United States
Environmental Protection
Agency
Water Engineering
Research Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/S2-86/081 Jan. 1987
Project Summary
Application of Recombinant
DMA Technology to
Methane Biosynthesis
John N. Reeve and David S. Cram
A project was conducted to clone the
genes encoding the polypeptide subunrts
of the enzyme methyl-coenzyme M methyl-
red uctase (methyl CoM-reductase). The
experimental approach was to purify the
enzyme (initially from Methanobacterium
thermoautotrophicum. and subsequently
from Methanococcus vannielii). produce
antibodies against the enzyme, and use
these antibodies to screen Escherichia coli
colonies for clones that synthesized
antigens with wNch the anti-methyl CoM-
reductase antibodies reacted. The E. coli
strains contained plasmids or were pre-
infected with bacteriophages that had
been constructed by in vitro DNA recom-
binant techniques to contain fragments of
either M. thermoautotrophicum or M.
vannielii genomic DNA's. The expectation
was that the E. coli clones that reacted
with the anti-methyl CoM-reductase anti-
bodies would contain cloned methanogen
DNA sequences encoding part or all of the
methyl CoM-reductase polypeptides. How-
ever, this technique frequently generated
false positive signals. Most of the study
period was used in improving the tech-
nology to decrease the number of artifac-
tually positive signals and in screening and
analyzing positive clones that ultimately
were found to contain none of the desired
genes. Because enzyme purification and
antibody production were very time-
consuming and artifactual results were
being obtained, alternative approaches
were investigated, and experiments were
undertaken to identify the sequences in
methanogen DNA's that act as regulatory
signals for gene expression. This informa-
tion will be needed in the next stage of this
project — the construction of DNA mole-
cules containing methyl CoM-reductase
sequences that can be manipulated to
direct the synthesis of the enzyme when
reintroduced into methanogens.
Although the specific goal of cloning the
genes encoding methyl CoM-reductase
has yet to be reached, the experiments
completed have produced valuable infor-
mation describing the structures and or-
ganization of methanogen genes and the
structure of methanogen messenger
RNA's.
This Project Summary was developed
by EPA's Municipal Environmental Re-
search Laboratory, Cincinnati, OH, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see Pro-
ject Report ordering information at back).
Introduction
The anaerobic fermentation of waste
biomass to methane concentrates 90% of
the solar energy entrapped in this material
by photosynthesis into a convenient
energy source. As already demonstrated
for many other bioprocesses, this bio-
process should be amenable to genetic
manipulation using the techniques of
genetic engineering. Researchers should
be able to delineate the enzymology of
methane biogenesis, identify and modify
the genes responsible for methane
biogenesis, and ultimately introduce such
a capability into microbial species cur-
rently incapable of methane biogenesis.
The substrates used for methane bio-
genesis are very limited (ag., acetate, for-
mate, methanol, methylamines, carbon
dioxide, and hydrogen), and often the
supply of these substrates to methan-
ogens is the rate-limiting step in methane
ane production into species capable of
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converting a more extensive range of
substrates to methane would be a major
achievement. Alternatively, genetic infor-
mation for increased substrate use might
be introduced into currently existing
methanogens to increase their capabilities.
The long-term goals of our research pro-
gram are to obtain an understanding of the
structure and activity of enzymes involved
in methane biogenesis and to create
improved methanogenic species for use in
conversion of biomass to methane. Our
immediate objective is to isolate biosyn-
thetic genes from methanogens and to
determine their structures and the mecha-
nisms of their regulation. The most abun-
dant enzyme in methanogenic bacteria is
methyl-coenzyme M methyl-reductase.
This enzyme is responsible for the terminal
step in methane biogenesis, in which a
methyl group (CH3.) bound to a cofactor
known as coenzyme M is reduced to
methane (CH4). This document sum-
marizes our experiments designed to clone
and characterize the genes that encode
the subunit polypeptides of methyl-
coenzyme M methyl-reductase.
Procedures and Results
Enzyme Purifications
Methyl-coenzyme M methyl-reductase
(methyl CoM-reductase) comprises approx-
imately 10% of the protein of methano-
gens. The holoenzyme form of methyl
CoM-reductase is a complex of 3
differently-sized polypeptide subunits (y 2
/? 2 « 2) with a combined molecular weight
of approximately 300,000 daltons. It was
purified following a published procedure
that required the development of anaer-
obic column chromatography facilities. An
anaerobic glove box was modified to
accommodate columns and a fraction col-
lector. A refrigeration unit and a spec-
trophotometer were located adjacent to
but outside the anaerobic compartment.
Conduits were built that allowed anoxic
cooling fluid or column eluates to be cir-
culated through the refrigeration unit or
through the spectrophotometer and then
be returned to the anaerobic environment.
Purification of the enzyme was followed
by polyacrylamide gel electrophoresis
(PAGE) of column extracts. The enzymatic
assay for the enzyme, namely methane
generation from methyl-CoM, was not
routinely used. The abundance of the
enzyme and known sizes of the subunit
polypeptides made identification by PAGE
a practical assay. Figure 1 shows the elu-
tion profile and a sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-
PAGE) analysis of the material in the col-
umn fractions that results from chroma-
tography of methyl CoM-reductase
through a DEAE-sephadex column. The
three subunit polypeptides (a ,/3 ,y) of
methyl CoM-reductase are clearly discer-
nible with molecular weights of approx-
imately 68,000, 45,000, and 38,000
daltons, respectively. Methyl CoM-
reductase contains tightly bound nickel
atoms, and in one purification, the culture
was grown in the presence of 63Ni to pro-
vide a specific radioactive label for the en-
zyme. Purification of the labeled enzyme
using the PAGE assay confirmed that
these procedures resulted in purification
of methyl CoM-reductase as the 63Ni
2.5
2
I 1.5
^
§ /
.5
0
•0 5 10 15 20 25 30
Fraction No.
11121314151617181920
-Y
Figure 1. Elution profile and SOS-PAGE
analysis of material in column
fractions that result from chro-
matography of methyl CoM-
reductase through a DEAE-
sephadex column.
copurified with the polypeptides identifii
as the subunits of methyl CoM-reductaj
The enzyme was initially purified fro
Methanobacterium thermoautotrophicL
(year 1), which required the use of
French pressure cell to rupture the eel
Later, enzyme preparations (years 2 ai
3) were obtained from Methanococa
vannie/ii. This species has only a pr
teinaceous cell wall and was much me
readily lysed. In addition, this species hi
become the species of choice in exp<
iments designed to develop a genetic e
change system for methanogens. Futu
work with the cloned methyl Cof
reductase genes will need this DN
transfer technology, and therefore it w.
decided to concentrate all methanoge
related research on this species.
The enzyme preparations obtained fro
both M. thermoautotrophicum and /
vannie/ii were shown by PAGE to I
almost free of contaminating polype
tides. But even with the use of silver stai
ing to visualize polypeptides in polyacr
lamide gels, it was never possible to o
tain enzyme preparations entirely free
other polypeptides. Several modificatioi
to the purification procedure we
evaluated (e.g., different column materie
and changes in salt concentrations us<
in eluting solutions). Although improv
ments were obtained, the preparatioi
were never absolutely free of contar
inating polypeptides. For this reaso
preparative PAGE was eventually used i
the final step in purification. The enzyn
subunits were separated by SDS-PAG
and the regions of the gels that contai
ed the individual subunits were cut fro
the gel slabs. These gel fragments we
then passed through a fine-bore syrinc
needle to fragment the gel. The resultir
gel slurries were incubated in a smi
volume of buffer to allow the polypeptidi
to elute from the gel. These solutions we
then used to vaccinate rabbits for antibo<
production.
Production of Antisera
Antisera were obtained by vaccinatic
of rabbits. The first antigen used was cor
plete methyl CoM-reductase from M. tht
moautotrophicum. This material contain*
all three subunit polypeptides, and ther
fore the antisera obtained contained an
bodies that reacted with all three polype
tides. In later experiments, the SDS-PAG
separated subunits were used as antigen
Antibodies raised against the separate
subunits have the advantage that
positive signal can be related directly
the gene encoding the polypeptide us<
as the antigen. However, they also ha'
-------
the potential disadvantage that the sepa-
rated polypeptides are denatured during
SDS-PAGE and therefore may not have all
the same antigenic determinants as the
native enzyme.
A standard vaccination regime was used
in which the initial inoculation was fol-
lowed 10 weeks later by a booster vacci-
nation. Sera were taken at weekly inter-
vals following the second vaccination, and
the presence of anti-methyl CoM-
reductase antibodies was assayed. Very
variable antibody titers were observed. In
some animals, high titers were rapidly ob-
ained and maintained, whereas in other
animals, very little useful antiserum was
produced. In particular, we obtained high-
iter antibody preparations for the two
smaller subunits (/} ,y) of the M. vannielii
enzyme, but we were unable to obtain a
high-titer antibody preparation from
animals vaccinated with the largest (a)
subunit polypeptide of this enzyme.
Titration and Evaluation of
Antisera
Antisera preparations were analyzed by
the standard enzyme-linked immunosor-
bant assay (ELISA) plate procedure to de-
termine their titers. Sera that contained
high titers of anti-methyl CoM-reductase
antibodies were then evaluated by Wes-
tern blotting to determine which polypep-
tides were recognized by the antibodies
contained in the antisera. Lysates of
Escherichia coli, M. thermoautotrophicum,
and/or M. vannielii were subjected to
electrophoresis alongside preparations of
the purified methyl CoM-reductases. Fol-
lowing electrophoresis, the separated
polypeptides were blotted onto nitrocel-
lulose paper, which was then immersed in
the antiserum being analyzed. Antibodies
in the serum bound to the appropriate anti-
gen bound, in turn, to the nitrocellulose.
These antibody-antigen complexes were
identified by using 125l-labeled sheep-anti-
rabbit antisera to bind to the rabbit anti-
bodies in the complexes. The locations of
the 125I were then determined by autora-
diography. Using this procedure, it was
possible to demonstrate that antisera pro-
duced by vaccination with the native
enzyme contained a mixture of antibodies,
including antibodies specific for each of
the three* polypeptide subunits. As ex-
pected, antisera raised against a purified
subunit contained antibodies directed
against only that polypeptide. The West-
ern blotting experiments also demon-
strated that antibodies raised against the
M. thermoautotrophicum polypeptides
recognized and bound to the equivalent
(small, medium, or large) polypeptides in
preparations of methyl CoM-reductase
from M. vannielii and vice-versa. These
two enzymes must therefore have con-
served amino-acid sequences and con-
served secondary and tertiary structures.
This cross-reactivity is potentially very
valuable in that antibody preparations
raised against the enzyme from one
methanogen can be used to screen for
genes cloned from other methanogens.
We determined that the cross reactivity
extends beyond M. thermoautotrophicum
and M. vannielii, since anti-methyl CoM-
reductase antibodies prepared against the
enzymes from these two species also bind
to methyl CoM-reductase polypeptides in
extracts of Methanobrevibacter smith/'/',
Methanosarcina barken, Methanococcus
voltae, and Methanococcus thermolitho-
trophicus.
Unfortunately, the Western blotting
experiments also demonstrated that all
antisera contained antibodies that reacted
with £ coli proteins. This was presumably
due to the £ coli population present in the
environment and the gut of the rabbits
used to produce antisera. The presence of
anti-£ coli antibodies made it impossible
to use the antisera preparations directly to
screen for methanogen antigens synthe-
sized in £ coli. Antisera were therefore
mixed with lysates of £. coli so that the
antibodies that bound to £ coli proteins
would be removed by adsorption to the
proteins in the lysates. This procedure had
to be repeated several times before all anti-
£ coli antibodies were removed from the
anti-methyl CoM-reductase antisera prep-
arations. Most of the DNA cloning pro-
cedures employed A-bacteriophage-based
vectors. The £ coli lysates used to remove
£ coli antibodies were therefore obtained
from A-infected £ coli cells to obviate
problems caused by anti-A antibodies in
rabbit antisera. Such anti-A antibodies
were detected in Western blotting experi-
ments. Following removal of the anti-£ coli
antibodies, the titer of anti-methyl CoM-
reductase antibodies was again deter-
mined. The antisera also had to be
evaluated using the experimental proced-
ures for screening £ coli recombinant
clones. A preparation of purified methyl
CoM-reductase was serially diluted, and
an aliquot of each dilution was spotted
onto a nitrocellulose filter. The filter was
then submerged in the antiserum being
tested, and following an incubation period
for antigen-antibody interaction, the filter
was removed and washed, and the pres-
ence of antigen-antibody complexes was
determined. Two related procedures for
this determination were used. In both
cases, the antigen-rabbit-antibody com-
plex was bound by sheep anti-rabbit an-
tibody. The presence of the sheep anti-
body was then detected by its being either
covalently linked to 125I or linked to horse-
radish peroxidase. The presence of 125I
was detected by autoradiography, and the
presence of horseradish peroxidase was
detected by adding a chromogenic sub-
strate for this enzyme. The 125l-based
assay was found to be approximately
10-fold more sensitive (recognizing as little
as 10 pg of antigen) than the enzyme-
based assay. In the majority of screening
experiments, 126l-labeled sheep anti-
rabbit antiserum was used. To screen the
plaques on lawns of £ coli, we used dilu-
tions of rabbit antiserum that could detect
less than 0.1 ng of methyl CoM-reductase
concentrated in a plaque-sized spot on a
cellulose nitrate filter.
Construction of Gene Libraries
The concept of a gene library is that of
a population of recombinant DNA mole-
cules in which every gene of the genome
of the organism being studied is present
in one or more of the DNA molecules. The
number of DNA molecules needed for a
complete library is determined by the size
of the organism's genome and the size of
the individual fragments of DNA cloned
into hybrid recombinant molecules. We
decided to use A-based vectors that allow
the cloning of large 10 to 30 kilobase (kb)
DNA fragments and thereby decrease the
number of clones needed to constitute a
gene library. In the case of methanogens,
such a gene library should be less than
1,000 different recombinant molecules.
Our initial experiments were to obtain
A1049 and ACharon 30 libraries of M. ther-
moautotrophicum DNA (genome size of
1»1x109 daltons). This required producing
restriction fragments of M. thermoauto-
trophicum genomic DNA within the size
range of 15 to 30 kb. A major problem was
that the only mechanisms available to lyse
this species incorporated a physical rup-
turing procedure such as sonication,
French pressure cell, or cryoimpacting.
With these procedures, it was impossible
to obtain sufficiently high-molecular-
weight genomic DNA. Without such DNA,
we could not produce the large restriction
fragments needed for A cloning. Some
recombinant clones were obtained, but it
was unlikely that a fully representive gene
library was produced. To circumvent this
problem, libraries were produced using the
plasmid vector pUCS, in which much
smaller fragments of DNA (less than 10
kb) generated by a range of restriction
-------
enzymes, could be cloned. Expression of
genes cloned in pUC8 can be controlled
by the £ colilac promoter also present on
pUC8. Later cloning experiments also used
plasmid pMF4, in which very small DNA
fragments (50 to 200 base pairs) are
cloned in a site located between the
amino-terminal portion of the Ac/gene and
the carboxyl region of the lacZ gene. If
cloning produces an inframe, open-reading
frame (probability of 1 in 6), then a poly-
peptide is synthesized that contains the
amino acids encoded by the cloned meth-
anogen DNA sandwiched between the
amino terminus of Ac/ and the carboxyl ter-
minus of lacZ. Fusion polypeptides are
generally stable and not subject to pro-
teolysis. This stability has been shown to
faciltate their detection in lysates of £ coll
using antibodies to screen gene libraries.
The problem of obtaining large DNA
molecules from M. thermoautotrophicum
was bypassed when cloning experiments
were undertaken with DNA from M. van-
nielii. Cells of this species can be lysed by
simply adding 1% SDS. This procedure
avoids mechanical cell disruption and thus
the concomitant breakage of DNA. Gene
libraries were constructed from M. van-
nielii genomic DNA using the bacteri-
ophage vectors ACharon 35 (ACh35) and
Agt11 and the plasmid vectors pUC8 and
pMF4. The A-based molecules were
packaged in vitro into A particles and used
to infect £ coli. The Agt11 recombinants
could be shown to contain inserts by using
X-gal indicator plates. Insertion of DNA
into the cloning site of Agt11 inactivates
the lacZ gene of this vector and gives
white plaques on X-gal plates. £ coli in-
fected with the vector alone produces blue
plaques. The number of recombinants
obtained using the bacteriophage vectors
ACh35 or Agt11 to clone M. vannielii DNA
varied between 5x103 and 1x105 clones;
plasmid libraries of either M. vannielii or
M. thermoautotrophicum DNA contained
approximately 1x105 to 1x106 different
clones. Based on the number of clones in
each library and the average size of the
insert DNA, all libraries of M. thermo-
autotrophicum or M. vannielii genomic
DNA were expected to contain copies of
all gene sequences.
Screening of Gene Libraries
Plaques produced by infection of E. coli
with A recombinant phages or colonies of
E coli containing plasmid-based recombi-
nant molecules were screened for the
presence of antigens that could bind the
anti-methyl CoM-reductase antibodies.
The first experiments resulted in very large
numbers of positive clones; however, this
result was quickly recognized as stemming
from the presence of anti-£ coli antibodies
in the antisera preparations (see above).
When the anti-£ coli antibodies were
removed, only a few recombinant clones
in each A library gave positive signals.
These were chosen for further study.
Analysis of Positive Clones
Phages from plaques containing anti-
gens that bound anti-methyl CoM-
reductase antibodies were plaque-purified
and then produced as high-titer phage
stocks. DNA was purified from these
phages, and restriction enzyme analyses
were perfomed to determine the size and
restriction maps of the cloned DNA's.
Although several completely different
recombinant phages were obtained from
both the M. thermoautotrophicum and M.
vannielii libraries, we also obtained several
independently constructed phages that
contained common and overlapping re-
striction fragments. The latter result was
expected because partial restriction
digests were used in the construction of
the A recombinant phages. We also obtain-
ed phages containing common restriction
fragments of M. vannielii DNA when dif-
ferent preparations of antisera were used
in the screening protocol. These results
were encouraging, as they demonstrated
that antisera raised against methyl CoM-
reductase facilitated the consistent
isolation of the same fragments of
methanogen-derived DNA's. Figure 2 pic-
tures an electrophoretic separation of DNA
fragments in restriction enzyme digests of
Agt11-based recombinant phage DNA's.
The large DNA fragments at the top of the
gel are the A vector DNA's; the smaller
DNA fragments that migrate further into
the gel during electrophoresis are the
cloned fragments of methanogen DNA's.
Southern blotting procedures were used
to demonstrate that the cloned DNA's had,
in fact, originated in the genomes of the
methanogens used as the sources of
DNA's. However, isolation of additional,
unrelated phages was also a concern in
that it was clear that many of these could
not contain the desired genes, even
though they gave strong positive signals
in the antigerrantibody screenings. Many
attempts were made to identify the
polypeptides that were synthesized in £
coli following infection with the different
A recombinant phages that interacted with
the anti-methyl CoM-reductase antibodies.
Western blotting of infected cells, immune
precipitation of infected cell lysates, and
PAGE analysis of proteins synthesized in
infected minicells all failed to unambig-
uously identify the polypeptide(s) that
gave the positive signals in the plaqu
screenings. Infection of minicell
demonstrated that many of the recombir
ant phages did direct the synthesis c
novel polypeptides in E. coli; with thi
procedure, however, only the molecule
weights (not the functional activities) c
these pahge-encoded polypeptides coul
be determined.
In spite of the uncertainty as to whicr
if any, of the recombinant phages cor
tained the methyl CoM-reductase gem
we decided to concentrate our efforts o
a DNA fragment from M. vannielii foun
cloned in several different recombinar
phages. This DNA fragment was shown b
Southern blotting to be highly conserve
in the genome of the related methanoge
M. voltae. This was expected for a DN,
sequence encoding what appears to be
highly conserved enzyme. A researc
group in Marburg, West Germany, ha
already reported cloning the genes er
Figure 2. Restriction analysis of hgt1
recombinant phage DNA.
-------
coding methyl CoM-reductase from M.
voltae. We therefore argued that if the
sequence of the DNA we had cloned from
M. vannielii was present in the methyl
CoM-reductase genes cloned by the Mar-
burg group, we would be able to assume
that we had also cloned a methyl CoM-
reductase gene, but from M. vannielii. The
DNA sequence we determined (see full
report) contains an open reading frame of
122 codons. Unfortunately, it does not
appear in the sequence of the M. voltae
methyl CoM-reductase genes. Our an-
tibody screening indicated it should be
part of the gene encoding the small
subunit of methyl CoM-reductase. Com-
parison of the M. vannielii and M. voltae
sequences indicated no similarity between
our sequence and theirs for the smallest
polypeptide of the M. voltae enzyme. We
must therefore conclude that although this
DNA, when cloned in E coli, causes the
synthesis of an antigen that specifically
reacts with antibodies raised against the
purified, smallest subunit polypeptide of
methyl CoM-reductase of M. vannielii, the
DNA probably does not encode this poly-
peptide. Discussions of this anomolous re-
sult with several investigators experienced
in using the antibody screening procedure
have elicited many descriptions of similar
artifactually positive results. The conclu-
sion appears to be that although this tech-
nique does allow the identification and
isolation of desired genes, it frequently re-
quires extensive screening of positive
clones before the correct positive is found.
A second, independent screening proce-
dure is therefore very useful to sort
through the clones identified as positives
by the antibody screen. The most fre-
quently used second screening employs a
synthetic DNA probe.
Determination of an Amino Acid
Sequence for Design of a DNA
Probe
In 1983, we first recognized the serious-
ness of the problem of false positives
generated by the antibody screening pro-
cedure. We therefore decided to obtain an
amino acid sequence of the amino ter-
minus of one of the methyl CoM-reductase
polypeptides. This sequence could then be
used to design a DNA probe that would
be used by DNA:DNA hybridization to
screen our gene libraries for the homolo-
gous DNA sequence. Two immediate prac-
tical problems were: (1) the need to obtain
sufficiently large amounts of the purified
enzyme for amino acid sequencing, and
(2) gaining access to an amino acid se-
quencing facility. Neither of these prob-
lems could be solved at The Ohio State
University (O.S.U.), as facilities for growth
of large cultures of methanogens and
equipment for amino acid sequence deter-
minations were not then available. Large
cultures were therefore grown in collabor-
ation with the University of Iowa, and
purified polypeptides were sent to the
amino acid sequence-determining facility
at the University of Michigan. We visited
both of these institutes to help with and
learn procedures. Unfortunately no useful
sequence information was obtained. The
University of Michigan facility was unable
to obtain a satisfactory amino acid se-
quence. We have therefore developed the
facilities to repeat this approach at O.S.U.
and have begun to prepare sufficient
enzyme for use in amino acid sequence
determination.
Isolation of mRNA for Use as a
Probe or Synthesis of cDNA
Methyl CoM-reductase constitutes ap-
proximately 10% of the total protein in
methanogens, and therefore we assumed
that the mRNA encoding the polypeptides
should be abundant. If such mRNA mole-
cules could be purified, they could be used
to locate complimentary DNA sequences
cloned in a gene library by functioning as
probes in DNA:RNA hybridizations. Alter-
natively, if these mRNA molecules had
3'poly-A sequences, they might serve as
templates to synthesize cDNA using re-
verse transcriptase. This cDNA could then
be cloned and used as a probe in
DNA:DNA hybridization screenings of the
genomic DNA libraries. A project was
therefore initiated to isolate and
characterize mRNA molecules from M.
vannielii. This effort required development
of purification protocols and procedures to
determine the size, stability, and poly-
adenylation status of mRNA's from this
species. The results of this work demon-
strate that methanogen mRNA's closely
resemble eubacterial mRNA's. These
mRNA's do not offer the opportunity of
synthesizing cDNA's, as there is only
limited polyadenylation. Gel electrophor-
esis of purified mRNA's did not show
bands of enriched mRNA's as was ex-
pected of mRNA's encoding very abun-
dant polypeptides. We have therefore
begun to develop an in vitro, translation
system so that extracted mRNA's can be
translated to facilitate identification of the
polypeptides they encode. Electrophoresis
and/or sucrose gradients will be used to
subdivide mRNA preparations into frac-
tions containing differently sized mRNA's.
The products of translation of each frac-
tion will be assayed for the presence of
methyl CoM-reductase antigens using the
anti-methyl CoM-reductase antibodies. If
we can identify an mRNA fraction that is
enriched for the mRNA encoding methyl
CoM-reductase, the mRNA's in this frac-
tion will be made radioactive. This labeled
mRNA preparation will then be used as a
probe using DNA:RNA hybridization to
screen the recombinant clones that gave
positive signals in the antibody:antigen
screening protocols already completed.
Structural Analysis of Cloned
Genes
In parallel with the attempts to clone the
genes encoding methyl CoM-reductase,
we have characterized methanogen genes
that, when cloned in £ coli or Bacillus sub-
tills, complement auxotrophic mutations
in these eubacterial species. The goals of
these experiments are: (1) to obtain precise
details of the structure of regulatory ele-
ments such as promoters and ribosome-
binding sequences, and (2) to determine
the overall organization of genes within
the genomes of methanogens. This infor-
mation will be essential for designing log-
ical genetic engineering approaches to ma-
nipulating the genes encoding methyl
CoM-reductase once these genes are
available.
Studies on these methanogen genes
that complement auxotrophic mutations
have shown that these genes resemble
eubacterial genes in being organized into
operons and having strong ribosome bind-
ing sequences. Codon usages differ
radically from both E coli and B. subtilis.
We have identified conserved methanogen
sequences that may be promoters. Com-
parisons of DNA sequences of related
genes cloned from different methanogenic
species have allowed us to estimate evolu-
tionary divergence and suggest taxonomic
relationships. Details of the procedures
and results obtained in these studies are
provided in publications from our labora-
tory listed in the reference section of the
full report.
Conclusions
The specific goal of cloning the genes
that encode the subunit polypeptides of
methyl-coenzyme M methyl-reductase has
not been reached. We have obtained a
number of positive clones in terms of their
ability to synthesize antigens that interact
with antibodies raised against the subunits
of methyl CoM-reductase. These clones
must be screened further to determine
whether any contain the desired recom-
binant DNA molecules. The experiments
-------
undertaken to isolate and characterize
methanogen mRNA's have provided the
first detailed description of archaebacterial
mRNA structures and have generated the
RNA substrates for development of an in
vitro translation system. Analyses of
cloned methanogen genes, which comple-
ment auxotrophic mutations in £ coli and
B. subtillis, have resulted in publications
containing the first DNA sequences of
methanogen-derived, protein encoding
genes. In addition, we have isolated and
characterized the first methanogen inser-
tion element (ISM1), and we have provided
the first description of regulatory elements
used in expression of methanogen genes.
Although it is disappointing that the
methyl CoM-reductase genes of M. van-
nielii are not yet in hand, we feel that the
technical problems encountered can be
overcome. Nonetheless, the results of our
research to date have already provided a
firm foundation for future applications of
recombinant DNA technology to methane
biogenesis.
The full report was submitted in fulfill-
ment of Contract No. CR810340 by The
Ohio State University under the sponsor-
ship of the U.S. Environmental Protection
Agency.
John N. Reeve and David S. Cram are with Ohio State University, Columbus. OH
43210.
Albert D. Venosa is the EPA Project Officer (see below).
The complete report, entitled "Application of Recombinant DNA Technology to
Methane Biosynthesis," (Order No. PB 87-102 265/AS; Cost: $9.95, subjectto
change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Water Engineering Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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United States
Environmental Protection
Agency
Official Business
Penalty for Private Use S300
EPA/600/S2-86/081
Center for Environmental Research
Information
Cincinnati OH 45268
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