts u;ill ^e made to establish groajth on one substrate anc
corcetabolism of a second suostrata. The degradation product of the
resulting comet =»bolic process mill oe identified. The involvement of the--
isolates, and their cometabolic capabilities, in the degradation of the
mixture of alkyl benzenes will be assessed ~>y determining, during
degradation of the mixture, the groutn response of the isolate in the
microbial community, the relative r 3 ,n o v a 1 rate of the hydrocarbon
components from the mixture and the appearance of the same degradation
product detected in the pure cultur? studies. This protocol can be
inter atively jsed to assess tne fat? of several alkyl benzenes. In
addition, cnanges in the ratio of the alkyl benzenes in the mixture u/ill be
tested to determine their effect on the microbial community response.
Efforts will focus on manipulating the "nicrobial community through multiple
inductions and biom^ss increases to mar? efficiently degrade the
hydrocarbon mixture, Microbial communities from polluted and pristine
estuarine sites mill alloui a comparison of aetabolic diversity. The
detactian of degradative plasmids in tha pure cultures tuill potentially
alloiu us to examine tnair involvement and d/namics in the degradation
processes mediated by the microbial communities.
STATUS : Cn Schedule
ACCOMPLISHMENTS : The continuous flooj biodegradation test system for mixtures
of volatile hydrocarbons has bean succpssfully tested uiitn toluene as tne
model substrate. Use of the system for the water soluble fraction of jet
fuel has besn tasted; A method of feeding a constant concentration of the
water soluble fraction has been developed bat methods are still being
tested for analysis of the hydrocarbons in th« affluent. Further tests
have shou/n that biodegradation of tne hydrocarbons in the water soluble
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , f=LORIDA 32561
FY '86 W03K PLANS
DATE : 7/ 2/36
WORK
PLAN CODE
Q195
fraction can be studied in sealed batch systems as uiell. A continuous
vapor feed enrichment system has bean used to isolate hydrocarbon
dagraders. Pure cultures uihich will degrade toluena have been isolated
from Range Point salt marsh» 3ayou Chico and 3scambia River and the
substrate specificities of tha cultures characterized. One isolate has
been tested for its ability to degrade the hydrocarbons in the j/ater
soluble fraction. Toxicity of toulenef xylenes and trimethyIbenzenes to
natural microaial communities has also been tested. Next quarter pure
cultures uihicn degrade other aromatic hydrocarbons will be isolated and
characterized for substrate specificity.
MILESTONES (DATES) :
Literature search aid
preliminary development of
experimental systam completed
Bottle test completed;
continuous flow experimental
system completed. WS^
degradation experiment initiated
Enrichment and characterization
of two aromatic hydrocarbons
dagraders. W5F degradation
experiment completed.
TARGET
12/35
03/86
REVISED REVISED REVISED
I
ACTU
12/81
03/86
Characterization
pathway for each
completed
of cometabolic
isolate •
06/86
09/86
Comparison of pjre culture
degrade tive activity ujith
microbial community degradative
activity
Output - Midterm Report: Air
Force Interagancy Agreement
Output - Journal Article!
Biodegradation of Hydrocarbon
Mixtures by Aquatic Communities
Output - Journal Article:
Cometabolism of Alkyl 3enienes
by Pure Cultures ^rom Estuarine
w aters
01/86
06/86
01/37
D6/36
11/36
Report on the Role of
Cometabolism in the
04/89
-------
ENVIRONMENTAL ^S^ARCH LABORATORY
SULF 3REEZE » FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q195
Biodegradation of Xenobiotics in
Aquatic Environments C7^36A)
-------
^ * <
I
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , PLORIOA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q277
DU CODE L. TITLE : L104 Chemical Testing 6 Assessment
OBJECTIVE CODE & TITLE : M Validated Exposure Assess Mthds Related to Eco
Risk
PPA CODE 6 TITLE : 03 Evaluation of Exposure Assessment Methods
PROJECT CODE £ TITLE : 07 Microcosms and Field Applicability
,^O.RK PLAN TYPE : Ihouse/Contract
INVESTIGATOR : 8 Ganthner
=»HONE NO : 636-9011
WORK PLAN TITLE : Anaerobic Degradation of Toxic Chemicals
WORK PLAN OBJECTIVE : 1) To obtain anaerobic microorganisms capable of
degrading chlorinated organic compounds. 2) To develop a medium for
isolation of anaerobic dechlorinating organisms. 3) To isolatet identify
and characterize thase-microorganisms. 4) To study the biochemical
pathways and enzymes involved. 5) To study the genetics of anaerobic
dehalogenation. 6) To determina the usefulness of these organisms in
detoxifying hazardous uiaste.
WORK PLAN APPROACH : Anaerobic enrichments mill oe prepared containing 2-,3-
or 4-C1 benzoatei or 2-,3- or 4-C1 phenol. Inoculum preexposed to
chlorinated organic compounds u/ill be used. A variety of terminal electron
acceptors (nitrate* sulfatet or C02) mill be used to increase our potenti
for obtaining degradation. Enrichments mill be analyzed for loss of pare__
compound. Parameters (rate* nutritional requirements* optimium conditions
of degradation mill bo determined. An anaerobic medium mill be developed
to isolate the anaerobes responsible for dechlorination. Isolated
organisms mill be characterized and identified. Enzymes and enzymatic
pathuiays involved mill be investigated. Genetic regulation of pathways
will be studied.
STATUS : On Schedule
ACCOMPLISHMENTS : All specialized anaerobic equipment and supplies have been
received. After problems rnith anaerobic gases and leaks due to manufactor
errorf 3 fully functional anaerobic chamber is in operation.
Anaerobic enrichments urars prepared from inocula from the Navel Air
Station, Lotue's Lagoon, Bayou Chico* Escambia River, and Monsanto Plant.
The WAS sampla >uas sewage sludge; all others mere sediment. The Monsanto
sample mas sediment previously exposed to PC3s. Tha enrichment conditions^
were: 1) nitrate-reducing* 2) sulfate-reducing* 3) methanogenic and 4)
nonmethanogenic• The nonmethanoganic enrichments contained 1.0 uM
bromoethanesulfonic acid C3ESA), a potent inhibitor of methanogenesis,
including in the event that reductive dechlirination and methanogenesis arm
competitive processes. Hotuavar* it mas recently learned that 3ESA also
inhibits dehalogenation by DC3-1* the only anaerobic dehalogenator now in
pure cultura. Tharefora* tne success of this set of anrichments may be irl
doubt. Eight compounds mere usad as energy sources. These included
b
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BRE5ZE , FLORIDA 32561
FY '86 WORK PLANS
DAT£ : 7/ 2/86
WORK PLAN CODE : 0277
Library investigations have lead to the formulation of a number of testable
media. The first approach is based on a change in pH that results from the
formation of HC1. The indicator bromcresol mill change from purple to
yelloui beloa pH 6.8 (normal pri-7.0). A yellow zone around a dechlorinating
colony may result. A sacond approach- is to incorporate CaC03 into the
medium. This compound is insoluble at, neutral pH» but solubilizes ar acid
prl» resulting in a clear zone around a dechlorinating colony. A third
variation mill incorporate aosin into the msdium. At acid pH this compound
is solubilized and taken up* resulting in a red colony. A final variation
mill incorporate fluorescein in the medium which displays an intensive
y ellouj-green fluorescence under ultraviolet light* which is extremely
sensitive to acid. At acid pH the fluorescence dissappears. A
nonfluorescant colony or zone should result from a dehalogenating colony.
To prevent color changes due to acid production from other catabolic
processes* a defined m-edium with 3.01% yeast extract mill be used whenever
possible. To prevent acid formation from metabolism of the resulting
phenol or benzoate» an inhibitor of methanogenesis can be incorporated.
Under anaerobic conditions in the absence of sulfata or nitrate? phenol and
benzoate generally require methanogens to utilize the hydrogen formed and
make the degradation thermodynamically feasible.
A second approach to developing a dahalogenation medium will rely on the
detection of the chloride ion CC1-) released. A Cl- free medium mill be
prepared using double distilled water and acetate salts. Sulfate salts
cannot be used because sulfate inhibits dehalogenation by strain DCB-1» a
sulfate reducar. The only source of chloride in this medium mould be the
chlorinated aromatic under study. Silver nitrate (AgNOB) incorporated into
the medium would result in white zones CAgCl) around the dechlorinating
colonies. If A.gN03 is too toxic* a filter paper disc containing K2Cr03
could bs pressed on a replica plate* than sprayed with AgN03 solution. If
Cl is raleased AgCl would form* if not AgCr03» a red compound mould form.
A fluorascein indicator will also be used to detect C1-. At a pH of 7-8»
fluorescein changes color depending on which ion, Ag+ or C1-, is in excess.
If AgN03 is incorporated into the medium containing fluorescein, a color
change mill occur if Cl- is released. This assay could also be used with
filter paper discs.
A third approach to the dahalogenating medium would be to use colored
halogenat^d compounds which loss thsir color when dehalogenated. These
include a numoer of pH indicators: bromochlorophenol blue, bromcresol
grean, brDmcrssol purple* bromophenol blua, bro^pyrogallol red,
bromoxylenol blue, bromothymol blue* dichloroindophenol*
dichlorofluorescein and tatrabromofluorescein. The color variations depend
on the side groups prasent on the aromatic ring* th? pH and the electron
potential (Eh). It is uncertain whether these compounds will be
d ahalogenated by the organisms. However, the approach would be most useful
for phenol dehalogenation as th£ structure contains phenol residues. A
number of structurally less complicated haloganated compounds are colored.
Among toese is 2-bromo-phenol* a red compound. Debromination mould result
in a colorless phanol.
A final approach usas recent information about strain DCB-1. A medium will
be prepared with a chlorinated aromatic compound with and without BES4.
which inhibits dehalogenation* and an additional anargy source (i.e.
acetate* lactate, pyruvate). Growth in the absence of SESA, but not in its
presence could indicate a dahalogantor which is physiologically similar to
DCB-1.
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY "86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q277
The relationship betuieen sulfate—reduction and dehalogenation is intriguing
and is being investigated. We have seven strains of sulfate-reducers H
representing three genera: Qesulfovibrio, Desulfabactjr and Oasulfobulbus.iJ
If dehalogenation is an alternative election sink for sulfate, a medium
lacking sulfate but containing an appropriate energy source and a
chlorinated aromatic may support growth. Growth and dehalogenation of
these seven strains mill be followed overtime in nedia a»ith and without
sulfate.
MILESTONES (DATES) : TARGET REVISED REVISED REVISED ACTUftT
Train technician; equip a 03/86 03/3-J
functioning anaerobic laboratory
Obtain and maintain cultures for 06/86
anaerobic culture collection;
prepare anaerobic enrichments of
2-,3-and 4-C1 benzoate snd
2-»3-and 4-C1 phenol under
sufate-reducing, n
itrat 9-reducing, met h an ogenie
and nonmethanogenic conditions;
determine initial level of
compound in enrichments;
determine level of compound at
monthly intervals
Devise anaerobic medium to 09/86
isolate dehalogenating bacteria;
biochemically characterize
degradation of compound in
enrichmants shoeing activs
degrade ti on.
Charactariz3 dehalogenating 12/85
activity in cultures obtained
fro in outside laboratories;
characterize dahslogenation and
degradation of compounds by
cultures i3olat?d from our
enrichments
Journal Article: Degradation of 09/86 12/86
chlorinated organic compounds
under a variety of enrichment
c ondition s
Journal Article: Anaerobic 12/86
medium for the isolation of
dehaloganating bacteris
Final Report: Isolation, 12/83
identification, biochemical and
genetic characterization of
anaerobic dehalogenation
processes in bacteria
-------
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q278
DU CODE & TITLE : L104 Chemical Tasting & Assessment
OBJECTIVE CODE & TITLE : M Validated Exposure Assess Mthds Related to Eco
Risk
PPA CODE I TITLE : 03 Evaluation of Exposure Assessment Methods
PROJECT CODE & TITLE : 07 Microcosms and Field Applicability
WORK PLAN TYPE t Inhouse/COCP
INVESTIGATOR : P Chapman
PHONE NO : 686-9011
WORK PLAN TITLE : Cometabolism Machanisms in Siodegradation
WORK PLAN OBJECTIVE : To elucidate mechanisms responsible for the process of
cometabolism. 3actaria able to use aromatic hydrocarbons will be used to:
1) Define the conditions responsiole for cometabolism. 2) Exemplify how
specific growth substrates direct the routes by which a bacterial
population transforms nongrowth substrates. 3) Obtain regulatory mutants
uihich accomplish specific transformation. 4) Show how fortuitous
metabolism and cometabolism can lead to Cregulatory) mutations in cultures.
5) Show how successive acquisitive mutations can lead to the evolution of
biodegradation strains with constitutively expressed enzymes.
WORK PLAN APPROACH : Cultures of Pseudomonas putida able to grow both with
p-cymene and with toluene (and also benzene and ethylbenzene) are available
and will be used. As controls P. putida cultures able to grow only with
p-cymene Cand r.ot with toluene) and with toluene (plus benzene and
ethylbenzene) but not with p-cymene mill be acquired or isolated. Initial
grouith experiments will confirm their ability to grow with the substrates
indicated and with related hydrocarbons and will be followed by assays of
enzymes characteristic of the p-cymene- and toluene-pathways to ensure the
pathways employed are as previously described and are inducible. Control
cells able to grow uiith only ona of the aromatic hydrocarbons will of
course not grow with the other and should demonstrate no activity towards
intermediates of the absent,pathway. Using washed cells grown with the
substrates compounds such as indole, naphthalane» trifluorotoluenef and
dibenzofuran dill bs tested as substratas for the toluene-induced pathway.
The spectral properties of these products will be detarmined to throw light
on their structures and to provide suitable wavelengths for measurement of
their ratas of formation. Comparisons will then be made of the rates at
which known dry weights of cells produca thesa products and how these rates
vary with growth substrata. por the p-cy,nene pathway 4-biphenylcarboxylie
acid* pseudocymane, and 2-chloro-p-xylene have been identified as compounds
converted to colored reaction products. Tha colored products accumulated
by toluene- and p-cyraene-grown cells can be used as a measure of the cells
aoility to cometabolize them. For example it will be possible to measure
the rate at which cells convert trifluorotoluane to a yellow reaction
product when it is added to cells growing witn either toluene* p-cymene or
lactate to demonstrate whether there is a r?pid conversion with any of the
above growth substrates. Once a number of compounds have been identified
as serving as noninducing substrates for ona or mora of the enzymes of the
toluene- and p-cymene-pathways uith P. putida cultures* similar lines of
investigation will be pursued with Rhodococcus cultures.
These studies will be followed by experiments with ,nixed cultures of P.
putida strains and Rhodococcus strains to ascertain whether the results
obtained with pure cultures have predictive value for mixed cultures.
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q278
These in turn mill lead to studies with mixed cultures from different
environments such as marine* frash and estuarine waters. The goal here is
to show whether addition of a specific growth substrate can promote
dagradation of a compound in a predictable fashion. It is overly
optimistic to predict the outcome of such experiments but this investigato'-i
anticipates that toluene addition to mixed and environmental communities '
mill specifically promote conversion or cometabolism of dibenzofuran and
that cymene addition mill lead to cometabolism of 2-chloro-p—xylene.
Certain other compounds such as p-chlorotoluenep p-bromotoluene and
p-iodotoluene mill also bo examined as cometabolizable substrates by our
Pseudomonas and Rhodococcus cultures. The products formed by each mill be
rigorously characterized and ara expected to be 3-halo-6-methyl catechols
(by the toluene pathway) and 4-halo-2 »3-dihydroxybenzoates (by the cymene
pathway). With these products colorimetric methods will be developed for
their distinction and measurement. These will then be applied to
determining their rates of formation by calls grouin with different
substrates. These examples are chosen to show how with pura cultures a
grouith substrate may determine what products are formed and therefore play
a directing role especially where the transformed compounds cannot induce
enzymes for their own attack.
The systems chosen for study have tne advantage that selection of
spontaneously occurring regulatory mutants of pseudomonads is a straight
forward procedure. Thus muta.nts expressing constitutively the initial
enzymes of toluane catabolism or of p-cymana catabolism can be readily
isolated. Furthermore it can ba shown that the selective pressures used t.o_
obtain constitutive mutations are brought about by cometabolic processes
and lead to mora versatile acquisitive mutants. Understanding the
mechanisms involved in the selection of more versatile* degrading
organises* as in the selection of biphenyl-utilizing bacteria from the
original cymene/toluene-utilizing strains* can lead to an understanding of
how similar procedures may be used to construct other more versatile
strains of bactaria with enhanced degradative potential.
STATUS : On Schedule
ACCOMPLISHMENTS : Isolation of additional bacterial isolates able to utilize
either p-cynene or toluane (but not both) has ba
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q278
fashion* the early reaction sequence uihich converts cymene to its
carboxylic acid oxidation product. Why this occurs in deregulated mutants
is presently not clear but doss suggest that in these strainst contrary to
previously published work, the cymene pathway is encoded by at least tuio
regulated clusters of ganes.
A convenient blocked mutant selection is currently being employed to obtain
organisms uiith specific lesions in the cymene pathway in order to
accumulate motabolites and cometabolites uihich can be characterized and
used as enzyma substrates. In tha course of this uiork, differences in
strains are evident from the type and frequsncy u/ith mhich mutants are
obtained.
MILESTONES (DATES) : TARGET RSVISED
Isolation and characterization 03/86 09/86
of mutants
REVISED REVISED ACTUAL
Accumulation and identification
of cometabolities
Competent mixed cultures
constructed
08/86 12/86
01/87
Environmental Studies 07/87
Project report of 2-years work 09/87
Journal Article: A comparison of 06/87
tne cometabolism of halogenated
aromatic hydrocarbons by
pseudomonads able to utilize
p-cymana and those possessing a
TOL plasmid.
Journal Article: Evolution of a 06/87
bipnenyl degradative pathway in
pseudomonads by recruitment of
enzymes of p-cymene and
ethylbenzene degradation
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE t FL3RIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q279
3U CODE & TITLE : L104 Chemical Testing & Assessment
OBJECTIVE CODE I TITLE : M Validated Exposure Assessment Methods Related to
Eco Risk
PPA CODE & TITLE : 03 Evaluation of Exposure Assessment Methods
PROJECT CODE & TITLE : 07 Microcosms and Field Applicability
WORK PLAN TYPE : Inhouse/Contract
INVESTIGATOR : P Pritchard
PHON5 NO : FTS 686-9011
WORK PLAN TITLE : Isolation and Characterization of Microoganisms Which
Degrade Trichlorosthylene
WORK PLAM OBJECTIVE : The purpose of this projact is to assess the feasibilitB
of utilizing microbial metabolism as a means of removing trichloroethylene
(TCE) from contaminates! grounduiater. The first major goal of the project
is to establish pure cultures or defined consortia of microorganisms
metabolize TCE to innocuous products (referred to herein as "complete
metabolism") and to define tha mechanisms by jihich this metabolism occurs.
The second goal of the projact is to establish a bench scale
c ontinuou s—f loiu system as a model to demonstrate the feasibility of
continuous biological treatment of contaminated grounduiater•
WORK PLAN APPROACH : The most commonly detected organic contaminants of
grounduiater are the volatile, chlorinated aliphatic hydrocarbons and these
compounds have become of major concern as potential health hazards in
drinking mater. Ona of the most prevalent contaminants in this group is
trichlorathylene.
At present, relatively little is known about microbial metabolism of TCEJ
evidence, houi-ever, suggests the compound may not be readily metabolized by
the indigenous microflora of soils when no additional carbon sources are
added to samples. Metabolism of TCE does occur under anaerobic conditions^
when acatata is addad as a primary substrata but some of the metabolites
include chlorinated ethanes,and vinyl chloride which ara as much or mora of
a concern as grounduiater contaminants as is TCE. Wilson snd Wilson,
suggest that aerobic metabolism of TCE may also occur under certain
conditions. They found TCE mineralization. to C32 in continuous-floui
columns filled juith sandy soils and their associated indigenous microflor^
exposed to natural gas in air. They hypothesized that an enrichment of
mathanotrophs containing nonspecific monooxyganasa nay have baen
rasponsible for degradation of the TCE, but no direct evidence supporting
this uias presented. All reports on trichloroethylene metabolism to date
have dealt with undefined mixed populations of microorganisms. Neither the
microorganisms nor the mechanisms by which thay metabolize TCE have been
d afined.
The present study proposes to investigate TCE metabolism further as
follows: 1) A pura culture or a defined consortium of microorganisms mill
ba astablished which metabolizes TCE to C02 or some other nonchlorinatedf
innocuous compound such as ethanol or acetate! 2) The optimum conditions
for TCE metabolism in batch culture will be dafined including a
determination of required cosubstrates and other nutrients mhich may
stimulate the process; 3) The macnanisms by which TCE are metabolized uiiljl
be investigated at the biochemical level. Tha information obtained may
allow further optimization of TCE metabolisit by manipulating the metabolisjn
at the molecular level. Also, information obtained about the biochemical
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE t FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q279
mechanisms of dechlorination "nay be useful in developing microbial methods
for dehalogenation of other hazardous halo-organic compounds. 4) A bench
scale continuous-flow system -juill be set up and optimized to demonstrate
t ne feasibility of treating TCE contaminated grounduater u/ith the TCE
metabolizing microorganism(s) that have been isolated and charact3rized as
dascribadabove.
STATUS : On Schedule
ACCOMPLISHMENTS : Strain G4 has been shoum to release 3 Cl ions during the
complata mineralization of TCr. .Efforts to characterize the pathway of
degradation have so far been unsuccessful. Procedures for preparing active
resting cell suspensions have been developed and oxygen uptake studies have
verified the nead for an aromatic hydrocarbon for TCE metabolism. A
manuscript describing the isolation of S-4 has been accepted for
publication in Applied and Environmental Microbiology. A second manuscript
is also being prepared. Research rasults were presented at the Annual ASM
meeting. Next quarter the ability of G-4 to attack other chlorinated
'aliphatics 'Jiill b? determined.
MILESTONES (DATES) :
Isolation of a Pure Culture that
metabolizes trichloroethy lene
Methods for culture and storage
of strain G4
Initial charactarization of-end
productCs) of TCE metabolism by
strain j4
Charact?riz?tion af raquirement
of strain G4 for cosubstrate
TAPGET REVISED REVISED REVISED ACTUAL
10/85
10/85
10/85
12/85
12/35 02/35
10/95
10/35
12/35
02/86
Identification and stoichiometry
of products of TCE metabolism by
strain G4
Optimum parameters for TCE
metabolism in batch cultures
01/86
04/86
04/36
03/86 36/86
Assays for dechlorinating
enzymes involved in TCE
metabolism by strain G4
Develop-nent of bench-scale TC;
treatment system
Detarmination of products and
co—substratas for dachlorinating
06/86 09/86
09/86
12/86
12/86
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE * FLORIDA 32561
FY *86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE
enzymes of strain G4
Inducibility of key enzymes of 03/87
TCE metabolism
Optimization of bench-scale 06/87
biological traatment system
Journal Article: Aerobic 06/87
degradation Trichloretyhlene
(7436C)
Isolation of a Pure Culture that 10/85
Metabolized of Trichloroethylane
(74363)
Report on Role of Co-metabolism 04/89
in the Biodegradation of
Xenobiotic Chemicals (7436A)
0279
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : Q096
OU CODE & TITLE : E104 Pesticides
OBJECTIVE CODE & TITLE : F Develop & Validate Techniques/Exposure
PPA CODE & TITLE : 02 Predictive Techniques for Env Exposure
PROJECT CODE I TITLE : 05 cstuarine Exposure Model and Field Applicability
WORK PLAN TYPE : Inhouse
INVESTIGATOR : L Muellar v
PHONE NO : FTS 636-9011
WORK PLAN TITLE : Mechanisms of Pesticide Siodagradation in Aquatic Sediments
WORK PLAN OBJECTIVE : To determine the mechanism by tuhich microbial
communities associated with sediments degrade a selected pesticide. To
determine the metabolic potential of the sediment-associated communities to
degrade different structural analogs of a base pesticide. To determine the
effects of bioturoation in intact sediment cores on the biodegradation
mechanisms and metabolic potentials.
WORK PLAN APPROACH : Our previous laboratory studies on the biodegradation of
pesticides in aquatic environments has made it very clear that the
microbial communities associated dith sedimants play a critical role in
determining the fate of pesticides in these aquatic systems. We have
shown, for example* that some pesticides are not readily biodegraded in the
water column but are rapidly degraded if sediments are present. Further*
in estuarina, wetland and saltmarsh areas, mhera pesticide runoff from
agricultural lands is likaly to occur, the potential exposure of the
pesticides to the active microbial communities in sadiments is no» known to
be quite highi irrespective of the partition coefficient of the pesticide.
In our attempts to extrapolate this laboratory-derived information to field
situations it has become apparent that me know very little about a) the
mecnanisms by which the microbial communities in sediments degrade
pesticides* b) the metaoolic potential which exists in these communities
and c) the relationship betwean these activities and those of
sediment-reworking benthic invertebrates. Very feiu studies have
intansively dealt u»ith aspects, particularly from the standpoint of
daveloping comprehensive exposure assessments and formulating regulatory
criteria. Many important questions remain, unanswered and no predictive
fra.nework exists. For example, we do not knou» if the mechanism of
pasticide degradation in sediments is a function of higher concentrations
of dagradar organisms on the sediment surface or a surface-concentrating
effact of bacteria and pesticide. We hava only preliminary information
regarding the biodegradability of pasticides which are sorbed to the
sediments. Likewise we know virtually nothing about the interactive
mechanisms by which the microbial communitias degrade or transform
pasticides; i.e.* «hat affect does tha biodagradation of one chemical have
on tha degradation rate and pathway of another (cometabolism) and can the
interactive effects ba manipulated through such factors as induction*
bioiiass increases or plasmid transfer to possibly enhance (adaptation)
pesticide degradation?
The metabolic potential of the .uicrobial communities associated with
sadiments is also thought* based on a relatively small data base* to be
extensive. Considerably more work is needed to verify this observation,
particularly as it relates to the impact that small changes in chemical
structure will have on biodegradation or biotransformation processes in
sediments. What affect will prior exposure to pollutants, such as in a
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I V i K -J il :'i C. i< I •« L. f\ >- J _ « <\ V. i I L.
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/36
WORK PLAN CODE : Q096
contaminated estuary? have on this metabolic potential and hom is the
potential related to the transfer of natural organic material in sediments
of prestine estuaries?
STATUS : On Schedule
ACCOMPLISHMENTS : A continuous floui sediment layer test system has been
successfully developed for the study of biodegradation mechanism at the
sediment u/ater interface. The system has been partially characterized for
general metabolic activity of the associated sediment microbial community —
A analogous glass bead column system has also been designed and tested as™
simpler model for the sediment layer system. An active microbial biofilm
has been shown to develop on the glass beads. Para-chlorodiphenyl ether,
our original test compound, did not biodegrade in standard sediment-mater
test systems; therefore it is not a suitable test chemical. Next quarter,
characterization of se'diment and glass beads systems mill continue and
2,4-D u/il 1 be checked for biodegradability in sediments and its potential
use as our principle test compound.
MILESTONES COATES) : --.. TARGET
Selection of pesticide 11/85
Analytical chemistry 12/85
Test System Design 03/86
Initial biodegradation study - 06/86
identification of primary
degradation product(s)
Enrichment -juith Cosubstrates 09/85
Characterization of pure 11/86
cultures.
Relationship of pure culture to 03/87
natural sediment community
Relation of pesticide sorption 96/87
to degradation rate
Initiation of metabolic 09/87
potential assessment
Report on Mechanisms of 39/88
Biodegradation of Pesticides in
Sediments C7373A)
Role of Microbial Biomass in the 09/87
Enhanced Degradation of
REVISED REVISED REVISED ACTU
ll/'
01/86
09/86
01/1
03/36
ft
I
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ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE , FLORIDA 32561
FY '86 WORK PLANS
DATE : 7/ 2/86
WORK PLAN CODE : 0096
Pesticide Products in Sediments
(7373O
Relating Microbial Metabolism
Studies to the Fate of
Pesticides in Aquatic Systems:
A Case Study tuith Fenthion
(abstract) (73730)
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