PB89-110282
Determination and Enhancement of
Anaerobic Dehalogenaticn
Degradation o"l Chlorinated
Organics in Aqueous Systems
3attelle Col_.abus Div., OH
Frepared for
Environmental Protection Agency, Cincinnati, Oil
Sep 88
3
wrftMRt of CMMwrce
iTuIhSaaI
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EPA/600/2-88/054
September 1988
eoo»-110<.o•
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NOTICE
This study has been funded wholly or in part by the United States
Environmental Protection Agency under cooperative agreement (CRS11120-02-4)
to (5attelle Columbus Division). It has been subject to the Agency's review,
and it has been approved for publication as an EP*\ document. Mention of
trade names or commercial products does not constitute endorsement or
recommendation for use.
i 1
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FOfcfcWtJhL'
rapid!* dsveloDinq and changing technotoqies ana
i r'liuP *" r i ji 1 j'.iJi'cts arid pract t Cc»vs terns. 'Under a
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r.iMmn df t i .• i ~ : f>i and the at:: lit.; of natural s v?-te««* to
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V-.- -1 Si-.'m;- t i on Slrv, ir ®er in.-j Laborator v is responsible tor
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*r •*! demon-str at : rr. r>r oa-*. i » i n iuctor t tit the policies, proar-nns, and
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>l i • .-a--. i.-r-.-»-e-it tn in.«.,>rob i •: Qi.iO jupnnr on.iier. t s.
E. T i mcth v Opuel t, Acting Director"
Pi '•:! red' • _ f h.ti Fnoirieer i •• :q Laborator v
I i i
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ABSTRACT
Anaerobic degradation has the potential of providing efficient
processes for destroying or detoxifying many environmental pollutants. Much
interest 1s currently focused on the area of anaerobic degradation of
halogenated organic compounds because of the toxicity of many of these
compounds. This report summarizes Initial efforts to isolate
microorganisms capable of anaerobic dehalogenatlon; to examine the
nutritional requirements of dehalogenatlng enrichments and a dehalogenating
consortium; and to study the genetics of dehalogenatlon. T'-.l1 ultimate goal
is to understand better the genetics of anaerobic dehalogenatlon and to use
this information to develop engineered microorganisms with improved
anaerobic dehalogenetlon capabilities.
Enrichments were made using secondary anaerobic dlgestor sludge (from
Columbus, OH) as an Inoculum. Anaerobic enrichments containing either 3- or
4-chlorobenzoate were established with a variety of terminal electron
acceptors. Degradation of 4-chlorobenzoate was not observed. Degradation
of 3-chlorobenzoate (3C8), as measured by High Pressure Liquid
Chromatography (HPLC), was observed after 10-23 weeks, first in the nitrate
enrichment and later in the methanogenlc enrichments.
A 3CB degrading consortium, supplied by Dr. J. M. Tledje, was examined
to determine the effect of media composition on the consortium's population.
These experiments were an effort to Increase the proportion of the
population which was capable of dehalogenatlon to aid In Isolating the
dehalogenator 1n pure culture. It was determined that when the consortium
was grown 1n basal medium with 800 jtM 3CB and 15 mM sodium nitrate, the
microorganisms capable of benzoate degradation and methanogenesls were lost
but dechlorination continued.
Genetic studies were begun using a pure culture of strain 0C8-1
(obtained from Or. J. M. Tledje), the dehalogenatlng microorganism from
1v
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Tledje's 3CB degrading consortium. Strain DCB-1 was examined for plasmld
DNA using two different methods, alkaline lysis and salt-cleared lysis, but
no plasmids were found. Therefore, It was presumed that the dehalogenase
activity was chromosomally encoded. Genomic DNA was extracted and purified
using a modified version of procedures previously used with anaerobes. A
partial library was generated by cloning large DNA fragments Into the cosmid
pHC79 and small fragments into the plasmid pUC8. To aid 1n Isolating
recombinants with dehalogenase activity a rapid dehalogenase assay, based on
the reaction which occurs between Iodine and starch, was developed.
In summary, enrichments capable of degrading 3CB were Isolated. It was
shown that the population of a 3C8 degrading consortium could be altered, by
manipulating the growth medium, so that the fraction of the population
capable of dehalogenation Increased relative to the original consortium.
Chromosomal DNA from strain DCB-1 was purified and found to be suitable for
cloning manipulations. Finally, work began on the construction of a strain
DCB-1 genomic library.
This report was submitted 1n fulfillment of CR811120-02-4 by Battelle
Columbus Division under the (partial) sponsorship of the U.S. Environmental
Protection Agency. This report covers a period from
October 17, 1933 tj April 30, 1987.
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CONTENTS
Foreward 111
Abstract 1v
Figures v11
Tables vi1
Acknowledgements v111
1. Introduction 1
2. Conclusions and Recommendations 4
3. Materials and Methods 5
4. Results and Oiscussion 20
References 46
vi
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FIGURES
Number Page
1 Battelle protocol for Isolation of DCB-1 chromosomal DNA 12
2 Dechlorination of 3CB by various cultures 27
3 Photographs of the variety of cell types isolated
in pure culture 33
4 Agarose gel electrophoretic analysis of pUC8: DCB-1
in host E. coli 40
5 DCB-1 DNA run on a 10-40% (wt/vol) sucrose gradient 41
6 Construction of genomic library in cosmid vector pHC79
and screening of the library for gene expression
in E. coli 43
7 Construction of genomic library in pUC8 and screening
of the library for gene expression in E. col 1 45
TABLES
Number Paqi
1 Composition of basal medium 6
2 Volatile fatty acid mix 7
3 Microorganisms aiid vectors 8
4 Anaerobic dilution solution 9
5 Development of 3-C1-benzoate degrading consortium
under various enrichment conditions 26
6 Growth and dechlorination of 3-Cl-benzoate
by dechlorinatlng consortium under various conditions 31
7 Comparison of dehalogenase activity by standard HPLC
methods and the rapid spot screening assay 37
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ACKNOWLEDGEMENTS
We would like to thank Darlene T. McCallum for her technical work on
this project. We thank Dr. J. Tiedje for his generosity in supplying us
with his dehalogenating consortium and strain DCB-1. We thank Dr. John
Reeve for many useful discussions. We thank Janet Knutson for her procedure
and her adv!.» on DNA isolation. We thank Dr. Albert Venosa for his help on
this project.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completingi
1 R£PORT NO. 2.
EPA/600/2-88/054
3 RECIPIENT'S ACCESSION NO
PE89 il0282iAS
4 0 'eTE W I°! ATf6 NL 'AN D EM HAM CEMENT OF ANAEROBIC
DEHALOGENATIOT: Degradation of Chlorinated
Organics in Aqueous Systems
5 REPORT DATE
September 1988
6. PERFORMING ORGANIZATION COOE
7 AUTHOR(S)
D. T. Palmer, T. G. Linkfield, J. B. Robinson,
B. R. Sharak Genthner, and G. E. Pierce
a. PERFORMING ORGANIZATION REPORT NO
9 PERFORMING ORGANIZATION NAME AND AOORESS
Battelle Columbus Division
Columbus, OH 43?^1
10. PROGRAM ELEMENT NO.
CBR D1A
11. CONTRACT/GRANT NO.
CR-8111?0-rv>-A
12. SPONSORING AGENCY NAME ANO AOORESS
RisV Reduction Engineering Laboratory--Cincinnati, OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH
>3. TYPE OF REPORT ANO PERIOD COVERED
Proj* Rnt./Surnary
14. SPONSORING AGENCY CODE
EPA/^nn/lA
IS. SUPPLEMENTARY NOTES
Project Officer: Albert D. Venosa FTS 68rt-7F*P, Comm.
16. ABSTRACT
TMs report suirmarizes initial efforts to isolate microorganisms capable of
anaerobic dehalogenation; to examine the nutritional requirements of dpbalogen^ting
enrichments and a dehalogenating consortium; and to stuHy thp genetics of dpbalonen-
ation. The ultimate goal is to understand better the genetics of anaerobic dehalo-
genation and tp use this information to develop capabilities. T*e important results
froi this wor1, are surmarized below.
Anaerobic enrichments were established in whicb .''-chlorobenzoate '?CB^ Kut not
4-chlorobenzoate was degraded. Studies using a 3CB degrading consortium showed that
specific manipulations of the,growth medium could eliminate some members of the
consortium while maintaining the organisms capable of dehalogenation. Such manipula-
tions arc? useful in efforts to isolate organisms in pure culture. Genetic studies
were begun using a pure culture of an anaerobic dehalogenator. No plasmids were
found in this strain; therefore, it was presumed that the dehalogenase activity was
chromosomally encoded. Genomic DMA was extracted and purified. A partial library
was generated by cloning DNA fragments into the cosmid pHC70 and into the plasmid
p'JCS. A rapid dehalogenase assay was developed for the purpose of screening
recombinants for dehalogenase activity.
17. KEV WORDS ANO OOCUMENT ANALYSIS
a. DESCRIPTORS
b.lOENT»FlERS/OPEN ENDED TERMS
c. COSATi Field/Croup
is. Distribution statement
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
20 SKCURtTY CLASS iTIlll pa/tl
UNCLASSIFIED
22. price / yiy/r
CP* f—m 2230-1 (*•». 4-77) »m»cog» «oitia» n omoliti ^
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SECTION 1
INTRODUCTION
Many halogenated organic compounds can pose a serious environmental
problem because of their toxicity and the tendency for accumulation 1n
sediments and soils affecting both flora and fauna (Edwards, 1973; Chapman,
1978; Schneider, 1979). Microbial degradation has the potential to remove
these halogenated compounds either in situ or in conjunction with above
ground waste treatment. Aerobic degradation has been extensively studied.
Some organic compounds do not appear to be degraded aerobically (DIGeronimo
et al., 1979; Bouwer, et al., 1981) and others are degraded in a manner in
which highly toxic intermediates are formed (DIGeronimo, et al., 15/9;
Horvath and Alexander, 1970; Evans, et al., 1971a; b; Gaunts and Evans,
1971; Ahmed and Focht, 1973; Relneke and Knackmuss, 1980; Hartman, et al.,
1979). Relatively little 1s known about anaerobic degradation of these
compounds. However, recent Investigations indicate that anaerobic
degradation of a number of halogenated organic compounds such as halogenated
benzoates (Horowitz, et al., 1982; Suflita, et al., 1982; Horowitz, et al.,
1983), halogenated phenols (Ide, et al., 1972; Murthy, et al., 1979; Boyd,
et al., 1983; Boyd and Shelton, 1984) and halogenated short chain aliphatic
compounds (Bouwer, et al., 1981; Bouwer and McCarty, 1983 a; b) does occur
in the environment. These studies have shown that some compounds which are
not degraded aerobically are readily degraded anaerobically. In contrast to
aerobic degradation, the first step of anaerobic degradation 1s
dehalogenation (Suflita, et al., 1982; Horowitz, et al., 1983) leading
immediately to the formation of a less toxic, more biodegradable compound.
The overall objective of the program 1s the development of engineered
microorganisms to cause the destruction of hazardous organic compounds
(e.g., chlorinated organics) under anaerobic conditions. An understanding
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of microblally mediated anaerobic dehalogeriatlon and the exploitation of
this process may result in the significant reduction of toxic and hazardous
wastes in the United States. Therefore, the success of this program would
greatly assist the EPA in detoxifying many recalcitrant compounds which in
the past have not been biodegradable or accessible to other chemical a.id
physical destruction techniques.
If the dehalogenase activity 1s encoded by a gene or genes carried by
the plasmid, then plasmid genes specifying the anaerobic degradation or
biotransformation of chlorinated organlcs can be Introduced Into suitable
hosts using genetic engineering techniques. Strains so modified could then
be examined to study and enhance the degradation of organic chemical
contaminants present 1n hazardous waste sites.
If dehalogenase activity 1s a chromosomally encoded activity, then a
genomic library will have to be constructed for each dehalogenating
microorganism. At present, since there are no suitable probes for the
dehalogenase enzyme gene, or the enzyme itself, the gene will have to be
isolated using a screening method that detects the activity of the enzyme.
In orde" to study the genetics and biochemistry of dehalogenation and
to begin a cloning effort, a pure culture of a dehalogenating organism was
needed. Dr. J. M. Tiedje and associates developed the first anaerobic
dehalogenating consortium and Isolated the first anaerobic dehalogenating
microorganism in pure culture (Shelton and Tiedje, 1984 a; b). When this
study began, neither Tiedje's consortium nor pure culture of DCB-1 were
available for study. It was not until later in the program that the
consortium and then DCB-1 became available. This study, therefore, was
divided into three phases. The first phase of this study was an effort to
detect the presence of anaerobic dehalogenators in Columbus sewage and to
isolate a pure culture of a dehalogenating microorganism. In the second
phase of this study, we obtained the 3-chlorobenzoate (3CB) degrading
consortium of Dr. Tiedje. This consortium was used as a model system for
the Isolation and identification of the organisms responsible for
dehalogenation. In the third phase of this study, a pure culture of DCB-1,
the organism responsible for dehalogenation in Tiedje's consortium, was made
available to us and genetic studies on this dehalogenating bacteria were
begun.
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The work described in this report is an initial effort to examine
anaerobic dehalogenation by applying some of the expertise qained in
studying aerobic degradation processes.
3
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SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
The ultimate goal of the work with anaerobic dehalogenating bacteria is
to develop microorganisms capable of degrading hazardous organic compounds
(eg., chlorinated organics) to environmentally safe forms under anaerobic
conditions. This work will also provide information on the mechanism of
anaerobic dehalogenation. To achieve this goal, we recommend completing the
generation of the DC8-1 library and the cloning of the dehalogenase gene(s).
When Columbus sewage was used as an inoculum for the various
enrichments discussed in this report, dehalogenation of 3-chlorobenzoate
(3CB) was detected. The Battelle laboratory was the second laboratory to
report this activity. Anaerobic dehalogenation is an activity found in
multiple sewage samples; it is not an isolated activity (Shelton and Tiedje,
1984 a;b, our work). Qehalogenation of 4-chlorobenzoate (4CB) was not
detected in the primary enrichment from Columbus sewage; this result is in
agreement with the work of others (Horowitz, et al., 1982).
A hypothesis that H2 at a concentration of 10% would aid in the
establishment of a 3CB degrading consortium was shown not to be true for the
conditions we used. Experiments with the dehalogenating consortium of
Tiedje showed that varying the growth conditions (change in the terminal
electron acceptor) could enhance the growth of the dehalogenating bacteria
relative to other bacteria originally present in the consortium.
Pure cultures of OCB-1, the microorganism responsible for the
dehalogenation of 3CB in the Tiedje consortium, were examined for the
presence of plasmid DNA by a variety of methods (Hansen and 01 sen, 1978 and
Birnboim and Doly, 1979); no plasmids were observed. The absence of plasmid
DNA indicated that a total genomic library of OCB-1 needed to be generated
in order to clone the gene or genes responsible for anaerobic
dehalogenation. Our efforts have shown that genomic-ONA Isolated from OCB-1
can be cloned in and banked in E. coli.
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SECTION 3
MATERIALS AND METHODS
ANAEROBIC METHODS AND MEDIA
Media and Strains
The anaerobic techniques used for the handling of the Inocula,
preparing the media, and handling of enrichments and cultures were
essentially those of Hungate (1950) as modified by Bryant (1972) and Balch
and Wolfe (1976). For the work with the enrichments and the consortium, the
basal medium contained rumen fluid, B-vitamins, minerals, NaHC03, Na2S
reducing solution, resazurin redox indicator, and a 90% N2-10X C02 gas phase
(final pH 7.0) (Table 1). NaHCOj solution was prepared by dissolving 5.0 g
in distilled water to make X00 ml, fiIttr-ster1l1z1ng the solution,
aseptlcally bubbling it with an 80 percent N2:20% CO2 gas mixture for 30
min. and then dispensing the solution under the same gas mixture in
appropriate amounts into sterile 18 x 150 mm anaerobic culture tubes fitted
with standard black butyl rubber stoppers (No. 1). Cysteine-sulfide
solution contained 1.255 each of cysteine/HCl and Na2S.9H20 and was prepared
as previously described (Bryant and Robinson, 1961). Gas mixtures were
obtained by using a gas proportioner with FM112-02G flow tubes (Aalborg
Instruments and Controls, Inc., Monsey, NJ) which in turn was connected to
the gasing apparatus described by Bryant (1972). Yeast extract (0.1%, w/v),
or other growth supplements (I.e., volatile and branched-chaln fatty acids
[Table 2]) can be used as substitutes for rumen fluid. The terminal
electron acceptor was C02 for methanogenlc media, while 20 mM Na2S04, 15 mM
KNO3 or 20 mM sodium fumarate was added for sulfate, nitrate or fumarate
enrichments, respectively, fermentative enrichments were prepared by
including the carbohydrates similar to those used 1n the Complex
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Carbohydrate medium (CM) of Leedle and Hespell (1980). The basal medium was
used, but 1n addition, the medium contained 800 jtM 3CB, Neopeptone (0.12,
Difco), Tryptone (O.lt, Difco), and 0.05X (vol/vol) each of cellulose
(Sigmacell, type 100), cellobiose, glucose, maltose, pectin (citrus fruit),
soluble starch (potato), xylan (larchwood) and glycerol.
TA8LE 1. COMPOSITION OF 8ASAL MEDIUM
Component Percent
Clarified rumen fluid
5.0
Mineral solution
S
Trace mineral solution
4
Vitamin solution
5.0
0.1
0.5
Resazurin (0.1% wt/vol)
0.1
NaHC03 solution
7.0
Cysteine-sulfide solution
2.0
Gas phase (N2:C02)
80:20
•*
All concentrations were 1n percent (vol/vol) unless
otherwise indicated.
+Mineral solution contained (g/Hter): KH2P04, 10.0;
MgCl2.6H20, 6.6; NH4C1, 8.0; CaCl2.2H20r 1.0
§
Trace mineral solution 1s the trace element solution
listed in Aragno and Schlege 1. 1981. (p. 874) with
the addition of (g/liter:FeC12.4H20, 1.5; Na2Se03, 0.1.).
A
Vitamin solution contained (mg/liter): 20 mg each of
biotin and folic acid; 10 mg of pyridoxal-hydrochloride;
60 mg lipoic acid; and 50 mg each of riboflavin, thiamine
hydrochloride, calcium-D-pantothenate, cyanocobalamin,
{j-aminobenzoic acid and nicotinic acid.
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TABLE 2. VOLATILE FATTY ACID MIX*
Component Volume (ml)
Acetic Acid 17
Propionic Acid 6
Butyric Acid 4
Isobutyrlc Acid 1
Valeric Acid 1
Isovaleric Acid 1
DL-Alpha-Methyl Butyrate 1
Adjusted pH to 7.5 with 10 N NaOH. Brought to final
volume of 200 ml with distilled water. Store sealed
1n refrigerator.
Stock cultures of DCB-1 were maintained in basal medium (Shelton &
Tiedje, 1984b) containing 10-20% (v/v) clarified rumen fluid and 0.2% (w/v)
pyruvate. Rumen fluid was obtained from fistulated cows at the Ohio
Agricultural Research Development Center. The crude product was filtered
through cheese cloth and centrlfuged at 9820 x g, autoclaved and stored at
4°C. Prior to use, the liquid was further clarified by centrlfugatlon. In
order to increase cell yield and to establish a larger Inoculum, DCB-1 was
transferred to basal medium containing 0.2% pyruvate, 0.1% yeast extract,
and 10 mM Na2S£03. For DNA extraction, DCB-1 was grown 1n one liter round
bottom flasks in basal medium consisting of 20% clarified rumen fluid, and
0.2% pyruvate. All cultures were grown 1n an atmosphere of 80% N2:20% C02.
The E. col 1 strains and vectors used In this work are listed 1n
Table 3. E. col 1 strains were grown on LB plates, LB broth (Miller, 1972)
or nutrient broth (Dlfco) with the appropriate antibiotic, 1f necessary, for
selecting recombinants. (Amp1c1111n was used at a final concentration of
30-40 /ig/ml and tetracycline was used at a final concentration of 15 |tg/ml).
7
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TABLE 3. MICROORGANISMS AND VECTORS
Strain
Genotype
Source
DCB-1
J. Tledje
E. coVj,
AC80
thr leu met hsdR-hsd+
L. Bopp
JM83
ara A (lac-pro) strA thi
(80 dlacIqZA M15)
MM294
endAl thi-1 hsdR17 supE44
B. Bachmann
DH5«
endAl hsdR17 supE44 th1-l recAl
gyrA96 relAl 80dlacZAM15
BRL*
Vectors
ApR TcR^
PBR322
BRL
pHC79
ApR TcR cos
BRL
pUC8
ApR lacZa
BRL
*Bethesda Research Laboratories
6 p o
ampicmin resistance, Ap ; tetracycline resistance, Tc
Tests for redox growth range and dehalogenation capability of the E. col 1
recombinants carrying DCB-1 DNA inserts were performed in basal medium
supplemented with 20% clarified rumen, 0.2% pyruvate, plus the necessary
antibiotic to maintain selective pressure for the plasmld. In addition,
AC80 required 0.05% casamino acids in the medium to satisfy its auxotrophic
requirements. The bacteria were incubated at 37°C.
Most Probable Numbers (MPN) Analysis
The MPN analysis procedure was used 1n experiments studying the 3CB
degrading consortium. Ten-fold serial dilutions were prepared with
anaerobic dilution solution (Table 4). One ml of each dilution was
Inoculated into triplicate tubes containing nine ml of the desired medium.
8
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The MPN was estimated from the number of tubes In each triplicate set which
were positive for a desired characteristic (Rodlno, 1972).
TABLE 4. ANAEROBIC DILUTION SOLUTION*
Component Percent
Mineral Solution 5.0
Trace Mineral Solution 0.1
Sodium Bicarbonate (5%) 2.5
Cysteine HCl/Sod1um Sulfide (1.25% ea.) 2.0
Resazurin (0.1%) 0.1
01 stilled H20 To 100 ml
Gas Phase (N2/C02) 90:10
Final pH 7.2
Prepare as described for basal medium and all quoted
In 4.5 ml amounts in septum-11pped tubes.
Source and Collection of Inoculum
The source of Inoculum for the enrichments was secondary anaerobic
digested sludge (Jackson P1ke Plant, Columbus, Ohio). Inoculum was
collected in a sterile 2 liter glass carboy containing a stir bar for mixing
and sealed with a black rubber stopper fitted with a one-way gas valve to
allow release of gas pressure.
Enrichments
Enrichments were prepared by adding sterile anaerobic solutions of 3CB
or 4CB to the basal medium with 10% sewage sludge as Inoculum. Some
enrichments were set up 1n order to determine the effect of hydrogen on the
development of actively degrading consortia by adding 10% H2 to the
atmosphere. Enrichments were Incubated at 35°C. Degradation of the
halogenated compound was followed over time by HPLC analysis. Once
9
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degradation was observed, the enrichments were transferred to fresh media
and passed several times to stabilize the activity.
Sample Collection, Storage and Analysis
Zero time samples of the gas phase and culture fluid were collected
with a syringe. The gas sample was analyzed immediately by gas
chromatography (GLC) for the presence of CH4 and CC^. The culture fluid was
filtered (0.45 ^m) or centrifuged to remove cellular material and stored
frozen until it was analyzed. Initially, weekly samples of the gas phase
and culture fluid were taken. Sampling intervals were altered when
indicated as necessary by the data. Degradation of the parent compound and
appearance of intermediates was followed using GLC or HPLC analysis.
Redox Studies
To determine the potential expression of a dehalogenase gene Isolated
from an obligate anaerobe and cloned into a facultative anaerobe, it was
decided to evaluate the clones for dehalogenase activity over a wide range
of redox conditions. Normally, DCB-1 is grown in a medium with an of
approximately -570 mV while the host E. coll strains JM83 and AC80 are grown
under normal aerobic conditions. Each recipient was first grown under
varying conditions of E^ to establish the limits of growth. JM83 and AC80
grew both aerobically and at Ej, values as low as -570 mV. Additional
tests with strain JM83 showed that growth occurred in the presence of sodium
thioglycolate (<-100 mV), cysteine-HCl (-210 mV), dithiothreitol (-330 mV),
and anaerobically with no extraneous reductant.
DEHALOGENASE SCREENING ASSAY
A dehalogenase screen was developed as a method to examine a large
number of recombinants rapidly. Using a glass pi pet, 1 drop each of the
following reagents were added to the well of a white porcelain well plate:
0.2% KNO2, 0.4% starch, 235 ZnC^ solution, and 1.9N HC1. A drop of liquid
culture medium was then added to the well containing the reagents. A bluish
purple color Indicated a positive reaction for the presence of iodide.
Iodide was present 1f the 3-iodobenzoate originally present had been
dehalogenated. To confirm the validity of the spot test for dehalogenatlon,
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DC8-1 was grown in the presence of-800 pM 3-fodobenzoate and analyzed for
dehalogenation by both HPLC and the spot test described above over a 23 day
period.
GENOMIC ONA ISOLATION AND PURIFICATION
In order to obtain sufficient quantities of purified genomic DNA, a
modification of the Marmur (1961) procedure was used (J. Knutson, Michigan
State University personal communication, 1985). Additional modifications
were required and were made as follows: Proteinase K (Sigma) was used at a
final concentration of 1 mg/ml to optimize cell breakage. RNase (Sigma) at
a final concentration of 20 #tg/ml was used to reduce the high concentration
of contaminating RNA; ethylenedlaminetetraacetlc acid (EDTA) concentrations
1n both the storage buffer and the dialysis buffer were increased to 10.0 mM
to minimize nuclease activity; and the TgES storage buffer (6 mM Tris pH
7.4, 0.1 mM EDTA, 10 mM NaCl), was replaced with TE buffer (10 mM Tr1s, pH
7.4, 10 mM EDTA) to decrease the salt content. The complete procedure 1s
shown in Figure 1. ONA of a specific size range was Isolated on a sucrose,
gradient usually 10 to 40% (w/v) (Hohn and Collins, 1980).
11
-------
Cell Harvest
a. Add NaCl to 1 M (final concentration)
b. To collect cells, centrifuge 10K, 10'
c. Wash once with TE buffer
d. Weigh pellet (initially pellet in pre-
weighed tube)
I
Cell Lysis
a. Resuspend cells in TE (1 ml TE/gm cells)
b. Freeze overnight - 70°C
c. Thaw cells
d. Add 5 ml TE/gm cells
e. Transfer to centrifuge tube and measure volume
f. Add 1/10 volume lysozyme (3 rog/ml in TE)
g. Incubate on ice 15'
h. Add 1/16 volume Proteinase K (Predlgested
37°C, 30') to a final concentration
of 1 mg/ml.
1. Add 1/9 volume 10% Sarkosyl; Mix well
j. Incubate 37°C; 3 hours
i
Phenol Extractions
a. Add equal volume phenol saturated with TE
b. Mix vigorously by vortexing
c. Spin 10K, 10'
d. Remove supernatant and save
I
Figure 1. Battelle Protocol for Isolation
of DCB-1 Chromosomal DNA.
12
-------
Back Extraction
a. Add TE to bottom layer (equal volume)
b. Mix (vortex)
c. Spin 10K, 10'
d. Remove supernatant and add to first supernatant
Continue phenol extractions until there is a clear
interface between top and bottom layer
4
Ether Extraction
a. Extract final aqueous phase with ether until both
layers are clear (4 times)
b. Mix and spin 10K, 10'
c. Blow off excess ether with air
Dialysis
a. Put DNA in dialysis tubing
b. Dialyze against 3 changes of 2L of:
TE = 10 mM Tr1s, pH 7.4, 10 mM EDTA
I
Measure A2go for concentration
Store at 4°C
i
Add RNase (stock 10 mg/ml) to final concentration
1n solution of 20 /tg/ml
I
Let stand at room temperature 1 hour
Figure 1, (Continued).
13
-------
ENZYMES
Restriction enzymes and terminal deoxynucleotidyl transferase (TdT)
were purchased from BRU Klenow fragments were purchased from Boehringer
Mannheim Biochemicals (BMB). Ligations were done overnight at either 12°C
or 4°C using standard procedures [International Biotechnologies, Inc. (IBI)
catalog]; DNA ligase was purchased from New England Biolabs, Inc. (NEB).
Cells were made competent and transformed using standard procedures such as
that in the IBI catalog. Competent DH5ot cells were purchased from BRL. In
vitro packaging kits were purchased from Stratagene. Reagents were used
according to the manufacturers' instructions.
GAS CHROMATOGRAPHY
Carbon dioxide, hydrogen, methane and nitrogen content of the gas phase
of the enrichments were analyzed using a Varian Model 3700 Gas Chromatograph
(Varian Instrument Group; Palo Alto, Calif.) in conjunction with a Hewlett
Packard Model 3390A Integrator (Avondale, Penn.). A Carbosieve S-ll
(100/12C, 10 ft. x 1/8 in.; Supelco, Inc.; Bellefonte, Penn.) column was
used with a helium carrier gas. Prior to use, the column was conditioned at
225°C with helium at a flow rate of 15 ml/min for 12 hours. Subsequent
analyses ^ere performed using helium at a flow rate of 30 ml/mln. Gas flow
rate was determined using a gas flowmeter. Gases were detected by thermal
conductivity. Detector and Injector port temperatures were 1S0°C and 200°C,
respectively. Sensitivity was 160 mA at an attenuation of 4X. An
autolinear program was used with an initial temperature of 35°C for 7
minutes followed by an increase in temperature at a rate of 25°C/min. to a
final temperature of 175°C. Unknown gases were identified by comparison of
retention time to the retention times of standard gases (99% purity).
Quantification of gases was achieved by comparing the area under the curve
produced by 0.5 ml of the sample to that produced by 0.5 ml of a 99% pure
sample gas. Percent composition of each sample was determined by this
method. Sample size was 0.5 ml for all injections.
Samples were collected from enrichments aseptically acid anerobically.
Sterile 1 ml gas-tight glass TB syringes (Becton Dickinson & Co.,
Rutherford, NJ) fitted with 22 gauge (1 in.) sterile needles were used to
14
-------
remove samples of the gas phase. The 1 ml glass syringes were flushed with
sterile anaerobic gas (N2) before removing the gas sample from enrichments.
The sterile anaerobic gas was delivered to a sterile 18 x 150 mm glass test
tube via a 17 gauge bent needle attached to a sterile cotton-plugged 2 ml
syringe. The gas was made anaerobic by passing over a heated (350°C)
reduc : copper column and passed through the gas proportlonator before
reaching the 2 ml glass syringe (Shelton and Tledje, 1984a). The stoppers
in the cultures were flamed before sampling and an excess of gas sample
(1e., greater than the 0.5 ml desired for Injection) was removed from the
culture. As the syringe was pulled from the culture vessel and stabbed into
a black rubber stopper, the excess gas was slowly forced out in order to
Insure that atmospheric gas did not contaminate the sample during this
transfer. Gas samples were analyzed Immediately. All samples were manually
injected. Due to the large gauge needle required to remove samples from the
enrichments, a high temperature white septum (3/8", All tech Associates,
Inc., Oeerfleld, IL) was used. This septum was chosen because it displayed
low bleed after repeated injections with large gauge needles.
HIGH PRESSURE LIQUID CHROMATOGRAPHY
Benzoate, 3C8 and 4CB were separated, Identified and quantified by high
pressure liquid chromatography (HPLC). A reverse phase C18 Lirhrosorb
column (10 p, 4.6 mm [ID] x 25 cm, Alltech Associates, Inc., Deerfleld, IL)
was used Initially with a liquid phase of water:methanol:acet1c acid
(6:4:0.5) at a flow rate of 1.5 ml/min. The method used for the separation
of benzoate and the chlorinated benzoate compounds was modified early in the
study. The ratio of the solvent components was changed from 60:40:5
H20/methanol/acetic acid to 60:40:5 methanol/H20/acetic acid and the flow
rate was decreased from 1.5 m1/rn1n to 0.7 ml/min. This modification
permitted us to decrease the flow rate without an Increase in the retention
times of compounds, and 1t eliminated the pressure control problems
associated with the former solvent mixture. Under these modified
conditions, the retention times for benzoate and 3CB were approximately 15.6
and 8.5 min. respectively. In addition, the sample volume was decreased
from 250 ^1 to 100 /tl, resulting 1n an increased lifespan of the guard
column. The ratios of the solutions 1n the solvent system and the flow rate
15
-------
were changed when necessary to increase the resolution of the data. Sample
detection was achieved by U.V. adsorption at a wavelength of 284 nm using a
LDC/Milton Roy Spectromonitor DUV detector (laboratory Data Control Riviera
Beach, FL). Sample peaks were integrated with the aid of a Hewlett Packard
Model 3380A Integrator (Avondale, PA). Solvent flow was controlled with a
Altex Model 420 Microprocessor (Beckman/Altex, Berkeley, CA) and a Beckman
Model 11CA pump. Water was purified for HPIC use by the M1111-Q Water
Purification System (Millipore Corporation, Bedford, MA). HPLC-grade
methanol (Burdlck & Jackson Laboratories, Muskegon, MI) and acetic acid
(99+%, Aldrich Chemical Co., Milwaukee, WI) were used for the liquid phase.
All glassware used in the preparation of the solvent was rinred with HPLC
water prior to use. Solvent components were measured and mixed in the
proportions described and were then filtered and degassed under vacuum using
a Millipore GS type filter (0.22 ^m). The filtrate receptacle was rinsed
with three 100 ml aliquots of filtrate before retaining the remaining
filtrate for use with HPLC. Filtered solvent was stored in brown glass
screw cap bottles and refiltered if not used within one week. Storage
bottles were also rinsed three times with filtered solvent before being
used.
Fluid culture samples for HPLC analysis were collected aseptlcally and
anaerobically as described above using a 5 ml sterile plastic disposable
syringe (Becton Dickinson & Company, Rutherford, NJ) with a 20 gauge (1 in.)
sterile disposable needle. Samples were placed in plastic screw cap tubes
and stored frozen until analyzed. After thawing, samples were prepared for
analysis by filtering 1 ml of the sample through a 0.2 /*m nylon-66
disposable filter cartridge (Al1 tech Associates, Inc., Deerfield, IL) into
new disposable glass test tubes. Compounds of interest in the samples were
identified by comparison of retention times to the retention times of
authentic (high purity) compounds. These compounds were then quantified by
comparing the integrated area under the curve produced by the compound in
the culture sample to that produced by a 500 pM sample of the authentic
compound.
16
-------
NITRATE REDUCTION MEASUREMENTS
In cultures containing nitrate as the terminal electron acceptor the
amount of nitrate reduction to nitrite was determined using Method 1, 1n the
ASM Manual of Methods for General Bacteriology (p. 419 & 439). Nitrate was
quantified by comparing the absorbance obtained with the sample to that of a
standard curve determined with potassium nitrate. The concentration of
nitrate still remaining 1n the fluid was determined by adding zinc powder to
each assay to convert the unreduced nitrite to nitrate.
DETERMINATION OF SULFATE REDUCTION
A medium similar to the basal anaerobic medium was prepared to
determine the presence of sulfate reducing organisms In the sulfate
enrichments. In addition to components previously listed, (Table 1) the
medium also contained (per liter) 0.1 g yeast extract, 2.0 g sodium lactate,
1.0 g sodium acetate, 0.1 g FeSO^, 0.5 g sodium sulfate (as an electron
accepter) and 2.0 g Bacto-?gar. The medium was prepared anaerobically using
standard Hungate technique, distributed Into 13 x 100 mm glass test tubes
and allowed to solidify at an angle to form a slant. Fluid from the
anaerobic enrichments containing sulfate was aseptlcally and anaerobically
removed as with a 1 ml sterile glass TB syringe fitted with a 22 gauge (1
in.) sterile needle. The sulfate slant medium was Inoculated by stabbing
the needle into the medium and injecting a small amount of culture. Air was
excluded from the tubes of medium during inoculation by passing a stream of
sterile anaerobic gas (N2/C02,* 90/10) into the tubes. Cultures were
incubated at 35°C. Blackening of the medium, I.e. formation of FeS,
indicated the presence of sulfate reducing organisms.
PLATING TECHNIQUES
Isolation of Dechlorinatlnq Organism,
in Pure Culture
The basic plate medium for the isolation of dechlorlnating organisms
had the same composition as the basal medium (Table 1), except that rumen
fluid was replaced by 0.1% yeast extract and 2.OX agar was added. The
following additions were made to aid 1n the Isolation of these
17
-------
microorganisms; (1) 3CB (800 pM); (2) 3CB (800 plus sodium pyruvate
(0.3%); (3) 3CB (800 ^M) plus sodium pyruvate (0.3%), plus Volatile Fatty
Acid Mix (2.0%; see Table 2) and (4) 3CB (800 /iM) plus hydrogen (50%) and
CC>2 (10%). Control media containing the additions, but lacking 3CB, were
also prepared. The medium was prepared in a manner similar to basal medium,
but after bringing the medium to a boil under the anaerobic gas phase the
flask was sealed and autoclaved. The sterile medium was cooled to 50°C in a
water bath, opened under the anaerobic gas phase, and the sodium bicarbonate
and reducing solutions were aseptically added. The flask was resealed and
transferred into the anaerobic chamber. The mouth of the flask was swabbed
with 100% methanol before it was opened Inside the chamber. An automatic
pipetor was used to aseptically dispense 20 ml of medium into sterile
plastic petri dishes (15 x 100 mm). Petri dishes and all other plastic or
glassware were placed 1n the anaerobic chamber for at least 48 hours prior
to using in order to remove traces of oxygen. Extra desiccant was placed in
the chamber to aid in the drying of the plates. While still inside the
anaerobic chamber, plates were sealed in anaerobic jars. The anaerobic jars
contained open tubes of 2.5% sodium sulfide (placed in the jars 24 hours
prior to Inoculation) to insure reduction.
Cultures from which dechlorinatlng microorganisms were to be isolated
were diluted from 1 x 10~* to 1 x 10 ^ by serially adding 0.5 ml of culture
to 4.5 ml of anaerobic dilution solution (Table 4). One milliliter of the
1 x 10-4 dilution and of the 1 x 10~^ dilution was spread plated on
duplicate plates of each of the media described above. Uninoculated plates
were included as controls. After allowing the agar to dry, the inoculated
plates were placed into anaerobic jars containing an open tube of 2.5%
sodium sulfide. After sealing the jars, the anaerobic jars were taken out
of the chamber and 0.2 atmospheres of N2/CO2 (90:10) were added via the
gassing manifold to maintain a positive pressure 1n the jars. Jars
containing plates to be Incubated under hydrogen were pressurized to 0.25
atmospheres with hydrogen and additional CO2 was added to maintain a
concentration of 10%. Anaerobic jars were Incubated outside the chamber at
33°C.
18
-------
Isolation of Dechlorlnating
Organism in Co-culture or Defined Mixed Culture
There were slight differences In the method used to obtain a co-
culture versus a pure culture. The medium was the same, but the additions
to the medium were different. Media containing (1) 3CB (80C /iM) plus sodium
sulfate (20 mM), and (2) 3CB (800 pM) plus sodium pyruvate (0.3S) plus
sodium furnarate (25 mM) were prepared. Control media without 3CB were also
prepared. Medium containing sodium sulfate was used for the preparation of
a coculture with Desulfovlbrio desulfuricans (ATCC 27774) as the terminal
hydrogen-utilizing organism. Medium containing sodium furnarate was for the
preparation of a coculture with Wollnella succinoqenes (ATCC 29543) as the
terminal hydrogen-utilizing organism. D. desulfuricans was grown in basal
medium containing sodium lactate (22 mM) and sodium sulfate (25 mM). W.
succinoqenes was grown in basal medium containing 25 mM each of sodium
formate and sodium furnarate.
Anaerobic dilution solution was used to dilute cultures containing the
dechlorlnating organism. An inoculum of D. desulfuricans or W. succinoqenes
was provided for a coculture by replacing 1.5 ml of the dilution solution
with 1.5 ml of an actively growing culture of the appropriate species.
Dilutions of the dechlorlnating culture were prepared as described for pure
culture Isolation. The 1 x 10"4 and 1 x 10~6 dilutions, also containing D.
desulfuricans or W. succinoqenes. were plated on the appropriate medium as
described above for pure culture Isolation. Plates were placed Inside
anaerobic jars as described above and were Incubated at 35°C 1n the
incubator with the anaerobic chamber.
19
-------
SECTION 4
RESULTS AND DISCUSSION
In order to properly examine the genetics of anaerobic dehalogenatlon,
it was necessary that we have defined pure cultures. Several different
approaches were used to obtain a pure culture(s) of an anaerobic
dehalogenator. Because Dr. Ttejde's dehalogenating consortium and the pure
culture DCB-1 were not available at the Initiation of this project and
because it was of Interest to determine whether additional anaerobic
dehalogenators could be isolated from an area other than the location in
Michigan froir. which Dr. Tiedje obtained an inoculum source, an effort was
initiated to examine Columbus sewage for dehalogenatlon activity. The
occurrence of dehalogenation activity led to a further effort to isolate 1n
pure culture the microorganism responsible for this activity. When Dr.
Tiedje's consortium was made available to us, work was begun to isolate the
dehalogenating organism(s) from the consortium. Finally, the dehalogenating
organism strain DCB-1, isolated by Shelton and Tiedje, was sent to us.
Since DCB-1 was a pure culture, studies with DCB-1 assumed a position of
highest priority. The goal of producing a superior dehalogenating anaerobic
organism can be best approached by studying the genetics and biochemistry of
anaerobic dehalogenation. This sort of study can only be carried out using
a pure culture. The availability of DCB-1 increased the speed at which we
could move toward our goal.
ENRICHMENTS FROM COLUMBUS SEWAGE
While at the initiation of this program, there had been only one report
1n the literature of the isolation of an anaerobic dehalogenating
microorganism In pure culture (Shelton and Tiedje, 1984b), it was likely
that there would be other anaerobic organisms capable of similar activities.
Because 1t may be possible to select for different dehalogenators by varying
20
-------
the conditions of the primary enrichment,,five different enrichment
conditions were chosen for the experiments described In this report.
4-chlorobenzoate was chosen for one enrichment series, because Boyd and
Shelton (1984) reported that cultures adapted to 4-chlorophenol were more
versatile 1n their degradatlve abilities than either 2- or 3-chlorophenol
adapted cultures. This may also be true of halobenzoate cultures; and a
consortium containing more than one dehalogenatlng activity would be vsry
useful 1n genetic studies. Methanogenic enrichments were prepared because
the consortium capable of dehalogenatlon developed by Or. Tledje came from a
methanogenic environment. Anaerobic degradation of organic compounds had
been shown to occur under nitrate-reducing (Bakker, 1977; Evans, 1977;
Bouwer and McCarty, 1983), sulfate-reduclng (Pfenning et al., 1981;
Mountfort and Bryant, 1982; Wlddel, 1983) and fumarate-reduclng conditions
(Barik and Bryant, ASM, Ann. Abstr. Meet. 1984). Therefore, enrichments
were prepared 1n which these compounds served as terminal electron acceptors
Instead of carbon dioxide (the electron acceptor 1n methanogenesls). A
fermentative enrichment was also examined. All five conditions for
anaerobic degradation of organic compounds were Investigated to increase the
probability of finding a variety of anaerobic dehalogenating bacteria. The
Isolation of a diversity of anaerobic dehalogenating bacteria 1n pure
culture would have provided a valuable pool of genetic Information for
future studies.
The basal medium shown in Table 1 was*used for methanogenic
enrichments. Yeast extract (0.1%) or other growth supplements (1e. volatile
and branched-chain fatty acids) can be used as substitutes for rumen fluid.
(A 2.5% solution of sodium sulfide can be used to replace the
cysteine/sulfide solution as there 1s little chance of loss of 1n the
system [1e. sealed serum bottles] being used. Sulfide alone resulted 1n a
lower £q and could, therefore, be more stimulatory to growth of strict
anaerobes).
The nitrate reduction medium was similar to that described 1n Table 1.
Since the E0 of NO3/NO2 1s +433 mV, cysteine (2.5%) replaced the
cysteine/sulfide reducing solution. A minimum of 12 mM NaNOj would be
21
-------
required for the complete degradation of 800 /tM benzoate (800 iiM x 15 *
12 mM):
C7H502H + 12H20 •» 7C02 + 15H2
h2 + no"3 no"2 + h2o
The amount of NaNOj added depended upon the organic compound being used
as the energy source. The N2/C02 gas phase was retained as some strict
anaerobes known to reduce nitrate also require C02 (de Vrles, et al., 1974).
For the sulfate reduction enrichment, the basal medium in Table 1 was
used, except that 20 mM NaS04 and 20 mM NaCl were added. Although sulfate-
reducing anaerobes can be cultivated under 100 percent N2, 1t has been found
that sulfate-reducing anaerobes that degrade organic compounds are enriched
under a gas phase containing C02 (Pfenning et al., 1981). The addition of
NaCl and the reduction of the iron content to the level found in our medium
should help to enrich for these organisms. In this case, the sodium sulfide
reducing solution was used in place of the cysteine/sulfide solution.
At the time we were preparing our enrichment series, M. P. Bryant
(Ann. Meet. ASM 1984) reported that fumarate could serve as a terminal
electron acceptor for microorganisms that degrade benzoate. The use of
fumarate would eliminate the need for an additional H2 utilizing organism as
the terminal electron sink 1n an enrichment. Therefore, it was of interest
to determine if a dehalogenating degrader of benzoate could be obtained in
pure culture if given fumarate as a terminal electron acceptor. Since the
reaction of fumarate to succinate is a 2 electron pair reduction, 12 mM
fumarate(the same concentration as N03 •+ N02) would be required for complete
degradation of benzoate. The basal medium described for the methanogenic
enrichment was used.
The ability of fermentative organisms to dehalogenate 3CB was also
examined. A number of carbohydrates were added to the basal medium for this
enrichment.
Enrichment Controls
A suitable number of control enrichments were also included.
Inoculated media of each type were: (1) autoclaved as a sterile control;
22
-------
(2) transferred to a sterile flask and Incubated aeroblcally to determine
the need for anaerobic conditions and (3) Incubated with benzoate as the
energy source 1n order to obtain a dehalogenase negative benzoate-degrading
organism. Also included was a sterile enrichment containing titanium
chloride and an artificial electron donor (ie., reduced Iron porphyrins;
Zoro et ah, 1974) in order to determine 1f nonbiological reductive
dehalogenatlon would account for dehalogenatlon 1n these enrichments.
Effect of H? on Enrichments
The effect of H2 on the development of consortia capable of degrading
chlorinated organic compounds was of special Interest and was examined.
Degradation of chlorinated organlcs 1n anaerobic environments depends on the
activity of three organisms. (1) a dehalogenator, (2) an organic degrader
(proton reducer), (3) a H2-ut111z1ng organism. It has been observed that
the chlorine must be removed before further degradation of the ring can
occur (SufUta et al., 1983); thus, this may be an obligatory first step.
The degradation of the dehalogenated Intermediate requires that the H2
produced be used by a third organism, because H2 must be kept very low (less
than 1 x 10"5 atm) to have degradation become thermodynamlcally feasible
(1e. a negative Gibbs Free Energy) (Mclnerney and Bryant, 1981). H2 would
stimulate the growth of the terminal H2-ut1lizing organism and probably
stimulate reductive dehalogenatlon. However, the presence of H2 would
inhibit the degradation of the organic Intermediate.
Most studies have used a N2/C02 atmosphere while examining anaerobic
dehalogenatlon. Horowitz et al. (1983) used a gas phase containing 10
percent H2, but included a 24 hour preincubation period to remove H2. long
lag periods (2 weeks to 6 months) have been observed before dehalogenatlon
occurred 1n these studies. A possible explanation for these long lag
periods may have been a result of poor availability of reducing equivalents
for reductive dehalogenatlon. Thus, Including Just enough H2 to provide
reducing equivalents for dehalogenatlon might result 1n a decrease 1n the
lag phase before dehalogenatlon 1s observed without leaving excess hydrogen
to Inhibit further degradation of the compound. However, the dehalogenatlng
organism must be able to compete for hydrogen with the other H2-ut1l1z1ng
anaerobes 1n the enrichments. The Km for hydrogen during reductive
23
-------
dehalogenatlon has been determined to be 30-67 /tM (SufUta et al., 1983),
and the affinity of methanogens and sulfate-reducing anaerobes for H2 is 2-
12 /tM and 0.2-1.2 /tM, respectively (Hungate et al., 1970; Robinson, 1982;
Robinson and Tiedje, 1982; 1984). Thus, hydrogen would have to be added in
nonHmitlng amounts to be available for dehalogenatlon. The Initial
presence of the halogenated compound may also be favorable to reductive
dehalogenatlon since these compounds are known to Inhibit methenogenesls
(Horowitz et al., 1983). According to the following equation:
2C1C7H402~ + H2 + 2 H+ > 2C7H502 + 2 H+ + 2 CI"
the degradation of 800 /«mo1es of 4-chlorobenzoate would require 400 /imoles
H2 for complete dehalogenation (at 25°C, 1 atm). This equals 9.79 ml H2, or
16.3 percent H2 1n a 160 ml serum bottle containing 100 ml of medium.
Enrichment Results
The enrichments were periodically sampled during this study 1n order to
determine some of the biological processes that were occurring. The
enrichments were set up between 2/84 and 5/84. By 9/84, methane was
detected by gas-liqu1d-chromatography 1n the gas phase of all the
enrichments tested. Based upon gas production, the methanogenic enrichment
was superior for the enrichment of methanogens but the methanogenic activity
was almost as high when sulfate reduction enrichment medium was used.
Methanogenrc activity in the nitrate enrichment was very low.
HPLC analyses of culture fluid from those enrichments containing
benzoate indicated that the benzoate was completely transformed within one
month. In these analyses, no peak having a retention time Identical to or
similar to that of a reference sample of benzoate was detected. This
observation Indicated not total utilization but rather that all of the
benzoate 1n the system has been at least partially degraded or transformed.
The degradation of 3CB and 4CB 1n the enrichments was periodically
monitored. Enrichments inoculated on 8/20/84 with 10 percent Columbus
sewage sludge were analyzed at various times during an 11 month period by
HPLC to determine the amount of degradation of the halogenated compound. No
degradation of 4C8 1n any of the enrichments was detected (Table 5).
24
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Although 3C8 degradation had been reported 1n primary enrichments to take up
to 11 months to occur, 4CB degradation has not been reported in primary
enrichments. Because it was unlikely that dehalogenatlon would occur In the
4C8 enrichments after 11 months of being monitored, these enrichments were
not maintained after June 30, 1985.
The degradation of 3CB was also monitored over time. Enrichments were
begun on 7/13/84 with 10 percent sewage inoculum. Because a change in the
analysis procedure 1n November resulted in some inconsistencies 1n the data
when comparing degradation before and after 11/84, a large decrease 1n the
concentration of 3CB was the criterion for degradation 1n these Initial
samples. Large decreases in the 3CB concentration were apparent even with
the change in analysis procedure.
One of the nitrate enrichments Incubated without hydrogen was the first
enrichment to show significant 3CB degradation (Table 5). Once the initial
amount of 3CB was no longer detectable, the culture was fed with 800 fan 3CB
to confirm its ability to degrade this compound. The 3CB refed into the
enrichment was utilized within one week of its addition (Figure 2a).
Initially, the rate of disappearance of 3C8 was 54 /imoles/11ter/day, but
after the third day, the rate increased to 145 ^moles/1iter/day.
Degradation after the third day was linear (R=0.999). A Gram stain of the
enrichment was prepared. Most cells were embedded 1n an amorphous matrix.
Gram negative short rods and cocci were present as well as refractlle
bodies, (1e. spores). Gram negative, thin, extremely long rods, similar to
Methanospirill urn hunqatel. were also present. The cellular morphology of M.
hunqatei 1s quite unusual and easily recognized. The presence of M.
hungatei would suggest that a methanogenic enrichment had become
established. It was thought that the methanogenic enrichment had become
established after the nitrate had been utilized.
The fumarate and methanogenic enrichments showed the complete absence
of 3CB after 28 weeks of incubation (Table 5). There was no degradation of
3CB 1n either the sulfate or CCM enrichments. Enrichments showing
degradation were passed to fresh medium as a 50 percent Inoculum. The
methanogenic enrichments were passed to basal medium. The fumarate
enrichment was passed to both basal and fumarate medium 1n order to
determine 1f fumarate was required for degradation of 3CB. The nitrate and
-------
'fumarate enrichments were also passed to fresh media with and without
hydrogen. Passage of the nitrate enrichment to media with and without
nitrate has been previously discussed. Degradation of 3CB was followed in
these fresh transfers. Neither the terminal electron acceptor present in
the original enrichment nor hydrogen were required for degradation. After 6
weeks, the transferred fumarate enrichment showed completed degradation of
3CB in the presence or absence of fumarate. At 10 weeks, the methanogenic
enrichments showed complete degradation. Microscopic examination of the
enrichments revealed a mixture of Cram negative rods of varying shape and
lengths (from coccobacillus to long rod similar to M. hunqatei). These
enrichment experiments showed that an organism or several organisms capable
of dehalogenatlng 3C8 were present 1n the sewage inoculum.
TABLE 5. DEVELOPMLNT OF 3-C1-BENZ0ATE
DEGRADING CONSORTIUM UNDER VARIOUS
ENRICHMENT CONDITIONS
Enrichment Type
Time (Weeks)*
3-Cl-benzoate
no Hg
+h2
methanogenic
sulfate
nitrate
fumarate
CC fermentative
10-23+
NDOS
6-10
10-23
NDO
10-23
NDO
10-23
10-23
NDO
4-Cl-benzoate
methanogenic
sulfate
nitrate
fumarate
NDO
NDO
NDO
NDO
NDO
NDO
NDO
NDO
*
+Weeks of incubation before degradation observed
Degradation not observed at 10 weeks, but apparent at 23 weeks
§ND0 - no degradation observed
26
-------
1st refeeding
4th refeeding
1000
3
100
Hours
150
3CB + 15 mM NOa
3CB + 50 H2 4 15 mM NOj
3CB + 0.3% Pyruvate + 15 mM NC;
1000
s
3
500
960
720
480
240
S 1000
500
150
100
Hours
Figure 2. Dechlorination of 3CB by various cultures.
(A) 3CB/NO3 enrichment from Columbus sewage.
(B) Response of "Tiedje" consortium to various culture conditions.
(C) 3CB/NO3 subculture from "Tiedje" consortium.
-------
EXAMINATION OF 3-CL-BENZOATE CONSORTIUM
The microbial consortium capable of degrading chlorobenzoates was
received from Dr. James Tiedje on 10/8/84. The consortium was immediately
fed 800 yiA 3CB and degradation of 3CB was shown to be complete after four
days. Residual benzoate was not detected. The "Tiedje" consortium was
maintained by weekly feeding with 3CB and by 50 percent transfers monthly.
Published data on the "Tiedje" consortium (Shelton and Tiedje, 1984b)
indicated that the dehalogenatlng organism Isolated from this consortium
grew slowly in a medium containing pyruvate and that it reduced nitrate to
nitrite. This observation prompted the suggestion that 1t might bo possible
to Improve the growth rate of the dehalogenatlng organism by providing
nitrate as the terminal electron acceptor. Selectively improving the growth
rate of this dehalogenatlng organism would aid 1n our attempt to isolate
this organism 1n pure culture because of the potential enrichment of the
dehalogenatlng microorganism. In order to enrich for the dehalogenating
microorganism, the consortium was inoculated (10% v/v) into the following
three types of medium:
(1) Basal medium with 800 /iM 3CB and 15 mM sodium nitrate
(2) Basal medium with 800 /iM 3CB, 15 mM sodium nitrate, and 0.3%
sodium pyruvate
(3) Basal medium with 800 pM 3CB, 15 mM sodium nitrate, and 50%
hydrogen
In these experiments, yeast extract was used instead of rumen fluid in the
basal medium (see Materials and Methods). Pyruvate was added because 1t
could provide an energy source as well as reducing potential for the
reductive dechlorination of 3CB. Nitrate was added so that it could serve
as a terminal electron sink to produce energy for growth.
The 3CB concentration was determined at 0, 3, 16 and 44 days
(Figure 2b). At 44 days, the culture containing 3CB and 15 mM N03 showed no
detectable 3C8, but a large peak was observed with a lower retention time
(8.36 minutes) corresponding to 694 pM benzoate. This observation Indicated
that under these conditions 3CB was being dechlorlnated, but not degraded.
The entire 30 ml culture was transferred to 60 ml of fresh medium and the
3C8 concentration was again determined over time (Figure 2c). It was
28
-------
observed that as the 3CB concentration decreased the benzoate concentration
increased. 3CB was dechlorinated at a rate of 38 pM per hour. A Gram stain
of the culture showed that about 95% of the cells present were small Gram-
negative coccobacilH found mostly in pairs. There were also some large
Gram-negative rods. Cells with the morphology of M. hunqatel were not seen.
This suggests that under these conditions the benzoate degrader and the
methanogens were selected against. Therefore, the dechlorinatlng organism
was either one or both of the remaining cell types but was neither a
benzoate degrader nor a methanogen. The large Gram-negative rods that were
observed corresponded to the dechlorinatlng organism described by Tledje,
but the small coccobacllH were not described 1n Tiedje's report.
Since the small coccobaclllus was present 1n much greater numbers than
the large bacillus, we prepared a Most Probable Numbers (MPN) Dilution Assay
(Rodlna, 1972) of the culture. It was hoped that this method would allow us
to determine which organism was responsible for the dechlorination activity.
Dilutions of the culture were prepared from 1 x 1G through 1 x 10 .
Triplicate tubes of basal medium containing 800 pM 3CB and 15 mM N03 were
Inoculated with each dilution. The optical density was followed With time
Q
to determine growth. MPN data Indicated that there were 1 x 10 total
cells/ml 1n this enrichment culture.
Dehalogenatlon only occurred in the lower dilutions (1 x 10"3 or less)
indicating that the dehalogenator was present at a level less than 1 x 104
cells/ml. In this enrichment the dehalogenator represented only 0.01% of
the total population. Direct isolation of this organism, therefore, could
be very difficult. Because no purified anaerobic dehalogenase enzyme
existed and no analogous enzyme was known, probes could not be constructed
which would facilitate the isolation of the anaerobic dehalogenator.
Microscopic examination of the MPN cultures revealed a Gram-negative
coccobaclllus, commonly found 1n pairs and chains, as the dominant cell type
in all dilutions; however, some large Gram-negative rods were present at the
lower dilutions and a few smaller Gram-negative rods were present at all
dilutions. The microorganisms 1n the lower dilutions were observed only
after extended Incubation to allow further growth of the culture. This
Incubation was followed by concentration by centrlfugatlon. The presence of
29
-------
the large gram negative rod correlated with dehalogenase activity 1n these
d11ut1ons-
A series of media were prepared in which 3CB, NO3 and yeast extract
(YE) were added in different combinations. This series was inoculated with
one drop of the dechlorinating culture. In this manner we hoped to obtain
pure cultures of the organisms or further enrich for the dehalogenating
organism. The series of media included the basal medium lacking yeast
extract with the following additions: 5 mM KNO3 only; 5 mM KNO3 + 50% H2;
5 mM KNO3 + 0.1% yeast extract; 5 mH KNO3 + 50% H2 + 0.1% yeast extract; 5
mM KNO3 + 0.3% lactate; 5 mM KNO3 + 0.3% lactate + 0.1% yeast extract; 0.3%
lactate + 0.1% yeast extract; 3CB (800 jtM) + 0.1% yeast extract; 3CB + 0.1%
yeast extract + 50% H2; 50% H2 + 0.1% yeast extract; 0.1% yeast extract
only; 3CB + 0.1% yeast extract + 5 mM KNO3; 3CB + 5 mM KNO3; and 3CB only.
Hydrogen was used as a possible energy source for reductive
dechlorination. Lactate was used as an energy source for the nitrate
reducer. Triplicate tubes of each medium were inoculated and the optical
density was followed. The results of this experiment are shown in Table 6.
Only tubes containing YE showed growth after one week. The optical density
in tubes containing both H2 and YE was greater (A600 » 0.42) than that in
tubes with YE alone (A^qq * 0.22), indicating that one or both of the
organisms can utilize hydrogen. The presence of lactate inhibited growth
even in the presence of yeast extract. Growth was best in media containing
hydrogen in addition to yeast extract but nitrata tended to inhibit growth.
After 6 weekc of incubation, dechlorination and a concomitant rise in the
benzoate concentration wer?detected in all the media containing 3CB, except
3CB + KNO3 and 3CB only. Apparently, yeast extract is necessary for
dechlorination when KN03 is present. Dechlorination occurred in the defined
media containing H2 and 3CB despite a Tack of visual apparent growth. To
determine the population make-up of the cultures, it was necessary to streak
the liquid culture onto plates. This work is described in the following
section.
Isolation of Dehalogenating Organisms in Pure Culture
Since dehalogenation did not occur in the MPN cultures containing only
the coccobacillus, but did occur at the lower dilutions containing the large
30
-------
Gram-negative rod, 1t was likely that the rod and not the coccobadllus was
the dehalogenatlng organism. Direct Isolation of the dehalogenatlng
organism was attempted from the defined H2/3CB medium because this culture
had the highest dechlorination activity per relative cell density.
TABLE 6. GROWTH AND DECHLORINATION OF 3-C1-BENZOATE BY
OECHLORINATING CONSORTIUM UNDER VARIOUS CONDITIONS
Additions
Growth+
3-Cl-Benzoate
(nM)
Benzoate
M)
KNO3
0.05
-
-
h2/kno3
0.05
-
-
YE/KNO3
0.22
-
-
h2/kno3/ye
0.32
-
-
Lactate/KNO3
0.08
-
-
Lactate/KNO-j/YE
0.05
-
-
Lactate/YE
0.07
-
-
3CB/YE
0.24
147.3
630.5
H2/3CB/YE
0.47
0
752.0
h2/ye
0.42
-
-
YE
0.22
-
-
3CB/YE/KNO3
0.32
384.4
422.6
3CB/KN03
0.05
815.9
65.8
H2/3CB
0.06
0
748.0
3CB
0.05
757.5
58.2
*Basal medium without yeast extract plus the additions Indicated (see text)
Maximum optical density (600 nm) after two weeks.
31
-------
The culture was streaked onto 3CB/YE medium and incubated for 2 weeks with
and without H2. After two weeks, over one hundred colonies were picked into
3CS plus yeast extract broth. These cultures were incubated for one week
and cellular morphology and dehalogenation were determined. The majority of
cultures contained the coccobaci11 us previously described (Strain BF19,
(Figure 3a), but five other morphological types were isolated. These
included two coccobacilli which differed slightly from the dominant cell
type. Strain 8G95 (Figure 3b) was more spherical and strain BG29 (Figure
3c) did not form pairs, while chains were only periodically observed. Three
Gram-negative rods were isolated, including: strain BG2, a medium rod with
pointed ends (Figure 3d); strain BG49, a medium rod with round ends (Figure
3e); and a long thin rod. The presence of H2 had little effect on the type
of cell isolated. The 3C8 concentration in cultures of each morphological
type was determined weekly for one month, but dechlorination was not
observed. A second attempt at direct Isolation of a dechlorinating organism
resulted in the isolation of two more types of Gram-negative rods, including
a medium rod (strain BG131) which was usually found in pairs (Figure 3f) and
a long oval rod (strain BG170) which tended to divide unevenly (Figure 3g).
After one week, 3CB had not been dechlorinated by either of these isolates.
At this point, a pure culture of the dehalogenating bacterium strain DCB-1
(Shelton & Tiedje, 1984b) became available and we could not justify
screening the remaining pure cultures for dehalogenase activity.
GENETICS OF DC8-1 DEHALOGENATION
The dehalogenating organism, strain DCB-1, was received from Dr. J. M.
Tiedje (Shelton & Tiedje, 1984b). DCB-1 is thought to be related to the
genus Desulfovibrio. This strain was originally isolated from anaerobic
digester sludge from a sewage treatment plant in Holt, Michigan. It is a
Gram-negative, non-sporeforming obligate anaerobe capable of dehalogenating
haloaromatic compounds by removing halogens (chloro, bromo, and iodo but not
fluoro) from meta-substituted benzoate compounds. This organism grows
fermentatively on pyruvate and has the ability to reduce sulfite (S02=) or
thiosulfate (S203=)- However, it will not dehalogenate in the presence of
these inorganic sulfur electron acceptors.
32
-------
Strain BG19.
3b.
Strain BG95.
3c. Strain 8G29.
Figure 3. Photographs of the variety of cell types
isolated in pure culture.
33
-------
f, ¦
f
•>
n
: k
' wf«.
• V-y*?
V iv -
v.- .
ir*-r /
vV<
V \
3d. Strain BG2.
3e. Strain BG49.
K \
i ^ - t ry
M ^
\ \
i
J \
\ x-v. -
3f. Strain BG131. 3g. Strain BG170.
Figure 3. (Continued).
34
-------
Because the purpose of this study was to Investigate the genetics and
biochemistry of anaerobic dehalogenatlon, the availability of this pure
culture allowed us to begin the genetic work on anaerobic dehalogenatlon,
without having to reisolate this dehalogenating organism from the Tiedje or
the Columbus consortia. The receipt of this strain saved us a great deal of
effort. Work with DCB-1 became the top priority.
DCB-1 represented the first anaerobic bacterium to be isolated in pure
culture that possessed dehalogenase activity. The dehalogenase activity of
DCB-1 is of intense interest because the mechanism and conditions of
dehalogenation are different from the mechanisms observed in many aerobic
dehalogenating microorganisms. Because of the very slow growth rate of
strain DCB-1 and because strain DCB-1 is a fastidious strict anaerobe, it
was decided that the best opportunity for studying anaerobic dehalogenation
would be achieved through the cloning and expression of the dehalogenase
encoding gene(s) from DCB-1 in an alternative host microorganism. Cloning
of the DCB-1 gene(s) encoding anaerobic dehalogenation would also permit us
to manipulate the environment of the enzyme 1_n vivo and determine effects of
various environmental conditions on the dehalogenatlon activity.
To facilitate our entry into the genetics and gene manipulation of the
anaerobic dehalogenators, Dr. John Reeve (The Ohio State University),
because of his successes with cloning methanogen DMA, was used as a
consultant. During our conversations with Dr. Reeve, 1t became apparant
that if any genetic engineering was to be successfully conducted, the
development of an effective selection/or screen would be imperative.
Rapid Screening Method for Dehalogenase Activity
When a large number of recombinant bacteria are made in an effort to
find a gene present only once in the genome, it is easiest if a screen is
developed so that single colonies on a agar plate can be checked for the
presence of the gene of interest. This type of testing allows a large
number of recombinants to be examined rapidly;, other methods are much less
efficient. Because of the limited amount of research that has been done on
the anaerobic dehalogenase enzyme(s), there was no way to make a DNA probe
or an antibody probe for use in screening. The only way to screen for the
enzyme is by expression of the dehalogenase activity in the recombinant.
35
-------
After examining the literature for methods that could be used to examine
large numbers of recombinant clones on agar surfaces, we were unable to
find a suitable method. We selected wet chemistry as the best alternative
to screening on an agar surface. We chose to test iodo-compounds in our
development of a screening method. Because most biological media contain
significant concentrations of chloride, the use of chlorine release, as
chloride, from a chloroaromatic compound 1s often difficult to assess.
Iodide or bromide measurements, however, usually do not present a problem 1n
biological solutions.
A rapid qualitative assay was developed that could detect the presence
of free Iodide ions in liquid culture. This assay permits the rapid
screening of recombinant clones for dehalogenase activity. The procedure
described is a modification of the starch-Iodide spot test for nitrites
(Skerman, 1967). The original test depends on the formation of nitrous acid
and its subsequent reaction with potassium iodide with the liberation of
iodine, which turns the starch blue. In the modified test, a source of
nitrite 1s provided as a 0.2 percent aqueous solution of KNC^. The presence
of iodide ions in the medium, due to the dehalogenation of the substrate 3-
iodobenzoate, can then be detected. Quantitative analysis of the loss of 3-
iodobenzoate and concomitant appearance of benzoate as determined by HPLC
was used as evidence of dehalogenation. The results of the HPLC analysis
were compared with the spot test reactions in order to determine the
sensitivity of the spot'test (Table 7).
After 23 days of incubation, all DCB-1 samples were positive for
dehalogenation using the rapid starch spot test. A very strong positive
reaction was evident in sample (a), the only sample shown to completely
dehalogenate the 3-iodobenzoate based upon HPLC data. The remaining samples
were all positive using the spot test, with 20.4 - 51.3 percent of 3
iodobenzoate remaining based on HPLC data. The development of a rapid
technique to assess anaerobic dehalogenation, using 3-iodobenzoate, was a
significant achievement. With this technique we will be able to screen
large numbers of clones under a variety of environmental conditions.
Without the development of this screen it would be extremely difficult to
assess anaerobic dehalogenation.
36
-------
Redox Studies
The presence of DC8-1 DNA encoding dehalogenase activity was to be
detected 1n the host organism after cloning by the detection of dehalogenase
activity. The'effect of oxygen and redox potential on the anaerobic
dehalogenase activity was not known.
TABLE 7. COMPARISON OF DEHALOGENASE ACTIVITY
BY STANDARD HPLC METHODS AND THE
RAPID SPOT SCREENING ASSAY.
Dehalogenatlon as
Measured by the
Sample Dehalogenatlon as Measured by HPLC Spot Test
3-1odobenzoate Benzoate Starch Iodine
(% remaining) % formed Reaction
0
(days)
9
23
0
(days)
9
23
(days)
0 9
23
DCB-1 a
100
74.3
0
0
NT+
100
_ _
+++
b
100
95.6
51.3
0
NT
43.3
-
+
c
100
89.4
20.4
0
NT
100
-
+
d
100
86.2
29.5
0
NT
53.8
-
+
e
100
88.9
28.1
0
NT
50.0
-
+
Each value represents the mean of duplicate samples.
*
A negative reaction 1s indicated by a minus sign and a positive reaction 1s
indicated by one or more plus signs. A three plus reaction 's one that
occurred rapidly and resulted 1n a very dark blue color.
+NT * not tested.
It was, therefore, Important to know that the chosen host organism, E. coli.
was capable of growth at the redox potential that DC8-1 was grown at when
dehalogenase activity was observed. Normally, DCB-1 is grown in a medium
with an Eh of approximately -570 mV. The host E. coll strains, JM83 and
AC80 were grown under varying conditions of Eh to establish the limits of
37
-------
growth. Both grew at Ej, values as low as -570 mV. In addition, tests with
JM83 showed that growth occurred in the presence of sodium thloglycolate (<-
100 mV), cysteine-HCl (-210 roV), dithlothreitol (-330 mV), and anaerobically
with no extraneous reductant. Thus, the host strains ability to grow at low
Ef, is not a limiting factor 1n the potential expression of a dehalogenase
gene(s).
Isolation of Plasmid DNA
Initially, It was hoped that DCB-1 might carry the genes encoding
dehalogenase activity on a plasmid; the cloning of a gene or genes carried
by a plasmid 1s generally much simpler than cloning a chromosome encoded
gene(s). However, all attempts to isolate plasmid DNA (Hansen and 01 sen,
1978; Birnboim and Doly, 1979) from DCB-1 were negative. There was no
evidence to suggest the existence of any plasmids 1n DCB-1. The only way to
clone the gene or genes responsible for the dehalogenase activity,
therefore, was to generate a complete genomic library of DCB-1 DNA and
search for the gene or genes of interest.
Isolation and Purification of DCB-1 Genomic DNA
In order to generate a complete genomic library, 1t was necessary to
isolate large quantities of DNA. Isolation and purification of DCB-1
genomic DNA required significant modification o. existing procedures
(Marmur, 1961). Initial DCB-1 genomic DNA preparations yielded low
quantities of DNA which were resistant to restriction endonuclease attack
and contained high levels of RNA. Two steps were taken 1n an effort to
increase the yield of DNA. First, cells were incubated with higher
concentrations of proteinase K to optimize breakage, and second, the EDTA
concentration of both the storage and dialysis buffer was Increased in order
to inactivate nucleases. An Increase In yield of genomic DNA from DC8-1 was
obtained following these modifications. We observed that these DNA
preparations could only be restricted by restriction endonucleases that
required high salt buffers. The level of salt in the final DNA preparations
was reduced by removing the sodium chloride from the storage and dialysis
buffers. The DNA preparations were then sensitive to the restriction
endonucleases required for cloning.
38
-------
Preparation of OCB-1 Genomic Library
The goal of the cloning effort was to generate a complete DCB-1 genomic
library and to screen the library for the gene (or gene complex) which
encodes the dehalogenase activity. The initial set of experiments were
performed in order to show that a DCB-1 genomic DNA library could be
constructed using an E. col 1 host and vector. For these experiments,
purified genomic DNA was digested with either Pstl or EcoRl and 11 gated to
Pstl restricted pBR322 and EcoRl restricted pUC8, respectively. Recombinant
E. coli with DCB-1 Inserts were Isolated (figure 4). These clones were
grown anaerobically and tested for dehalogenase activity using the
3-iodobenzoate screen. However, dehalogenase activity was not detected in
any of the clones.
The successful cloning of DCB-1 DNA (small fragments) indicated that
the DNA was suitable for a more extensive cloning effort. Because the
isolation of DCB-1 was a slow process and the yield 1s relatively poor, 1t
seemed more desirable to clone larger DNA pieces [DNA of a specific size
range was Isolated on a sucros* gradient (Figure 5)] into a vector such that
DCB-1 DNA could then be produced in the recombinant host, E. col 1, and
subclones could be made then from these large Inserts. A cosmid system was
chosen as the primary method of generating a genomic library. Cosmids
contain the cos (cohesive ends site) of the bacteriophage lambda. The
presence of the cos region allows the packaging of DNA inside of lambda
phage heads, which can then be further processed to become infectious
particles. To be effective, the DNA located between the two cos sites must
approximate the size of wild type lambda for packaging (37-52 kb).
Inserting DNA into a cosmid and packaging the DNA iji vitro selects for large
inserts (30-45 kb). By using a cosmid cloning system, as opposed to a
system requiring smaller inserts, a genomic library can be generated which
contains a significantly smaller number of recombinants. The use of cosmids
is also beneficial when cloning a gene complex. At this point, we do not
know if the dehalogenase activity 1s encoded by a single gene or a set of
genes. Enough recombinants must be isolated so that deletion of chromosomal
material from the library 1s minimized. (One difficulty seen with the
cosmid system is instability can occur, which can result 1n the deletion of
cloned material). A genomic library was partially generated in the host
39
-------
1 3 5 7 9 11 13
Figure 4. Agarose gel electrophoretic analysis of pUC8: DCB-1 clones
in host E. col i JM83. Hind III (track 1) [23, 130 base
pairs (bp), 9416 bp, 6557 bp, 4361 bp, 2322 bp, 2027bp];
plasmid vector pUC8 (track 2); 0UC8 restricted with EcoRI
(track 3); pUC8: DCB-1 clones unrestricted (tracks 4, 6,
8, 10 and 12); corresponds.g pUC8: 0C8-1 clones restricted
with EcoRI (tracks 5, 7, 9, 11 and 13).
40
-------
m 3
DCB-l DNA run on a 10-40 (wt/vol) sucrose gradient.
Lane 1- DNA restricted with Hind III; Lane 2- DNA
(48,502 bp); Lane 3- DCB 1 DNA; Lane 4-14 - first
11 fractions of DNA from the sucrose gradient.
41
-------
strain MM294 using the cosmld pHC79 (Hohn and Collins, 1980). The method 1s
outlined 1n Figure 6, In the first round of cloning, ten recombinants were
generated: these clones were stored by the standard procedure of freezing
at -80°C. These clones were not recoverable after freezing. This unusual
behavior may be the result of alterations in the hosts' physiology due to
the presence of DC8-1 DNA. The second round of cloning has generated fifty-
seven potential cosmid-containing clones, almost one genomic unit. In order
to generate a genomic library which has a 95% probability of containing any
particular single-copy gene, 380 recombinants (with an average insert size
of 35 kb) will have to be isolated. This is a library of about five genomic
units. If all of the recombinants are shown to contain large inserts, then
we have about 15% of the library. The potential recombinants must be
analyzed further to verify the presence of an insert and to determine the
size of the insert.
Banked cosmids can be tested for the presence of the gene(s)
responsible for dehalogenation by screening the recombinant bacteria for
expression of dehalogenase activity. Because of the large size of the
cloned DNA fragments, expression of these recombinants will depend almost
entirely on the ability of DCB-1 promoters and translation initiation sites
to function in E. col 1. The large size of the fragments makes it unlikely
that read-through from plasmid promoters will result in the synthesis of
mRNA from the entire cloned fragments. To determine that these sites are
functional, a complementation study must be done with the cosmid genomic
library. E. col 1 hosts with a number of auxotrophic markers will be
transformed using the pooled recombinant plasmids. The recombinant E. coli
can then be screened to determine if the 0C8-1 DNA can complement any of the
mutations. Successful complementation would suggest that DC8-1 promoters
and translation start sites can function in E. coli. Further experiments
would be needed to prove that such a correlation was real. Detection of
dehalogenase activity would not be limited by the need for an E. coli
promoter 1f the DCB-1 promoters and translation start sites were active 1n
E. coli.
Since 1t may be necessary to supply an E. coli promoter 1n order to
detect expression of DCB-1 DNA in E. col 1. our second strategy for cloning
addresses this problem. Smaller fragments (3-9 kb) can be isolated from the
42
-------
EcoRl
Pst 1
Bgl 11
Bam Hi
Total Genomic
DNA
Sucrose
gradient
Bgl 11
Large DMA fragments
Restrict with Pst 1
\and G tail / fi11 in
with
Klenow
Add C tail
anneal
Insert DNA
\
Pst 1
Package in vitro into infectious
particles; infect
Screen for recombinants (Tc^, ApS)
Isolate cosmids with inserts
Pool cosmids
..Transform auxotrophic
hosts
Test for dehalogenase
activity
Examine recombinants
for complementation of
auxotrophic mutations
Figure 6. Construction of genomic library in cosmid vector pHC79
and screening of the library for gene expression in
E. coli.
43
-------
cosmids and subcloned into a restriction site, such as EcoRI, of pUC8 by the
method outlined 1n Figure 7. Since the EcoRI cloning site 1s located within
the N-terminal portion of the lacZ gene, expression of fragments cloned into
this site may occur from the l_ac promoter site. Recombinant clones will be
pooled and screened for dehalogenase activity. The plasmids can then be
isolated and also used in complementation studies. Because the pUC8 plasmid
can be mobilized, complementation studies are not limited to an E. col 1
host. It should be possible to do complementation studies in Pseudomonas in
order to determine if enzymes from OCB-1, an obligate anaerobe, will
complement mutations in aerobic degradative pathways. These complementation
studies could provide a great deal of information some of which would
otherwise be provided only by mutation studies in DCB-1. Subcloning may
also be useful for generating recombinants which can be stored by freezing.
If It was the presence of large pieces of DCB-i DNA which made the first set
of cosmid recombinants sensitive to freezing, then If may be only possible
to store either recombinants with small inserts or store the cloned DNA as
purified plasmids. The first of these two methods is preferable to the
second.
The size fractionation of DCB-1 DNA for cosmid cloning yielded several
fractions of DNA which contained predominantly large pieces of DNA (>10kb)
and yet these were pieces too small for cosmid cloning «30kb). These
pieces were reserved for cloning Into pl)C8 in order to further increase the
size of the DCB-1 genomic library. These DNA fragments were partially
restricted with the enzyme Sau3Al to a size range of 2-5 kb and ligated into
the BamHI site of pl)C8. These ligation reactions were transformed into DH5oc
cells, and the recombinants are currently being analyzed.
Work with the recombinants will continue to verify the presence of
OCB-1 DNA and to determine the best method of storage of the cloned DNA.
44
-------
Total Genomic
DNA
Restriction
^^Sucrose gradient
3-9 kb DNA fragments
ligate
Hind III
Sm I Ace I Hind II
Bam HI
Sma I
Restrict
dephosphorylate with CIP
Insert DNA
Transform
select ApR
Screen for recombinants
Isolate plasmids and pool
Transform auxotrophic hosts'
Examine recombinants for
complementation of
auxotrophic mutations
(B-galactosidase negative)
Screen recombinants for
dehalogenase activity
Figure 7. Construction of genomic library in pUC8 and screening
of the library for gene expression in E. coli.
Recombinant bacteria can be distinguished by their ApR,
.l-galactosidase negative phenotype.
45
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