United States
                  Environmental Protection
                  Agency
Risk Reduction
Engineering Laboratory
Cincinnati OH 45268
                  Research and Development
 EPA/600/S2-88/054  Jan. 1989
f/EPA         Project Summary
                  Determination and
                  Enhancement of Anaerobic
                  Dehalogenation:  Degradation  of
                  Chlorinated Organics  in
                  Aqueous  Systems
                  Donna T. Palmer, Timothy G. Linkfield, Jayne B. Robinson,
                  Barbara R. Sharak Genthner, and George E. Pierce
                   Anaerobic degradation is  poten-
                 tially an efficient means to destroy or
                 detoxify many environmental pollu-
                 tants. Anaerobic  degradation of
                 halogenated organic compounds is
                 especially interesting, because many
                 of these compounds are toxic and
                 apparently  resistant to aerobic
                 degradation. The full  report sum-
                 marizes our initial  efforts to  isolate
                 microorganisms capable of anaero-
                 bic dehalogenation; to examine the
                 nutritional requirements of dehalo-
                 genating  enrichments  and  a
                 dehalogenating consortium and to
                 study the genetics  of  dehalo-
                 genation.
                   Anaerobic enrichments  were
                 established in which 3-chloro-
                 benzoate  (3CB but not 4-chloro-
                 benzoate  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  dehalo-
                 genation.  Such manipulations are
                 useful in efforts to isolate organisms
                 in pure culture. Genetic studies were
                 begun using the anaerobic dehalo-
                 genator, strain DCB-1 (obtained
                 from Dr. J. M. Tiedje).  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 pHC79 and into the
plasmid pUC8. A rapid  dehalogenase
assay was developed for the purpose
of screening recombinants  for
dehalogenase activity.
  This Project Summary  was devel-
oped by EPA's Risk Reduction Engi-
neering Laboratory, Cincinnati, OH, to
announce  key findings of  the re-
search  project that is fully docu-
mented in a separate  report of the
same title  (see  Project Report
ordering information at  back).

Introduction
  Many halogenated organic compounds
pose  a serious environmental problem
because of their persistence and toxicity.
It may be possible to employ microbial
degradation to detoxify or destroy these
compounds either in situ or during waste
treatment.  Recent  investigations  have
shown that many halogenated organic
compounds, including halogenated ben-
zoates and  halogenated phenols, can be
anaerobically degraded. Some of these
compounds are not known  to be aero-
bically degraded;  thus, evidence of
anaerobic  degradation  is extremely
important. In contrast to the mechanism
of aerobic degradation of many haloaro-

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  matics,  the first  step  in  anaerobic
  degradation involves the  removal of the
  halogen leading  immediately to  the
  formation of a generally less toxic, more
  biodegradable compound.
    The overall objective of the  proposed
  program  is  the  development  of
  engineered  microorganisms  capable of
  destroying hazardous  organic  com-
  pounds (e.g., chlorinated organics) under
  anaerobic conditions. An understanding
  of microbially  mediated  anaerobic
  dehalogenation and  the  exploitation of
  this process may result in the significant
  reduction of toxic and hazardous wastes
  in the  United  States.  Therefore,  this
  program would assist in  detoxifying
  recalcitrant  halogenated organic  com-
  pounds that  have not been biode-
1  gradable or accessible to chemical  and
  physical destruction techniques in  the
  past.
    In  order  to  study the genetics of
  anaerobic dehalogenation, it would  be
  useful  to  clone  the  gene  or genes
  responsible for this  activity.  If plasmids
  are found  in  dehalogenating  micro-
•  organisms and if dehalogenase activity is
  encoded by a gene or genes carried by
  the plasmid, plasmid genes  specifying
  the anaerobic  degradation  or  bio-
  transformation  of  chlorinated  organics
  could  be introduced into suitable hosts
,  using  genetic engineering techniques.
  These  modified strains could  then  be
  examined  to  study and enhance  the
  degradation of organic  chemical  con-
  taminants present in hazardous  waste
  sites.  However,  if  dehalogenase activity
  is chromosomally  encoded,  a  genomic
  library  must be constructed  in order to
  isolate the dehalogenase gene.
    In  order to  initiate the cloning effort
  and study the genetics and biochemistry
  of dehalogenation, a pure culture of an
  anaerobic dehalogenating organism  was
  needed. In the first phase of this  study,
  an  effort  was  made  to  isolate  a
  dehalogenating enrichment and a pure
  culture of  a  dehalogenating  micro-
  organism from a  secondary sludge in
  Columbus, OH. In  the second phase, we
  obtained  a 3-chlorobenzoate (3CB)
  degrading  consortium from  Dr.  J.  M.
  Tiedje. This consortium was  the  first
  anaerobic dehalogenating consortium to
  be reported.  It   served as  a model
  system for the isolation and identification
  of  the organisms  responsible  for
  dehalogenation.  In the final phase  of this
  study,  genetic  studies began on a pure
  culture of  strain DCB-1, the  organism
  responsible  for  dehalogenation in
  Tiedje's consortium. Since plasmid DNA
  was not detected  in DCB-1,  efforts were
undertaken to construct a  library of
DCB-1 genomic  DNA in  Escherichia
co//.  A partial  genomic  library  has
potentially been cloned using a  cosmid
cloning system. Further effort is  needed
to  characterize the recom-binants.

Materials and Methods

Growth Conditions and  Strains
  The  anaerobic techniques  employed
for the  handling of  the  inocula,
preparation of media,  and handling of
enrichments  and  cultures  had  been
previously established. Enrichments
were   prepared  by  adding  sterile
anaerobic  solutions of 3-chlorobenzoate
(3CB) or  4-chlorobenzoate  (4CB) to the
basal medium containing 10% secondary
anaerobic  digester  sewage  sludge
(Jackson  Pike Plant, Columbus,  OH) as
inoculum. For  the  work  with  the
enrichments  and the consortium, the
basal  medium contained rumen fluid (or
yeast  extract), B-vitamins,  minerals,
NaHCOa,  Na2S  reducing   solution,
resazurin  redox indicator,  and  a  90%
N2:10% C02  gas phase (final pH 7.0).
The terminal electron acceptor was C02
for methanogenic   media, while  20 mM
NaS04, 15 mM KNOa,  or 20 mM  sodium
fumarate was  added for sulfate, nitrate or
fumarate  enrichments,  respectively.
Fermentative enrichments were prepared
by adding the carbohydrates used in the
Complete  Carbohydrate  medium (CCM)
of  Leedle and   Hespell  (I980).
  Stock  cultures of  DCB-1  were
maintained  in  basal medium  containing
10-20% (v/v)  clarified  rumen  fluid  and
0.2%  (w/v) pyruvate. For DNA extraction,
DCB-1 was  grown in basal medium
consisting of  20%  clarified rumen  fluid,
and 0.2% pyruvate. The cultures  were
grown  in an atmosphere of 80%  N2:20%
CO2.
  The E. co//  strains and vectors  used in
this work  are  listed in Table  1. E. co//
strains were  grown on  LB plates,  LB
broth  or  nutrient broth with  the appro-
priate antibiotic,  as  necessary, for
selecting  recombmants. Antibiotics were
used  at a final  concentration of 30-40
ng/ml  for  ampicillin and 15 ng/ml  for
tetracycline. The  bacteria were incubated
at 37°C.

Dehalogenase Screening Assay
   Using a glass pipet,  1 drop each of the
following  reagents was added to  the well
of a  white porcelain  well plate:  0.2%
KNO2, 0.4%  starch, 2% ZnCI2 solution,
and  1.9N  HCI. A drop of  DCB-1  liquid
culture medium  (which  included 3-
iodobenzoate  as  a substrate  for  dehalo-
genation) was  then added  to  the
containing the  reagents. A bluish-pi
color indicated a positive reaction for
presence of  iodide  and,  theref<
evidence for dehalogenation.

Genomic DNA Isolation and
Purification
  In order to obtain sufficient quant
of purified genomic DNA, a modifies
of a published  procedure  was u;
Additional modifications  were made
follows:  Proteinase K (Sigma) was i
at a final concentration  of  1 mg/m
optimize cell breakage. RNase (Sigm.
a final  concentration  of 20  ng/ml
employed to  reduce  the high  com
(ration of contaminating  RNA; ethyl'
diaminetetraacetic acid  (EDTA)  con
trations in both the storage buffer anc
dialysis buffer were increased to 10
to minimize nuclease  activity;  and
TgES storage buffer (6 mM Tris pH
0.1  mM  EDTA,  10 mM  NaCI),
replaced  by TE buffer (10 mM Tris
7.4, 10 mM  EDTA) to  decrease the
content. For cloning,  DNA of a spe
size range  was  isolated on a suci
gradient,  usually 10% to 40% (w/v).

Cloning Techniques
  Restriction enzymes  (DNA modif
enzymes) were purchased from se>
manufacturers.  Ligations  were  d
overnight at either 12° or 4°C. Cells v
made  competent and transformed u
standard  procedures (as  in the  Ir
national  Biotechnologies, Inc.  catal
Competent  DH5a cells  were purchi
from BRL.  In vitro packaging kits \
purchased  from Strategene. Reag
were  used  according  to the  mi
facturer's instructions.

High Pressure Liquid
Chromatography
  Benzoate,  3CB,  and  4CB  w
separated, identified, and  quantifiee
high pressure  liquid  chromatogra
(HPLC). A reverse phase C18 Lichro
column  (10  n,  4.6 mm [ID] x  25
Alltech Associates, Inc., Deerfield, IL)
used.  The ratio  of the solvent  c
ponents used during most of the s
was 60.40:5 methanol/H2O/acetic
and the flow  rate was normally
ml/min.  Sample detection was  achii
by  U.V.  adsorption at  a wavelengt
284 nm.  These compounds  were  q
tified by comparing the integrated
under  the  curve  produced   by
compound  in the culture sample to
produced by a 500 pM sample of
authentic compound.

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Table 1. Microorganisms and Vectors

 Strain             Genotype
                                Source
 DCB-1

 E. coli
   AC80

   JM83
   MM294

   DH5o


 Vectors
   pBR322
   pHC79
   pUC8
thr leu met hsdR-hsd *

ara 4 (lac-pro) strA thi
(080 dlac rZA M15)

endAl thi-1 hsdflt7  sup£44

endAl hsdfll? sup£44 thi-1 recAl
gyrA96 relAl SOdlacZA M15
                                J. 7/ed/e
L Bopp

B. Bachmann
Ap* TcR cos
    /acZa
BRL
BflL
BflL
§ Bethesda Research Laboratories.BRL
" ampicillin resistance, ApR,; tetracycline resistance, TcR
6 Mention of trade names or commercial products does not constitute endorsement or
 recommendation for use.
Results and Discussion
  In  order  to properly  examine the
genetics of anaerobic  dehalogenation,
defined pure cultures  were  highly
desirable. Several different  approaches
were used to obtain a pure culture of an
anaerobic dehalogenator. Because  Dr.
Tiedje's dehalogenating consortium and
 he pure  culture DCB-1  (from  this
consortium) 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 Michigan  location  where  Dr.  Tiedje
obtained his inoculum source, an  effort
was  begun  to   examine   Columbus
sewage for  dehalogenation activity. The
successful demonstration of  anaerobic
dehalogenation by  Columbus enrich-
ments  led to an  effort to  isolate the
microorganism  responsible for  this
activity. When we received  Dr. Tiedje's
consortium, work  began  in  parallel to
isolate  the   dehalogenating  organisms
from the Tiedje and Columbus consortia,
and to study some of the  characteristics
of the  dehalogenating  microorganisms.
Finally, Tiedje's  dehalogenating  orga-
nism,  strain  DCB-1, was sent to us.
Since  DCB-1 was  a  pure  culture,
studies with  DCB-1  assumed highest
priority. The goal  of producing a superior
dehalogenating  anaerobic  organism
could  be best approached by studying
the genetics and  biochemistry of
anaerobic  dehalogenation.  This   study
required the use of a pure culture. The
ivailability of  DCB-1   increased the
                     speed at which we could move toward
                     our goal.

                     Enrichments from Columbus
                     Sewage
                       At the initiation  of this  program, there
                     had been only one  report of anaerobic
                     dehalogenation; it was highly  probable
                     that other  anaerobic microorganisms/
                     consortia capable of similar  activities
                     would also  be found. The parameters of
                     enrichment were  varied  in  an effort to
                     obtain these  other anaerobic  dehalo-
                     genating organisms in laboratory pure
                     cultures. The following enrichments were
                     undertaken.
                       Two compounds, 4CB and 3CB, were
                     used in the attempt to isolate different
                     anaerobic  dehalogenating  organisms.
                     Methanogenic  enrichments  were pre-
                     pared because the dehalogenating con-
                     sortium developed by Dr. Tiedje came
                     from a methanogenic  environment.
                     Enrichments were also prepared in which
                     nitrate, sulfate,  and  fumarate served as
                     terminal electron  acceptors  instead of
                     carbon  dioxide. A fermentative enrich-
                     ment was also examined.
                       The basal  medium was  used  for
                     methanogenic enrichments.  Nitrate  re-
                     duction enrichments  used  a  medium
                     similar to  the basal  medium.  Cysteine
                     (2.5%)  replaced  the cysteine/sulfide
                     reducing solution.  The  N2/CC-2  gas
                     phase was retained as some  strict
                     anaerobes known  to reduce  nitrate also
                     require  CC-2.  In the  sulfate reduction
                     enrichments, the basal medium with 20
                     mM NaS04 and 20 mM NaCI was used.
                              Also,  the  sodium sulfide  reducing
                              solution replaced  the  cysteine/sulfide
                              solution.
                                M, P. Bryant reported  that fumarate
                              could  serve as  a terminal  electron
                              acceptor  for  microorganisms  that de-
                              grade  benzoate.  Since  the  use  of
                              fumarate might eliminate the need for  an
                              additional  H2 utilizing  organism as the
                              terminal electron sink, an enrichment was
                              made  with fumarate added to the basal
                              medium.  The ability  of fermentative
                              organisms  to  dehalogenate  3CB  was
                              examined. The basal medium  was used,
                              with the addition of Neopeptone (0.1%,
                              Difco),  Tryptone  (0.1%, Difco),  and the
                              carbohydrates  used in CCM  medium.
                              Benzoate, 3CB, or 4CB  was added to  all
                              the enrichments.
                                The effect of Hg on the development of
                              consortia  capable of  degrading  chlori-
                              nated organic compounds was of special
                              interest because anaerobic dehalogen-
                              ation is a reductive process. It has been
                              observed  that the  chlorine  must  be
                              removed  before  further degradation  of
                              the ring can occur; thus, this may be  an
                              obligatory first step. The degradation  of
                              the dehalogenated  intermediate  requires
                              that the H2 concentration be  kept very
                              low  (less than 1  x 10-5  atm) to have
                              degradation become thermodynamically
                              feasible (i.e., a  negative Gibbs  Free
                              Energy [G]). Therefore, while the pre-
                              sence of some hydrogen might stimulate
                              reductive  dehalogenation,  the  presence
                              of too much hydrogen would  inhibit the
                              degradation of the organic intermediate.
                              Including just enough  \\%  to  provide

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reducing equivalents for  dehalogenation
might result in  a decrease in  the lag
phase before dehalogenation is observed
without  leaving  excess  hydrogen  to
inhibit further degradation  of the com-
pound.
  The enrichments were  periodically
sampled during this study in order  to
determine some  of the biological
processes  which were occurring.  HPLC
analyses of culture  fluid from  those
enrichments containing benzoate  indi-
cated that  the benzoate was completely
transformed within one month. The HPLC
results did  not  indicate total utilization,
but rather that all of the benzoate in the
system  had  been at least partially
degraded or transformed.
  The degradation of 3C8 and 4CB in the
enrichments was periodically monitored
by  HPLC  during an  11-month period.
No degradation  of 4CB was detected in
any  of the enrichments  (Table  2). A
nitrate enrichment incubated  without
hydrogen was  the  first  enrichment  to
show significant 3CB degradation (Table
2). Once the initial  amount of 3CB was
no longer  detectable, an additional 800
nmoles 3CB was added to the culture to
confirm its ability to  degrade this
compound.  The 3CB added to  the
enrichment was  utilized within one week.
Initially, the rate of disappearance of 3CB
was  54  iimoles/liter/day,  but after the
third day,  the  rate increased to 145
nmoles/liter/day.  Degradation after the
third day was linear (R = 0.999).
Table 2.  Develpment  of  3-CI-Benzoate
        Degrading Consortium Under
        Various Enrichment Conditions
Enrichment Type
3-CI-benzoate
methanogenic
sulfate
nitrate
fumarate
CCM
4-CI-benzoate
methanogenic
sulfate
nitrate
fumarate
Time
no H2

I0-23f
NDO§
6-10
70-23
A/DO

A/DO
A/DO
A/DO
A/DO
(Weeks)"
+ H2

70-23
A/DO
70-23
70-23
A/DO

A/DO
A/DO
A/DO
A/DO
* Weeks of incubation  before  degration
  observed.
t Degradation not observed at 10 weeks, but
  apparent at 23 weeks.
§ A/DO - no degradation observed
  A Gram stain of the enrichment  was
prepared. Gram-negative short rods  and
cocci were  present as well as refractile
bodies,  (i.e., spores).  Gram-negative,
thin, extremely long  rods,  similar to
Methanospirillum  hungatei,  were  also
present. The presence of  M. hungatei
suggested that a  methanogenic  enrich-
ment had become established.
  The  fumarate  and  methanogenic
enrichments,  after 28 weeks of incu-
bation, showed the complete absence of
3CB (Table  2). Further examination of the
enrichments  showed  that neither  the
terminal electron acceptor present in the
fumarate and nitrate  enrichments  nor
hydrogen was required for degradation.
Microscopic examination of the  enrich-
ments  revealed  a mixture  of  Gram-
negative rods of varying shape  and
lengths  (from coccobacillus to long rod
similar  to M. hungatei).  There was no
degradation of 3CB in either  the sulfate
or CCM  enrichments.

Examination of 3-CI-Benzoate
Consortium
  The microbial consortium capable of
degrading  3-chlorobenzoates  was sup-
plied by Dr. James  Tiedje. Previous
workers indicated  that the dehalo-
genating organism  isolated  from  the
consortium  grew  slowly in a medium
containing pyruvate and  that  it reduced
nitrate to nitrite. This  suggested  that it
might be possible  to improve  the growth
rate of  the  dehalogenating  organism by
providing nitrate as  a terminal electron
acceptor.   Selectively  improving  the
growth  rate of  this  dehalogenating
organism would aid  in  an  attempt to
isolate this organism in pure culture.  The
consortium  was inoculated (10% v/v) into
the  following three variations of basal
medium in  order  to establish  a pure
culture of the dechlorinating organism:

1. 800   uM  3CB  and 15  mM sodium
  nitrate,
2. 800  jiM  3CB, 15  mM sodium nitrate,
  and 0.3% sodium pyruvate,
3. 800  nM  3CB, 15  mM sodium nitrate,
  and 50% hydrogen.

The basal   medium contained  yeast
extract instead of rumen  fluid. Pyruvate
could serve as an  energy source and as
reducing potential  for  the  reductive
dechlorination of 3CB.  Nitrate  could
serve as a  terminal electron sink to  pro-
duce energy for growth.
  The  3CB concentration was  deter-
mined at 0, 3, 16, and  44 days. At 44
days, the culture containing 3CB and 15
mM NC>3 showed no detectable 3CB, t
a large peak was  observed with a low
retention time  (8.36 minutes)  corr
spending to 694.2 ^M  benzoate.  Tf
indicated that under these conditio
3CB was being  dechlorinated,  but r
degraded. A Gram stain of the  culti
showed that about  95% of the  ce
present  were  small  Gram-negati
coccobacilli found mostly in pairs. The
were  also  some  large  Gram-negati
rods.  Cells  with  the  morphology of
hungatei were not seen.  It appeared tt
under  these  conditions  the benzos
degrader  and  methanogens  we
selected  against  and were now  abse
Thus, the dechlorinating  organism  w
thought to  be one or both  of the c
types present. The large Gram-negat
rod  observed  corresponded  to t
dechlorinating  organism  described
Tiedje, but  the small coccobacillus w
not described in his report. Further  wt
indicated that it was most  likely  that t
rod and  not the coccobacillus was I
dehalogenating organism.
  At this  point  in  the  research, t
dehalogenating organism,  strain  DCB
was  received from Dr. Tiedje and t
isolation  effort  was discontinued.
seemed  likely   that  the  organi;
responsible for dehalogenation in the
experiments was  the same  as  or  vi
similar to strain  DCB-1.

Genetics  of DCB-1
Dehalogenation
  DCB-1  is thought to be  related to
genus  Desulfovibrio. This  strain  v\
originally  isolated  from  anaerol
digester sludge from a sewage treatm
plant in Holt, Ml.  It is a Gram-negal
non-sporeforming obligate  anaerc
capable of  dehalogenating  haloarom;
compounds  by  removing  haloge
(chloro, bromo, and iodo  but not flue
from  meta-substituted benzoate cc
pounds.
  DCB-1   is  the  first  anaerot
bacterium to be isolated in pure  cult
which  possessed  dehalogenase  activ
The  dehalogenase activity of DCB-1
interesting because the mechanism «
conditions of anaerobic  dehalogenat
are  different  from  the  mechams
observed  in  many  aerobic  deha
genating microorganisms. Because sti
DCB-1 grows very slowly and beca
strain  DCB-1 is  a fastidious sti
anaerobe, it was  decided that the  t
opportunity for  studying  anaero
dehalogenation   would  be  achie\
through the cloning and expression of
dehalogenase encoding  gene or ge

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from  DCB-1   in  an alternative  host
nicroorganism.

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  necessary  to  efficiently and  rapidly
screen the recombinant bacteria to find
those  recombinants carrying the gene of
interest.
  A rapid qualitative assay was devel-
oped  that could detect  the presence of
free iodide ions  in  liquid  culture.  The
procedure is a  modification  of the
starch-iodide  spot test  for nitrites.  The
otiginal test depends on the formation of
nitrous acid and its  subsequent reaction
with potassium iodide to liberate iodine,
which turns the starch blue. By providing
a source of  nitrite as a 0.2%  aqueous
solution of KNOa the presence of iodide
ions  in  the  medium  due  to  the
dehalogenation  of the  substrate 3-
iodobenzoate  can be detected.  Quanti-
tative  analysis of the  loss  of  3-iodo-
benzoate 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
jrder  to determine the sensitivity of the
spot test (Table 3).
  After 23 days of incubation, all DCB-1
samples were positive for dehalogenation
as determined by 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%
of 3-iodobenzoate remaining based on
HPLC data. The development of a rapid
technique  to  assess  anaerobic  dehalo-
genation, using 3-iodobenzoate,  was a
significant  achievement. With  this
technique, large numbers of clones can
be  screened  under  a variety of
environmental conditions.

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 carried by a plasmid
would be  much simpler than  cloning a
chromosome  encoded gene.  However,
all attempts to isolate plasmid DNA  from
DCB-1 were negative. Since there  was
no evidence to suggest the existence of
any  plasmids  in  DCB-1,   it  was
concluded that the dehalogenase activity
was chromosomally encoded. In order to
clone the gene or genes  responsible for
the  dehalogenase  activity,  it  was
necessary to generate  a  complete
genomic library  of   DCB-1  DNA  and
search for the gene or genes of interest.

Preparation of DCB-1 Genomic
Library--
  The goal of the cloning effort was to
generate a DCB-1 genomic library  and
to screen the library  for the  gene (or
gene complex)  that  encodes  the
dehalogenase activity. The initial set of
experiments was performed in order to
show that a  DCB-1  genomic  DNA
library could be constructed using an £
coli vector and host.  For these experi-
ments,  purified genomic DNA  was
digested with either  Pst1  or EcoR1  and
hgated to Pst1  restricted  pBR322 and
EcoR1 restricted  pUC8,  respectively.
Recombinant  E. coli with DCB-1  inserts
were isolated. These clones were grown
anaerobically and tested for dehalo-
genase  activity  using  the 3-iodo-
benzoate screen.  However, dehalo-
genase 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  was  relatively  poor,  it seemed
desirable to clone large DNA pieces into
a vector  so that DCB-1 DNA could then
be produced in the recombinant host, E.
coli, and  subclones could be made then
from these large inserts. Cosmid vectors
were designed for the purpose of cloning
large DNA fragments. A genomic library
was partially  generated  in the cosmid,
pHC79, by the method outlined in Figure
1.
  Banked cosrnids can  be  tested  for the
presence  of the gene 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  initi-
ation  sites to function in E. coli.  (A
complementation study,  as indicated  in
Figure  1, is  useful for determining  if
foreign promoters  and  translation  initia-
tion sites  function in £. coli).
  Since   an £.  coli promoter may be
needed to express DCB-1 DNA  in  E.
coli, we  plan to purify  DCB-1  DNA from
       Table 3. Comparison of Deba/ogenase Acf/v;fy by Standard HPLC Methods and the Rapid Spot Screening Assay.
                                      Dehalogenation as Measured by HPLC
                                           Dehalogenation as Measured by
                                                   the Spot Test
3-iodobenzoate
(% remaining)
Sample
DCB-1 a
b
c
d
e
0
roo
too
roo
roo
roo
(days)
9
743
95.6
89.4
862
88.9
23
0
51.3
20.4
29.5
28.1
0
0
0
0
0
0
Benzoate
% formed
(days)
9
NT§
NT
NT
NT
NT

23
100
43.3
100
53.8
50.0
Starch Iodine
Reaction"
(days)
0 9 23
— — + + +
— — -4-
— — +
— — +
— — -t-
       Each value represents the mean of duplicate samples.
       § Not tested; NT
       * A negative reaction is indicated by a minus sign and a positive reaction is indicated by one or more plus signs. A three plus
        reaction is one that occurred rapidly and results in a very dark blue color.

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                          EcoR1
              Pst 1 »
                                            Large DNA Fragments
                                                Fill in
                                                with
                                                Klenow
                                                AddC Tail
                          Restrict with Pst 1
                             and G Tail
               Total Genomic
                   DNA
                  f
                  Sucrose
                  Gradient
                                     Package in vitro into infectious
                                     particles, infect
                    Screen for Recombmants (Tcf
    Aps)
                                            Isolate cosmids with inserts
                                               Pool cosmids
                                               Transform auxotrophic
                                                   hosts
        Test for dehalogenase
        activity
        Examine recombmants
        lor complementation of
        auxotrophic mutations
Figure 1.    Construction of genomic library in cosmid vector pHC79 and screening of the
            library for gene expression in E coli
the cosmid    recombinants and then
subclone smaller  fragments of  DCB-1
DNA  (3-9 kb)  into the  vector  pUC8.
Expression of  fragments cloned  into
multiple restrictions site in the lacZ gene
may occur from  the  lac promoter  of
pUC8.  Recombmant  clones  will  be
pooled and screened  for dehalogenase
activity.
  In order to generate a genomic library
with a 95% probability of containing any
particular single-copy gene,  approxi-
mately  380  recombinants  (with DNA
inserts  of 35  kb) would have  to  be
isolated  (This  is a library of about five
genomic units). At this point we have 57
potential cosmids  containing clones
(about  15% of  the library)  These
potential recombinants must be analyzed
further to verify the presence of an insert
and to determine the size of the insert
We are somewhat concerned by the lack
of vigor shown by these  potential
recombinants These bacteria grow very
slowly and  some  were found to be
sensitive to preservation by freezing

Conclusions and
Recommendations
  The  ultimate  goal of  our  work  with
anaerobic dehalogenatmg bacteria  is to
develop  engineered  microorgamsr
capable of  degrading  hazardous orgar
compounds (e.g., chlorinated organics)
environmentally  safe  forms  under  s
aerobic conditions  This  work  will  al
provide information on the  process
anaerobic dehalogenation  To achie
this goal, we recommend completing t
generation  of the DCB-1  library and t
cloning of the dehalogenase gene
  When Columbus sewage was used
an inoculum for the various ennchmei
discussed in  this report,  dehalogenati
of 3-chlorobenzoate  (3CB)  was c
tected  The Battelle laboratory was t
second laboratory  to report this activi

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Anaerobic  dehalogenation,  therefore,  is
an activity found  in  multiple sewage
samples; it is  not  an isolated activity.
Dehalogenation of  4-chlorobenzoate
(4CB) was not detected in the primary
enrichment; this  result  is in  agreement
with the work of others.
  A hypothesis that Ha at a concentration
of 10% would aid in the establishment of
a 3CB  degrading consortium was  shown
not  to  be true.  Experiments with the
dehalogenating consortium  of  Tiedje
showed that varying the  growth  condi-
tions (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  DCB-1, the  micro-
organism  responsible  for the dehalo-
genation of 3CB in  the Tiedje  consorti-
um, were examined  for the presence  of
plasmids by a variety  of  methods; no
plasmids were  observed. The absence of
plasmid  DMA  indicated  that  a  total
genomic  library of  DCB-1 needed to  be
generated in order  to clone the  gene or
genes  responsible  for  anaerobic
dehalogenation. Our efforts  have shown
that genomic-DNA  isolated  from  DCB-
1 can  be cloned in and banked in E. coli.
  The full report was submitted in ful-
fillment of CR811120-02-4  by  Battelle
Columbus  Division  under  the spon-
sorship  of  the U.S.  Environmental
Protection Agency.

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                                             Robinson, Barbara R.
                                             Battelle  Columbus  Division,
Donna  T. Palmer,  Timothy G. Linkfield, Jayne B.
   Genthner,  and George E. Pierce are with
   Columbus, OH 43201.
Albert D. Venosa,  is the EPA Project Officer (see below).
The complete report, entitled, "Determination and Enhancement of Anaerobic
   Dehalogenation: Degradation of Chlorinated Organics in Aqueous Systems,"
   (Order No. PB 89-110 2821 AS;  Cost:  $15.95,  subject  to  change) will be
   available only from:
        National Technical Information Service
        5285 Port Royal Road
        Springfield, V'A 22161
        Telephone:  703-487-4650
The EPA Project Officer can be contacted at:
        Risk Reduction Engineering Laboratory
        U.S. Environmental Protection Agency
        Cincinnati, OH 45268
United States
Environmental Protection
Agency
                             Center for Environmental Research
                             Information
                             Cincinnati OH 45268
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