Previous Article | Next Article 
J Bacteriol, May 1998, p. 2623-2629, Vol. 180, No. 10
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The L-Isoaspartyl Protein Repair
Methyltransferase Enhances Survival of Aging Escherichia
coli Subjected to Secondary Environmental Stresses
Jonathan E.
Visick,
Hui
Cai, and
Steven
Clarke*
Department of Chemistry and Biochemistry and
the Molecular Biology Institute, University of California, Los
Angeles, California 90095-1569
Received 28 January 1998/Accepted 11 March 1998
 |
ABSTRACT |
Like its homologs throughout the biological world, the
L-isoaspartyl protein repair methyltransferase of
Escherichia coli, encoded by the pcm gene, can
convert abnormal L-isoaspartyl residues in proteins (which
form spontaneously from asparaginyl or aspartyl residues) to normal
aspartyl residues. Mutations in pcm were reported to
greatly reduce survival in stationary phase and when cells were
subjected to heat or osmotic stresses (C. Li and S. Clarke, Proc. Natl.
Acad. Sci. USA 89:9885-9889, 1992). However, we subsequently demonstrated that those strains had a secondary mutation in
rpoS, which encodes a stationary-phase-specific sigma
factor (J. E. Visick and S. Clarke, J. Bacteriol. 179:4158-4163,
1997). We now show that the rpoS mutation, resulting in a
90% decrease in HPII catalase activity, can account for the previously
observed phenotypes. We further demonstrate that a new pcm
mutant lacks these phenotypes. Interestingly, the newly constructed
pcm mutant, when maintained in stationary phase for
extended periods, is susceptible to environmental stresses, including
exposure to methanol, oxygen radical generation by paraquat, high salt
concentrations, and repeated heating to 42°C. The pcm
mutation also results in a competitive disadvantage in stationary-phase
cells. All of these phenotypes can be complemented by a functional
pcm gene integrated elsewhere in the chromosome. These data
suggest that protein denaturation and isoaspartyl formation may act
synergistically to the detriment of aging E. coli and that
the repair methyltransferase can play a role in limiting the
accumulation of the potentially disruptive isoaspartyl residues in
vivo.
| |
INTRODUCTION |
Spontaneous chemical reactions which
occur in the cytoplasm of a cell under physiological conditions often
result in damage to the cell's critical macromolecules, a process
which may be enhanced by exposure to environmental changes, such as
increased temperature, reactive oxygen species, altered osmotic
conditions, and various chemicals (32). While damage to DNA
and mechanisms for its repair have been extensively studied, many
chemical changes also occur in proteins (30). The
significance of such protein damage may be greatest for cells which are
not dividing or those in which protein turnover is curtailed by
nutrient limitation or environmental stresses.
Organisms ranging from bacteria to mammals and plants have developed
strategies for coping with one product of spontaneous protein damage,
the L-isoaspartyl residues which arise from aspartyl and
asparaginyl residues in proteins (5). These abnormal
residues can affect protein structure and enzyme activity (reviewed in reference 30) but are recognized and methylated by
L-isoaspartyl protein carboxyl methyltransferases (EC
2.1.1.77), such as the product of the Escherichia coli pcm
gene (8, 12). Methylation stimulates the reformation of an
unstable succinimide intermediate which can yield a normal
L-aspartyl residue upon hydrolysis, thus ultimately
catalyzing net repair of the damaged site (17). In plants,
methyltransferase activity is induced during seed production and in
response to dehydration and other stresses (19), while the
enzyme activity has been strongly correlated with aging in mammals by
the recent finding that mice lacking the gene accumulate damaged
proteins and die at an early age (11).
The construction of an E. coli strain carrying a
pcm deletion has been described previously (12).
This mutant strain showed reduced ability to survive for prolonged
periods in stationary phase and increased sensitivity to heat and
osmotic shock (12, 13). Similar, though more severe,
phenotypes have been associated with mutations in rpoS, a
gene located about 1.5 kb downstream of pcm and encoding
S, an alternative RNA polymerase sigma subunit crucial
to the cellular response to starvation and other stresses (reviewed in
reference 14). We recently determined that a
nonsense mutation located near the 5' end of rpoS and
present in many common laboratory strains (but not MC1000, the parent
strain for these mutants) had been inadvertently introduced into the
pcm mutant (31). The amber mutation reduced RpoS
activity (as measured by the activity of HPII catalase, induced at
stationary phase under the control of RpoS) to about 10% of normal.
This unexpected complication made it necessary to reevaluate the
pcm mutant to determine whether some or all of the
phenotypes previously observed (12, 13) might actually be
due to the secondary mutation in rpoS.
The experiments reported here demonstrate that the phenotypes
originally attributed to the pcm mutation could be
complemented by a functional copy of rpoS integrated
elsewhere on the E. coli chromosome but not by a functional
pcm gene. A new pcm mutant strain was constructed
which lacked the rpoS nonsense mutation; this strain
survived as well as its parent for 10 days in stationary phase or when
subjected to the other stresses which reduced the viability of the
original mutant. The new pcm mutant, however, did display
specific phenotypes when cells maintained in stationary phase were
subjected continuously or repeatedly to environmental stresses with the
potential to unfold proteins. These phenotypes were complemented by a
wild-type pcm gene and demonstrate the inability of aging
cells to cope with additional stresses in the absence of the
isoaspartyl repair enzyme.
| |
MATERIALS AND METHODS |
Strains and plasmids.
E. coli strains and plasmids
used in the phenotypic assays are described in Table
1. Strain JV1026 was constructed by first inserting a 3.8-kb BamHI-BclI fragment from pCL1
(12), carrying the surE-pcm operon and its
promoter, into pLDR10 (7) adjacent to the lambda
recombination sequence attP. A NotI restriction fragment of this plasmid lacking the origin of replication was then
self-ligated and introduced into E. coli WM2269, harboring pLDR8, a plasmid with a temperature-sensitive replication origin carrying the lambda int gene (7). Colonies
expressing the ampicillin resistance marker from pLDR10 when grown at
42°C (the nonpermissive temperature for pLDR8) were screened to
confirm the loss of the helper plasmid. The construction of HC1011 was
done similarly, except that pLDR11 (7) was used as the
integration vector and the integrated DNA was a 4.8-kb EcoRI
fragment carrying rpoS+ from a derivative of
pMMkatF2 (20). The resulting
attB::pcm+ or
attB::rpoS+ insertion was
transduced into strain CL1010 by using bacteriophage P1 (25)
and confirmed by PCR amplification with attB- and
gene-specific primers and by Southern hybridization.
Strain JV1068 was constructed with pKAS46, which is dependent on the
pir gene product for its maintenance and carries a kanamycin
resistance (Km
r) marker and a wild-type (Str
s)
rpsL gene (
26). A chloramphenicol resistance
(Cm
r) marker was used to replace nearly all of the
pcm coding sequence
(from
MluI to
ClaI) in pCL1 and was oriented in the same direction
as
pcm, paralleling the construction of the original mutant
strain
CL1010 (
12). An
XbaI fragment carrying the
marker and flanking
DNA was then inserted into pKAS46, and the
resulting plasmids
were maintained in CC118
(
pir+). For chromosomal gene replacement, these
plasmids were electroporated
(by standard methods with a Bio-Rad Gene
Pulser apparatus) into
strain MC1000 (which lacks the
pir
gene and is Str
r), and Km
r single recombinants
were screened for Cm
r. These colonies were then streaked on
plates containing streptomycin
(1 mg/ml) to select for loss of the
vector sequences, including
rpsL. Str
r colonies
were restreaked on the same medium and then screened
for the
Km
s Cm
r phenotype, which would indicate that
gene replacement had occurred.
The identities of the recombinant
strains were confirmed by transduction
and linkage analysis, PCR
amplification with primers complementary
to
pcm, and
sequencing of
pcm and
rpoS.
The complementing strain JV1083 was constructed by transducing the
pcm+ gene and the linked Ap
r marker
from strain JV1023 (MC1000
attB::
pcm+) into JV1068.
Strains TST1 (
malE52::Tn
10) and TSM7
(
malE57::Tn
5)
were obtained from the
E. coli Genetic Stock Center and used in
the construction of
reciprocally marked strains for the growth
advantage in stationary
phase (GASP) assay. Selection for Tc
r after infection of
MC1000 and JV1068 with bacteriophage P1 grown
on TST1 permitted the
isolation of strains JV1090 and JV1093;
strain JV1094 was constructed
similarly by transduction of MC1000
with phage grown on TSM7. Strain
JV1093 was then transduced with
phage grown on strain JV1023 and
selected for Ap
r to generate the complementing strain
JV1098.
PCR and sequencing.
Oligonucleotide primers were synthesized
with an Oligoassembler apparatus (Pharmacia Biotech, Piscataway, N.J.),
and PCR amplifications were performed with an MJ Research (Watertown,
Mass.) thermal cycler and Taq DNA polymerase (Promega Corp.,
Madison, Wis.). For longer PCR products, Taq Extender
(Stratagene Cloning Systems, La Jolla, Calif.) was used according to
the manufacturer's instructions. For sequencing, chromosomal DNA from
colonies or overnight cultures was amplified directly (9).
Sequencing was carried out with an Applied Biosystems 373A or 377 automated sequencing apparatus.
Enzyme assays.
Cytosolic extracts were prepared by gentle
sonication of E. coli grown to stationary phase (18 to
20 h in 5 ml of Luria-Bertani [LB] broth) and resuspended in a
0.5-ml solution of 5 mM potassium phosphate, 5 mM disodium EDTA, 10%
glycerol, and 25 µM phenylmethylsulfonyl fluoride (pH 7.0) as
described previously (31). The protein concentration was
estimated by the method of Lowry et al. (16) after
trichloroacetic acid precipitation. A vapor phase assay to measure
base-labile methyl esters produced by transfer of the methyl group from
S-adenosyl-L-[methyl-14C]methionine
to a specific isoaspartyl-containing peptide substrate (Lys-Ala-Ser-Ala-isoAsp-Leu-Ala-Lys-Tyr) by methyltransferase in the
extract was performed essentially as described previously (12). Methylation was carried out for 20 min at 37°C, 1.5 M Na2CO3 was used to release labeled methanol
from the methyl esters, and peptide-specific counts were determined by
subtraction of a control sample assayed in the absence of peptide
substrate. HPII catalase activity was determined by means of a
spectrophotometric assay for H2O2 breakdown in
extracts that had been heated to inactivate HPI catalase
(31).
Stationary-phase and stress survival tests.
Resistance to
lethal heat shock was measured by using cultures grown for
approximately 20 h in LB broth. Cells from these cultures were
spun down and resuspended to 1/10 their original density in 0.85%
NaCl, and 50-µl aliquots were then heated to 55°C for 10 min in a
thermal cycler, using a thin-walled polycarbonate 96-well plate. Viable
counts were performed by making serial dilutions in 0.85% NaCl and
plating them on LB agar (incubated for 16 to 20 h at 37°C) to
determine the number of bacteria that had survived; these results were
then compared to counts of the original cultures. Oxidative stress
testing was carried out similarly, except that H2O2 was added to the diluted cells to a final
concentration of 15 or 30 mM and the cells were incubated for 1 h
at 37°C. For osmotic stress, the culture aliquots were resuspended in
2.5 M NaCl and maintained at 37°C for 4 h.
Long-term stationary-phase survival was measured by growing cultures
for 20 to 24 h at 37°C in 1.5 ml of M9 minimal medium
or LB
broth, using 15-ml plastic screw-cap tubes and a roller
drum for
aeration. At this point, aliquots were removed for "day
0" viable
counts. The cultures were then maintained in the original
culture
medium for an additional 10 days on the roller drum at
37°C, and
aliquots were removed for viable counts every 1 to 2
days. To determine
the effect of methanol (0.5 or 1%), paraquat
(0.1 or 0.25 mg/ml), or
salt (0.5 or 1 M) on the stationary-phase
cells, long-term cultures
were grown and maintained in LB broth
as described above. The desired
agent was added to the indicated
concentration on day 0, following the
removal of aliquots for
counting. The effect of pulsed heating was
determined by transferring
cultures maintained as above to a 42°C
incubator for 1 or 2 h
every 24 h; in this case, aliquots
were removed for counting prior
to heating.
GASP assay.
The ability of aged cells to outcompete fresh
overnight cultures (termed the GASP phenotype) was determined
essentially as described previously (33, 34). Parental and
mutant strains to be tested were marked with chromosomal antibiotic
resistance genes, grown overnight in LB broth, and maintained for an
additional 10 days in the same medium, with aeration. After 9 days,
viable counts were performed to determine the numbers of living cells remaining; on day 10, the aged cultures were diluted 1:1,000, based on
the number of live cells, into fresh overnight cultures of a parental
strain marked with a different antibiotic resistance. The mixture was
maintained for an additional 10 days, and aliquots were removed every 1 to 2 days, diluted, and plated on appropriate antibiotic-containing
media in order to determine the number of viable cells of each strain.
| |
RESULTS AND DISCUSSION |
The original pcm mutant strain shows
rpoS-like phenotypes that can be complemented by
rpoS but not pcm.
Previously, a strain with
pcm deleted was shown to be defective in stationary-phase
survival and in resistance to heat and to increased osmotic strength
(12, 13). Similar, though more severe, phenotypes are
characteristic of mutations in rpoS (encoding the sigma
factor S, a key positive regulator of the cellular
response to stationary phase), which is located only 1.5 kb downstream
of pcm. As additional phenotypes, including resistance to
heat and osmotic stress, were investigated, a more significant overlap
was observed (Table 2) between the
phenotypes of the original pcm mutant (CL1010) and those of
an rpoS mutant (JV1012). The relatively small difference in
oxidative stress resistance between CL1010 and the Pcm+
parent strain, MC1000, noted previously (12) became much
more pronounced upon increasing the incubation time to 60 min and the peroxide concentration to 30 mM (Table 2). Lowered resistance to
peroxide is one of the characteristic phenotypes of an rpoS mutation, because S directs the transcription of the
stationary-phase catalase, HPII (15). We also observed
little difference between the pcm and rpoS
mutants in survival of lethal heat shock (55°C) or treatment with 2.5 M NaCl (Table 2).
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Enzyme activities and short-term stress survival of
original and new pcm mutants and complemented strains
|
|
We have recently determined that this previously characterized
pcm mutant in fact carries an amber mutation in
rpoS derived
from the JC7623 strain used in its construction
and inadvertently
cotransduced into the MC1000 background
(
31). This allele, now
designated
rpoS396,
reduces RpoS activity, as measured by
S-dependent HPII
catalase activity at stationary phase, to about
10% of that in MC1000
(Table
2). Complementation studies were
performed to determine whether
the phenotypes observed for CL1010
were due to the
pcm
mutation, the
rpoS mutation, or the combination.
Previously,
efforts at plasmid complementation had yielded ambiguous
results which
might be attributed to the difficulty of ensuring
plasmid maintenance
in long-term stationary-phase cultures, so
we inserted a wild-type copy
of
pcm or
rpoS into the bacteriophage
lambda
attachment site (
attB) on the
E. coli chromosome
(strains
JV1026 and HC1011, respectively) for these experiments. The
complementing
copy of
pcm restored isoaspartyl
methyltransferase activity to
at least 50% of normal without affecting
HPII catalase activity;
similarly, the complementing
rpoS
restored HPII to wild-type levels
but did not change the Pcm activity
(Table
2).
When cultures were grown to stationary phase in minimal medium and
monitored by plate counts of viable cells for 10 days (Fig.
1), the
pcm+
complementing strain (JV1026) did not show any significant improvement
in survival over the
pcm mutant (CL1010). However, the
introduction
of a functional
rpoS gene restored
stationary-phase survival to
wild-type levels (HC1011). Similarly, the
reduced survival of
heat, osmotic, and oxidative stresses observed for
the
pcm mutant
strain was not relieved by a functional copy
of
pcm (Table
2,
compare CL1010 to JV1026) but the
phenotypes were complemented
by the
rpoS+
construct (Table
2, HC1011). Complementation with
rpoS alone
thus appears to be sufficient to overcome the survival defects
of
CL1010. These results suggest that the amber mutation in
rpoS is responsible for the phenotypes previously reported
for
pcm mutants by Li and Clarke (
12).
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 1.
Stationary-phase survival of pcm mutants and
complemented strains. Cultures of the indicated strains were grown in
M9 medium for 24 h (day 0), and the number of viable cells
remaining was determined at intervals for the next 10 days. The number
of CFU is shown as a percentage of the maximum number of CFU; the
cultures did not always reach their maximum levels within the first
24 h. Survival of the original pcm deletion mutant
strain, CL1010 (-- --) is shown
compared to that of its parent, MC1000 ( ), to those of strains
complemented with a chromosomal wild-type pcm (JV1026) ( )
or rpoS (HC1011) ( ) gene, and to that of a newly
constructed pcm deletion mutant strain, JV1068
( ).
|
|
A newly constructed pcm mutant survives normally in
stationary phase.
In order to determine whether the pcm
defect made any contribution to the observed phenotypes, a new
pcm mutant was constructed which was free from the secondary
rpoS mutation. This was accomplished by using a suicide
vector to integrate a near-complete deletion of the pcm gene
directly into the chromosome of MC1000 (see Materials and Methods), a
strain in which the chromosomal rpoS had been sequenced and
shown to be wild type (31). The resulting strain (JV1068)
had no detectable Pcm activity (Table 2).
As shown in Fig.
1, the new
pcm mutant survived long-term
maintenance in minimal medium as successfully as the parental strain;
this was also the case when the cells were grown in LB broth or
when
the experiment was extended to 40 days (data not shown).
When tested
for viability after 55°C heat shock, treatment with
2.5 M NaCl for
4 h, or treatment with 15 or 30 mM H
2O
2
for 1 h,
the mutants again showed no survival defects (Table
2).
These
results demonstrate that the original phenotypes attributed to
the
pcm mutation were indeed due solely to the secondary
rpoS mutation. Furthermore, despite the physical proximity
of
pcm to
rpoS, its disruption had no effect on
S activity as judged by measurement of HPII catalase
(Table
2)
or by means of
rpoS-lacZ translational fusions
(data not shown).
Although we would expect isoaspartyl damage to reach significant levels
during this long incubation in stationary phase, inactivation
of the
only known repair mechanism results in no detectable loss
of viability
under these conditions. Thus, we must conclude that
unless this damage
is being repaired by some other means, the
formation of isoaspartyl
residues is not in itself terribly detrimental
to
E. coli
under otherwise stable conditions. We therefore became
interested in
determining whether the significance of the isoaspartyl
residues might
increase when the bacteria encounter changing environmental
conditions.
Susceptibility of pcm mutants to environmental stresses
in extended stationary-phase cultures.
When subjected to
short-term environmental stresses (Table 2) or to conditions which
reduce culture viability very quickly (e.g., desiccation, maintenance
at 42°C, or exposure to chloramphenicol [data not shown]), there
was no observable difference between the survival of the parental
strain and that of the pcm mutant JV1068. We therefore
concentrated on subjecting stationary-phase cultures to conditions
which reduced the viability of the cultures more slowly, allowing time
for isoaspartyl formation. Initially, we found two stresses which
differentially affected the survival of the parental and pcm
mutant strains: low concentrations of either methanol or paraquat.
To test the effect of methanol, strains were grown to stationary phase
in LB broth and then methanol was added to a final
concentration of
0.5% (vol/vol; 156 mM). Under these conditions,
the parental strain,
MC1000, survived nearly as well as it did
when no methanol was added
(Fig.
2). The
pcm mutant
(JV1068),
however, showed a distinct and reproducible drop in
viability:
within 2 days, viable cells in the mutant culture were
reduced
to about 40% of the wild-type level. Although both strains
declined
gradually over time, survival of the mutant cells remained at
about 20 to 40% of the level of wild-type cells for several days.
The
difference between the two strains was smaller toward the
end of the
10-day period, presumably because the concentration
of methanol (or
some toxic product) was gradually reduced by evaporation
and/or
metabolic activity.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 2.
Effect of methanol on stationary-phase survival of the
pcm mutant strain JV1068. Methanol was added to 0.5%
(vol/vol) to cultures grown overnight in LB broth, and the number of
viable cells remaining was monitored for 10 days thereafter. The points
are shown as the percentage of the day 0 CFU remaining viable and are
averages of five trials: the error bars represent 1 standard deviation.
The strains used were as follows: the untreated parental strain
(untreated MC1000) (), a methanol-treated parental strain (MC1000)
( ), a pcm mutant (JV1068) (), and a pcm
mutant complemented with
attB::pcm+ (JV1083)
().
|
|
This survival defect could be readily complemented either by a
wild-type
pcm gene integrated into the chromosome at
attB (Fig.
2, JV1083) or by a plasmid-encoded copy (data not
shown). Both
of these complementing strains survived as well as MC1000.
Increasing
the methanol concentration to 1% did not greatly affect the
outcome
of the experiment, while 5% methanol was sufficient to cause a
rapid loss of viability in both wild-type and mutant strains,
to the
extent that no viable cells were recoverable after about
5 days (data
not shown). Neither
n-butanol nor isopropanol had
a
differential effect on the
pcm mutant at the same
concentrations
(0.5 or 1%) as used for methanol (data not shown).
The longevity of the
pcm mutant was also significantly
compromised when we added paraquat to the long-term stationary-phase
cultures. Paraquat, a redox-cycling drug, results in the production
of
reactive oxygen species, including hydrogen peroxide and superoxide
and
hydroxyl radicals (
10). The advantage of this agent over
the
peroxide treatment we used in our short-term experiments is
that
paraquat can be repeatedly reduced and reoxidized and thus
produces
reactive oxygen species continuously, whereas oxidative
stress produced
by direct application of peroxide lasts only until
the peroxide is
inactivated by catalase.
When maintained in the presence of 0.1 mg of paraquat/ml (Fig.
3), the
pcm mutant (JV1068)
typically survived nearly as well
as the parental strain (MC1000) for
about 4 days, but then its
viability dropped dramatically to below the
limit of detection.
The parental strain was also affected by the
addition of paraquat
but could invariably be recovered from the
cultures after 10 days,
as could the
pcm mutant when
complemented with the
attB::
pcm+ construct
(JV1083). Increasing the paraquat concentration to
0.25 or 0.5 mg/ml
resulted in faster reduction in viability for
both strains, but the
mutant cultures again dropped rapidly to
undetectable levels while the
parental and complemented strains
remained recoverable for 2 to 4 additional days (data not shown).
A concentration of 1 mg/ml was
sufficient to completely kill both
cultures within 2 days (data not
shown). Paraquat had no effect
on either strain when cultures were
grown in tightly closed tubes
filled to the cap with growth medium to
limit oxygen (data not
shown), supporting the idea that paraquat's
effect is indeed related
to oxygen radical formation. These results
suggest that the
L-isoaspartyl
methyltransferase is
required for survival when cells are under
continual oxidative stress.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 3.
Effect of paraquat on stationary-phase survival of the
pcm mutant strain JV1068. Paraquat was added to a
concentration of 0.1 mg/ml to cultures grown overnight in LB broth, and
the number of viable cells remaining was monitored for 10 days
thereafter. The points are shown as logs of the percentage of the day 0 CFU remaining viable (averages of at least five trials) for the
untreated parental strain (untreated MC1000) () and for the
paraquat-treated parental strain (MC1000) (), a pcm
mutant (JV1068) (), and a pcm mutant complemented with
attB::pcm+ (JV1083) ().
Viable JV1068 cells remaining on days 8 and 10 were below the limit of
detection, which was approximately 50 cells/ml.
|
|
Interestingly, neither methanol (up to 1%) nor paraquat (up to 0.5 mg/ml) appeared to reduce the viability of the mutant when
grown in
minimal medium rather than in LB broth (data not shown).
This may be
attributable to the development of a generally higher
level of stress
resistance when grown in minimal medium (
24),
or the
phenotypes may be dependent on the more dynamic nature
of LB cultures
or the higher metabolic rates of stationary-phase
cells in LB broth
(
33).
One explanation for the effect of methanol would be toxicity resulting
from the formation of formaldehyde, one immediate oxidation
product of
methanol; however, we found no differential effect
of formaldehyde
(0.005 or 0.015%) on
pcm mutants (data not shown).
Another
possibility would be that methanol increases protein denaturation.
Like
ethanol (
28), whose known effects include protein unfolding
(
4,
21), methanol can induce the transcription of heat shock
genes in
E. coli (
29). This is an attractive idea
because denaturation
is also one of the effects of oxidative stress on
proteins (
6),
providing a link between the two phenotypes
described above. If
this hypothesis were correct, we would expect other
denaturing
stresses, such as ethanol, heat, or osmotic stress (
1,
21,
27), to have a stronger effect on
pcm than on the
parental strain.
The
pcm mutant did not show impaired survival when ethanol
was added to stationary-phase cultures at a concentration of 1
or
5%
indeed, ethanol-treated cultures of either MC1000 or JV1068
actually survived better than untreated cultures (data not shown).
Presumably, this occurs because alcohol dehydrogenase, expressed
at low
levels under aerobic conditions (
3), permits the
nutrient-limited
bacteria to obtain some carbon and energy by
metabolizing the
ethanol. However, stationary-phase cultures treated
with 0.5 M
NaCl (Fig.
4A) showed a
phenotype similar to that seen with methanol.
Viability of the
pcm mutant (JV1068) decreased by a small amount
relative to
that of the parent strain; the decrease was repeatable
and could be
complemented by a wild-type
pcm gene (JV1083). Increasing
the salt concentration to 1 M gave similar results (data not shown).
Although incubating the cultures continuously at 42°C resulted
in
rapid loss of viability for both strains, shifting them to
42°C for
2 h every day again produced a repeatable difference
between the
two strains (Fig.
4B), which could be complemented
by introducing a
functional copy of
pcm.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 4.
Effects of osmotic and heat stresses on stationary-phase
survival of the pcm mutant strain JV1068. (A) Survival curve
showing long-term maintenance in LB broth of the untreated parental
strain (untreated MC1000) () and the effect of adding NaCl to 0.5 M
on the parental strain (MC1000) (), the pcm mutant
(JV1068) (), and the pcm mutant complemented with
attB::pcm+ (JV1083) ().
The points shown are averages of six trials; the error bars represent 1 standard deviation. (B) Survival curve for the untreated parental
strain (untreated MC1000) () and the effect of heating the parental
strain (MC1000) (), the pcm mutant (JV1068) (), and
the complemented pcm mutant (JV1083) () to 42°C for
2 h per day. The points shown are averages of at least three
trials; the error bars represent 1 standard deviation.
|
|
When an isoaspartyl residue forms, it introduces a kink into the
polypeptide backbone, due to the routing of the backbone
through what
was formerly the side chain of an aspartyl or asparaginyl
residue and
the consequent placement of an extra methylene group
between two
adjacent residues (see reference
30 for a review).
This might alter the protein's conformation sufficiently to increase
its susceptibility to subsequent denaturation resulting from heat,
oxidative attack, or other stresses, or the isoaspartyl damage
might
inhibit refolding once the protein becomes denatured. Alternatively,
the presence of isoaspartyl residues might expose new sites to
oxidative or other damage, or conversely, partial unfolding by
environmental agents might make aspartyl or asparaginyl residues
which
were previously buried accessible to the solvent, increasing
the amount
of isoaspartyl damage. Further experiments will be
needed to evaluate
the ability of these hypotheses to account
for the synergistic effect
of stress and
pcm mutations.
Deletion of pcm affects the GASP phenotype.
E.
coli cultures aged for 10 days in LB broth acquire mutations which
appear to enhance their ability to maintain metabolic activity under
starvation conditions. When mixed with fresh overnight cultures of the
same strain, the aged cells outcompete the younger ones and take over
the culture within a few days, a phenomenon termed the GASP phenotype
(33, 34). We asked whether this competitive situation might
provide a means of identifying subtle defects in long-term survival
resulting from the pcm mutation.
The MC1000 parental strain, the
pcm mutant JV1068, and the
attB::
pcm complementing strain JV1083
were marked by transducing
the antibiotic resistance marker from
strains carrying either
a Tn
10 (Tc
r) or
Tn
5 (Km
r) insertion in the
malE locus
(see Materials and Methods) (Table
1). The resulting strains were grown
to stationary phase in LB
broth and then aged for 10 days. Aliquots of
the aged cultures
were added at a 1:1,000 ratio to fresh overnight
cultures of either
the Tc
r or Km
r derivative of
MC1000 so that each resulting culture contained
one Tc
r
strain and one Km
r strain. Plate counts were done to
monitor the number of viable
cells of each type.
As shown in Fig.
5A, aged parental cells
exhibited the GASP phenotype and became dominant in the cultures within
10 days,
as did the
pcm mutants with the complementing
attB::
pcm+ construction
(Fig.
5B). Although there was some variability in
the time required for
this shift to occur, the numbers of the
two strains reached equality in
3 to 6 days, and the aged cells
were invariably in the majority after
10 days. In a total of seven
trials for MC1000 and six trials for the
complementing strain,
no exceptions were seen. When the aged competitor
was the
pcm mutant, however, the results were much more
variable; three representative
outcomes from eight trials are shown in
Fig.
5C to E. In two of
the eight cultures, the aged
pcm
mutants demonstrated GASP phenotypes
similar to that seen for the wild
type (Fig.
5C). However, in
four cases, the mutants were delayed in
reaching equality with
the fresh MC1000 (Fig.
5D), and in two
experiments, they failed
to become the dominant strain at all (Fig.
5E).
View larger version (11K):
[in this window]
[in a new window]
|
FIG. 5.
Deletion of pcm affects the development of
the GASP phenotype. A culture of the competitor strain of interest
(open symbols) which had been maintained in stationary phase for 10 days was mixed in a 1:1,000 ratio with a fresh overnight culture of
JV1094 (Kmr parental strain; closed symbols). The
competitor strains were as follows: (A) aged JV1090 (Tcr
parental strain); (B) aged JV1098 (Tcr pcm
mutant complemented by
attB::pcm+); (C to E) aged
JV1093 (Tcr pcm mutant). Each panel represents
the result of a single experiment, representative of at least six
experiments (A and B) or typical of the three different types of
outcomes observed for the pcm mutants in eight experiments
(C to E). Results similar to those shown in panels C and E were
observed twice each, while results similar to those shown in panel D
were observed four times.
|
|
The reduced ability of
pcm mutants to display the GASP
phenotype suggests that although we were unable to detect a direct
effect of the Pcm repair mechanism on the viability of unstressed
cells
during stationary phase, it may nonetheless provide an important
competitive advantage. The need for strains lacking the
methyltransferase
to spend more metabolic energy in degrading and
replacing damaged
proteins in order to maintain key cellular functions
may affect
their competitive ability. Another possibility is that
wild-type
strains may be able to respond more rapidly to any nutrients
which
become available, damage to their key enzymes having already been
repaired. The variability noted in this experiment is not unexpected,
since the particular mutations which occur or are selected for
during
the aging period would affect the outcome of the experiment,
as would
the particular proteins which become damaged by the formation
of
isoaspartyl residues. In view of this inherent randomness,
the fact
that aged
pcm mutants did not reliably take over the
culture
while aged MC1000 never failed to do so seems to suggest
a clear
reduction in competitive ability which could certainly
provide a
significant selective advantage for Pcm
+ bacteria.
Summary.
Since a rapidly dividing E. coli cell can
produce enough material for an entirely new cell in as little as 20 min, there might seem to be little point in repairing any polypeptide
species that become damaged. However, protein synthesis represents a
significant expenditure of energy for E. coli and other
bacteria, and they apparently make a considerable investment in protein
repair systems as well. The best-known examples of such protein repair
are the refolding and disaggregation functions performed by chaperones (see reference 22 for a review) and the enzymatic
reduction of methionine sulfoxides to their original form by the
peptide methionine sulfoxide reductase (18). The necessity
for such mechanisms becomes more obvious when one considers the
oligotrophic environments in which E. coli spends most of
its time when not fortunate enough to encounter a vertebrate host. When
protein synthesis is constrained by limited nutrient availability,
suboptimal temperatures, or other environmental conditions, existing
proteins become much more valuable. Thus, repair of any protein damage which can be recognized and corrected enzymatically, as is the case for
isoaspartyl formation, may represent a more frugal use of scarce
resources than the synthesis of new proteins.
The phenotypes which we have demonstrated here, using newly constructed
pcm mutants, are subtle, but they nevertheless point
to a
role for the isoaspartyl methyltransferase in the stationary-phase
longevity of
E. coli. The
pcm mutants showed
reduced viability
in stationary phase, but only when exposed to an
additional environmental
stress, such as methanol or paraquat. In this
sense, the Pcm repair
system might be somewhat analogous to the yeast
chaperone Hsp104,
whose greatest effect on survival is observed upon
exposure to
both heat and ethanol rather than in response to the
individual
stresses (
23).
Our observations are consistent with the idea that isoaspartyl damage
indeed contributes to the loss of viability in the
pcm mutants. They also suggest that such damage may be relatively
innocuous
in a fully folded protein under normal conditions but
become
detrimental to the cell under potentially denaturing conditions.
Future
experiments will examine the possible link between protein
unfolding
and isoaspartyl damage and investigate whether there
are particular
substrates for the Pcm methyltransferase whose
repair is most crucial.
| |
ACKNOWLEDGMENTS |
We thank P. C. Loewen (pMMkatF2), W. Messer (pLDR
plasmids), K. A. Skorupski (pKAS46), and M. Berlyn of the E. coli Genetic Stock Center (MC1000, TST1, TSM7) for making strains
available to us. We are grateful to Jeffrey Ichikawa and other members
of the Clarke laboratory for reading the manuscript and for helpful discussions.
This work was supported by grant GM26020 from the National Institutes
of Health (S.C. and H.C.) and NIH Postdoctoral Fellowship AG05684
(J.E.V.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Chemistry and Biochemistry, Box 951569, Los Angeles, CA 90095-1569. Phone: (310) 825-8754. Fax: (310) 825-1968. E-mail:
clarke{at}ewald.mbi.ucla.edu.
| |
REFERENCES |
| 1.
|
Beckmann, R. P.,
M. Lovett, and W. J. Welch.
1992.
Examining the function and regulation of hsp 70 in cells subjected to metabolic stress.
J. Cell Biol.
117:1137-1150[Abstract/Free Full Text].
|
| 2.
|
Casadaban, M. J., and S. N. Cohen.
1980.
Analysis of gene control signals by DNA fusion and cloning in Escherichia coli.
J. Mol. Biol.
138:179-207[Medline].
|
| 3.
|
Clark, D., and J. E. Cronan, Jr.
1980.
Escherichia coli mutants with altered control of alcohol dehydrogenase and nitrate reductase.
J. Bacteriol.
141:177-183[Abstract/Free Full Text].
|
| 4.
|
Clark, D. P., and J. P. Beard.
1979.
Altered phospholipid composition in mutants of Escherichia coli sensitive or resistant to organic solvents.
J. Gen. Microbiol.
113:267-274[Abstract/Free Full Text].
|
| 5.
|
Clarke, S.
1987.
Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins.
Int. J. Pept. Protein Res.
30:808-821[Medline].
|
| 6.
|
Davies, K. J. A.,
S. W. Lin, and R. E. Pacific.
1987.
Protein damage and degradation by oxygen radicals. IV. Degradation of denatured proteins.
J. Biol. Chem.
262:9914-9920[Abstract/Free Full Text].
|
| 7.
|
Diederich, L.,
L. J. Rasmussen, and W. Messer.
1992.
New cloning vectors for integration into the attachment site attB of the Escherichia coli chromosome.
Plasmid
28:14-24[Medline].
|
| 8.
|
Fu, J. C.,
L. Ding, and S. Clarke.
1991.
Purification, gene cloning, and sequence analysis of an l-isoaspartyl protein carboxyl methyltransferase from Escherichia coli.
J. Biol. Chem.
266:14562-14572[Abstract/Free Full Text].
|
| 9.
|
Gussow, D., and T. Clackson.
1989.
Direct clone characterization from plaques and colonies by the polymerase chain reaction.
Nucleic Acids Res.
17:4000[Free Full Text].
|
| 10.
|
Hassan, H. M., and I. Fridovich.
1979.
Paraquat and Escherichia coli.
J. Biol. Chem.
254:10846-10852[Abstract/Free Full Text].
|
| 11.
|
Kim, E.,
J. D. Lowenson,
D. C. MacLaren,
S. Clarke, and S. G. Young.
1997.
Deficiency of a protein-repair enzyme results in the accumulation of altered proteins, retardation of growth, and fatal seizures in mice.
Proc. Natl. Acad. Sci. USA
94:6132-6137[Abstract/Free Full Text].
|
| 12.
|
Li, C., and S. Clarke.
1992.
A protein methyltransferase specific for altered aspartyl residues is important in Escherichia coli stationary-phase survival and heat-shock resistance.
Proc. Natl. Acad. Sci. USA
89:9885-9889[Abstract/Free Full Text].
|
| 13.
|
Li, C.,
J. K. Ichikawa,
J. J. Ravetto,
H.-C. Kuo,
J. C. Fu, and S. Clarke.
1994.
A new gene involved in stationary-phase survival located at 59 minutes on the Escherichia coli chromosome.
J. Bacteriol.
176:6015-6022[Abstract/Free Full Text].
|
| 14.
|
Loewen, P. C., and R. Hengge-Aronis.
1994.
The role of the sigma factor S (KatF) in bacterial global regulation.
Annu. Rev. Microbiol.
48:53-80[Medline].
|
| 15.
|
Loewen, P. C., and B. L. Triggs.
1984.
Genetic mapping of katF, a locus that with katE affects the synthesis of a second catalase species in Escherichia coli.
J. Bacteriol.
160:668-675[Abstract/Free Full Text].
|
| 16.
|
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr, and R. J. Randall.
1951.
Protein measurement with the folin phenol reagent.
J. Biol. Chem.
193:265-275[Free Full Text].
|
| 17.
|
McFadden, P. N., and S. Clarke.
1987.
Conversion of isoaspartyl peptides to normal peptides by coupled enzymatic/nonenzymatic reactions: implications for the cellular repair of damaged proteins.
Proc. Natl. Acad. Sci. USA
84:2595-2599[Abstract/Free Full Text].
|
| 18.
|
Moskovitz, J.,
M. A. Rahman,
J. Strassman,
S. O. Yancey,
S. R. Kushner,
N. Brot, and H. Weissbach.
1995.
Escherichia coli peptide methionine sulfoxide reductase gene: regulation of expression and role in protecting against oxidative damage.
J. Bacteriol.
177:502-507[Abstract/Free Full Text].
|
| 19.
|
Mudgett, M. B., and S. Clarke.
1994.
Hormonal and environmental responsiveness of a developmentally regulated protein repair l-isoaspartyl methyltransferase in wheat.
J. Biol. Chem.
269:25605-25612[Abstract/Free Full Text].
|
| 20.
|
Mulvey, M. R.,
P. A. Sorby,
B. L. Triggs-Raine, and P. C. Loewen.
1988.
Cloning and physical characterization of katE and katF required for catalase HPII expression in Escherichia coli.
Gene
73:337-345[Medline].
|
| 21.
|
Nguyen, V. T.,
M. Morange, and O. Bensaude.
1989.
Protein denaturation during heat shock and related stress: Escherichia coli  -galactosidase and Photinus pyralis luciferase inactivation in mouse cells.
J. Biol. Chem.
264:10487-10492[Abstract/Free Full Text].
|
| 22.
|
Parsell, D. A., and S. Lindquist.
1993.
The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins.
Annu. Rev. Genet.
27:437-496[Medline].
|
| 23.
|
Sanchez, Y.,
J. Taulien,
K. A. Borkovich, and S. Lindquist.
1992.
Hsp104 is required for tolerance to many forms of stress.
EMBO J.
11:2357-2364[Medline].
|
| 24.
|
Siegele, D. A.,
M. Almirón, and R. Kolter.
1993.
Approaches to the study of survival and death in stationary-phase Escherichia coli, p. 151-169.
In
S. Kjelleberg (ed.), Starvation in bacteria. Plenum Press, New York, N.Y.
|
| 25.
|
Silhavy, T. J.,
M. L. Berman, and L. W. Enquist.
1984.
In
Experiments with gene fusions.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 26.
|
Skorupski, K., and R. K. Taylor.
1996.
Positive selection vectors for allelic exchange.
Gene
169:47-52[Medline].
|
| 27.
|
Tanford, C.
1968.
Protein degradation.
Adv. Protein Chem.
23:121-282[Medline].
|
| 28.
|
VanBogelen, R. A.,
P. M. Kelley, and F. C. Neidhardt.
1987.
Differential induction of heat shock, SOS, and oxidation stress regulons and accumulation of nucleotides in Escherichia coli.
J. Bacteriol.
169:26-32[Abstract/Free Full Text].
|
| 29.
|
Van Dyk, T. K.,
D. R. Smulski,
T. R. Reed,
S. Belkin,
A. C. Vollmer, and R. A. LaRossa.
1995.
Responses to toxicants of an Escherichia coli strain carrying a uspA'::lux genetic fusion and an E. coli strain carrying a grpE'::lux fusion are similar.
Appl. Environ. Microbiol.
61:4124-4127[Abstract].
|
| 30.
|
Visick, J. E., and S. Clarke.
1995.
Repair, refold, recycle: how bacteria can deal with spontaneous and environmental damage to proteins.
Mol. Microbiol.
16:835-845[Medline].
|
| 31.
|
Visick, J. E., and S. Clarke.
1997.
RpoS- and OxyR-independent induction of HPI catalase at stationary phase in Escherichia coli and identification of rpoS mutations in common laboratory strains.
J. Bacteriol.
179:4158-4163[Abstract/Free Full Text].
|
| 32.
|
Watson, K.
1990.
Microbial stress proteins.
Adv. Microb. Physiol.
31:183-223[Medline].
|
| 33.
|
Zambrano, M. M., and R. Kolter.
1996.
GASPing for life in stationary phase.
Cell
86:181-184[Medline].
|
| 34.
|
Zambrano, M. M.,
D. A. Siegele,
M. Almirón,
A. Tormo, and R. Kolter.
1993.
Microbial competition: Escherichia coli mutants that take over stationary phase cultures.
Science
259:1757-1760[Abstract/Free Full Text].
|
J Bacteriol, May 1998, p. 2623-2629, Vol. 180, No. 10
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Oge, L., Bourdais, G., Bove, J., Collet, B., Godin, B., Granier, F., Boutin, J.-P., Job, D., Jullien, M., Grappin, P.
(2008). Protein Repair L-Isoaspartyl Methyltransferase1 Is Involved in Both Seed Longevity and Germination Vigor in Arabidopsis. Plant Cell
20: 3022-3037
[Abstract]
[Full Text]
-
Banfield, K. L., Gomez, T. A., Lee, W., Clarke, S., Larsen, P. L.
(2008). Protein-Repair and Hormone-Signaling Pathways Specify Dauer and Adult Longevity and Dauer Development in Caenorhabditis elegans. Journals of Gerontology Series A: Biological Sciences and Medical Sciences
63: 798-808
[Abstract]
[Full Text]
-
Rath, D., Jawali, N.
(2006). Loss of Expression of cspC, a Cold Shock Family Gene, Confers a Gain of Fitness in Escherichia coli K-12 Strains.. J. Bacteriol.
188: 6780-6785
[Abstract]
[Full Text]
-
Chourey, K., Thompson, M. R., Morrell-Falvey, J., VerBerkmoes, N. C., Brown, S. D., Shah, M., Zhou, J., Doktycz, M., Hettich, R. L., Thompson, D. K.
(2006). Global Molecular and Morphological Effects of 24-Hour Chromium(VI) Exposure on Shewanella oneidensis MR-1. Appl. Environ. Microbiol.
72: 6331-6344
[Abstract]
[Full Text]
-
Hicks, W. M., Kotlajich, M. V., Visick, J. E.
(2005). Recovery from long-term stationary phase and stress survival in Escherichia coli require the L-isoaspartyl protein carboxyl methyltransferase at alkaline pH. Microbiology
151: 2151-2158
[Abstract]
[Full Text]
-
Kern, R., Malki, A., Abdallah, J., Liebart, J.-C., Dubucs, C., Yu, M. H., Richarme, G.
(2005). Protein Isoaspartate Methyltransferase Is a Multicopy Suppressor of Protein Aggregation in Escherichia coli. J. Bacteriol.
187: 1377-1383
[Abstract]
[Full Text]
-
Xu, Q., Belcastro, M. P., Villa, S. T., Dinkins, R. D., Clarke, S. G., Downie, A. B.
(2004). A Second Protein L-Isoaspartyl Methyltransferase Gene in Arabidopsis Produces Two Transcripts Whose Products Are Sequestered in the Nucleus. Plant Physiol.
136: 2652-2664
[Abstract]
[Full Text]
-
Flashner, Y., Mamroud, E., Tidhar, A., Ber, R., Aftalion, M., Gur, D., Lazar, S., Zvi, A., Bino, T., Ariel, N., Velan, B., Shafferman, A., Cohen, S.
(2004). Generation of Yersinia pestis Attenuated Strains by Signature-Tagged Mutagenesis in Search of Novel Vaccine Candidates. Infect. Immun.
72: 908-915
[Abstract]
[Full Text]
-
Kindrachuk, J., Parent, J., Davies, G. F., Dinsmore, M., Attah-Poku, S., Napper, S.
(2003). Overexpression of L-Isoaspartate O-Methyltransferase in Escherichia coli Increases Heat Shock Survival by a Mechanism Independent of Methyltransferase Activity. J. Biol. Chem.
278: 50880-50886
[Abstract]
[Full Text]
-
Heinz, E. B., Streit, W. R.
(2003). Biotin Limitation in Sinorhizobium meliloti Strain 1021 Alters Transcription and Translation. Appl. Environ. Microbiol.
69: 1206-1213
[Abstract]
[Full Text]
-
Athmer, L., Kindrachuk, J., Georges, F., Napper, S.
(2002). The Influence of Protein Structure on the Products Emerging from Succinimide Hydrolysis. J. Biol. Chem.
277: 30502-30507
[Abstract]
[Full Text]
-
Chavous, D. A., Jackson, F. R., O'Connor, C. M.
(2001). Extension of the Drosophila lifespan by overexpression of a protein repair methyltransferase. Proc. Natl. Acad. Sci. USA
10.1073/pnas.251446498v1
[Abstract]
[Full Text]
-
Thapar, N., Kim, A.-K., Clarke, S.
(2001). Distinct Patterns of Expression But Similar Biochemical Properties of Protein L-Isoaspartyl Methyltransferase in Higher Plants. Plant Physiol.
125: 1023-1035
[Abstract]
[Full Text]
-
Riehle, M. M., Bennett, A. F., Long, A. D.
(2001). Genetic architecture of thermal adaptation in Escherichia coli. Proc. Natl. Acad. Sci. USA
10.1073/pnas.021448998v1
[Abstract]
[Full Text]
-
Mason, C. A., Dünner, J., Indra, P., Colangelo, T.
(1999). Heat-Induced Expression and Chemically Induced Expression of the Escherichia coli Stress Protein HtpG Are Affected by the Growth Environment. Appl. Environ. Microbiol.
65: 3433-3440
[Abstract]
[Full Text]
-
Cai, H., Clarke, S.
(1999). A Novel Methyltransferase Catalyzes the Methyl Esterification of trans-Aconitate in Escherichia coli. J. Biol. Chem.
274: 13470-13479
[Abstract]
[Full Text]
-
David, C. L., Keener, J., Aswad, D. W.
(1999). Isoaspartate in Ribosomal Protein S11 of Escherichia coli. J. Bacteriol.
181: 2872-2877
[Abstract]
[Full Text]
-
Berlyn, M. K. B.
(1998). Linkage Map of Escherichia coli K-12, Edition 10: The Traditional Map. Microbiol. Mol. Biol. Rev.
62: 814-984
[Abstract]
[Full Text]
-
Thapar, N., Griffith, S. C., Yeates, T. O., Clarke, S.
(2002). Protein Repair Methyltransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus. UNUSUAL METHYL-ACCEPTING AFFINITY FOR D-ASPARTYL AND N-SUCCINYL-CONTAINING PEPTIDES. J. Biol. Chem.
277: 1058-1065
[Abstract]
[Full Text]
-
Riehle, M. M., Bennett, A. F., Long, A. D.
(2001). Genetic architecture of thermal adaptation in Escherichia coli. Proc. Natl. Acad. Sci. USA
98: 525-530
[Abstract]
[Full Text]
-
Chavous, D. A., Jackson, F. R., O'Connor, C. M.
(2001). Extension of the Drosophila lifespan by overexpression of a protein repair methyltransferase. Proc. Natl. Acad. Sci. USA
98: 14814-14818
[Abstract]
[Full Text]
Top
Abstract
Introduction
