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Journal of Bacteriology, April 2001, p. 2165-2171, Vol. 183, No. 7
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.7.2165-2171.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Viability of rep recA Mutants Depends on
Their Capacity To Cope with Spontaneous Oxidative Damage and on the
DnaK Chaperone Protein
Marie-Florence
Bredèche,
S. Dusko
Ehrlich, and
Bénédicte
Michel*
Laboratoire de Génétique
Microbienne, Institut National de la Recherche Agronomique, Domaine
de Vilvert, F-78352 Jouy en Josas Cedex, France
Received 1 September 2000/Accepted 11 January 2001
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ABSTRACT |
Replication arrests due to the lack or the inhibition of
replicative helicases are processed by recombination proteins.
Consequently, cells deficient in the Rep helicase, in which replication
pauses are frequent, require the RecBCD recombination complex for
growth. rep recA mutants are viable and display no growth
defect at 37 or 42°C. The putative role of chaperone proteins in
rep and rep recA mutants was investigated by
testing the effects of dnaK mutations. dnaK756
and dnaK306 mutations, which allow growth of otherwise wild-type Escherichia coli cells at 40°C, are lethal in
rep recA mutants at this temperature. Furthermore, they
affect the growth of rep mutants, and to a
lesser extent, that of recA mutants. We conclude that both
rep and recA mutants require DnaK for
optimal growth, leading to low viability of the triple (rep recA
dnaK) mutant. rep recA mutant cells form
colonies at low efficiency when grown to exponential phase
at 30°C. Although the plating defect is not observed at a high
temperature, it is not suppressed by overexpression of heat shock
proteins at 30°C. The plating defect of rep recA
mutant cells is suppressed by the presence of catalase in the plates.
The cryosensitivity of rep recA mutants therefore results from an increased sensitivity to oxidative damage upon propagation at low temperatures.
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INTRODUCTION |
Interconnections between DNA
replication and homologous recombination have been observed in a number
of organisms and are likely to play an important role in the
maintenance of genome integrity (reviewed in references 19, 21,
and 29). An additional link was found with the observation that
recombination enzymes act in Escherichia coli to rescue
blocked replication forks (40). rep mutants
were used to study the fate of replication forks upon blockage. The
rep mutation causes a slow progression of chromosomal replication forks which suggests the occurrence of frequent pauses (6, 23). Because the Rep helicase is able to displace a
DNA-bound protein in vitro, it was proposed that in vivo Rep could
facilitate chromosomal replication by dislodging DNA-bound proteins
from the path of the replication forks (28, 46).
rep mutants require the recombination complex RecBCD for
viability (43), suggesting a link between replication fork
arrest and homologous recombination. RecBCD initiates homologous
recombination of linear DNA and is therefore essential for the repair
of DNA double-strand breaks. It binds to DNA double-strand ends and
opens while simultaneously degrading the DNA. Upon encounter with
a specific sequence named CHI, RecBCD promotes the formation of single-stranded DNA recognized by RecA (reviewed in references 20, 25, and 33). rep recBC mutant lethality
results from the occurrence of RuvABC-dependent DNA double-strand
breaks (30, 40). The RuvAB proteins bind to Holliday
junctions and catalyze branch migration. The RuvAB-bound DNA is
cleaved by RuvC, which resolves the recombination intermediates by
introducing nicks in strands of opposite polarity (reviewed in
reference 44). To account for the action of RuvABC at
blocked replication forks, a model was proposed in which, upon
replication arrest, a Holliday junction forms by annealing of the
two nascent strands (40). In the absence of RecBCD,
resolution of the RuvAB-bound DNA by RuvC leads to chromosomal
breakage. In cells proficient for homologous recombination,
reincorporation of the double-strand tail formed by replication fork
reversal into the chromosome allows replication restart from a
recombination intermediate. However, rep recA mutants defective for homologous recombination are viable. The viability of
rep recA mutants depends on the exonuclease V activity of
RecBCD (40, 43); therefore, we proposed that in rep
recA mutants RecBCD may degrade the double-strand tail formed by
replication fork reversal, allowing replication restart from a
Y-structure.
In this work we further analyzed the properties of the rep
recA double mutant. We tested the effects of dnaK
mutations on the viability of rep recA mutants. DnaK is a
member of the Hsp70 family of stress-induced proteins which are highly
conserved in procaryotes and eucaryotes (1). It is one of
the major chaperone proteins induced by a shift to a high temperature
in E. coli. Like several other chaperone proteins, DnaK is
involved in processes that protect cells against various stresses and
plays a role in DNA replication (reviewed in reference
12). We report that dnaK mutations that do not
affect the viability of wild-type strains affect the growth of
rep mutants and are lethal in rep recA double mutants. This indicates that rep recA mutants depend on DnaK
for growth.
A peculiar property of the rep recA mutants remains
unexplained. Liquid cultures grown to exponential phase at 30°C
exhibit a defect in plating efficiency (43). A normal
plating efficiency is spontaneously recovered when cells reach the end
of exponential phase or if cells are grown at 37 or 42°C. The best
characterized origin of double-strand breaks is the presence of
oxidative compounds, a natural consequence of aerobic growth. DNA is
the primary site of lethal damages (16, 17, 24). As a
first line of defense against oxidative stress, bacterially encoded
catalases and superoxide dismutases prevent the accumulation of
reactive oxygen species (reviewed in references 8 and 10).
A second line of defense is a set of DNA repair enzymes. DNA damages
include mainly base modifications and DNA single-strand and
double-strand breaks (24; reviewed in reference
17). Consequently, cells that lack enzymes required for
recombinational or base-excision DNA repair pathways (RecA, RecB, PolA,
Xth) are killed by low doses of H2O2
(16). In contrast, the rep mutants are not more
sensitive to H2O2 than wild-type strains
(16). We explored the reason for the plating defect of
rep recA mutant cells grown at 30°C. The plating defect was not suppressed by overexpression of heat shock proteins at 30°C,
whereas it was suppressed by the presence of catalase in plates,
suggesting that it results from oxidative damage.
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MATERIALS AND METHODS |
Strains and media.
Strains used in this work are described
in Table 1. dnaK756 carries
three mutations in dnaK (31).
dnaK306 is a single mutant (45). Strains were
constructed by P1 transduction. rep, recA, and rep
recA derivatives of the dnaK306 and dnaK756
mutants were constructed at 30°C, and the isogenic rep
recA double mutants were constructed at 37°C (with an MC1061
background for the dnaK306 mutant and a C600 background for
the dnaK756 mutant, in contrast with the AB1157 background
used for Fig. 3). Transductants obtained by introduction of the
rep::kan allele in the
dnaK756 mutant originally exhibited variable plating
efficiencies at 40°C, but acquired during propagation at 30°C the
capacity to form about 100% small colonies at 40°C in 24 to 48 h. One such clone, which was a rep (defective for M13
replication) and dnaK756 (defective for 
growth) mutant
but which may have acquired a compensatory mutation facilitating its
propagation at 30°C, was used to perform the experiments reported
below. All other double mutants were obtained with the expected
efficiency and had the expected phenotype (see below). P1 transduction
of the (recA-srl)::Tn10 mutation in
the rep dnaK306 mutant strain led to very few clones, and
only one (recA-srl)::Tn10 clone was
obtained with the rep dnaK756 mutant strain. This may result
from the poor plating efficiency of these strains (see below). Since
rep recA dnaK mutants exhibited the expected phenotype (UV
sensitive, with an M13 replication defect and a growth defect),
they were used for further experiments. The Rep
phenotype
was verified by transformation of CaCl2 competent cells with M13mp2 DNA on a lawn of Hfr indicator strain, and recA
mutants were verified by measuring their UV sensitivity. The
dnaK756 mutation was verified as preventing growth of
wild-type phages and replication of the mini-F plasmid. The
dnaK306 mutation was verified as preventing mini-F
replication. Plasmids pMob45 (dnaKJ carried by pMob vector; McMacken Laboratory) and pNRK416 (dnaK under lacUV5 promoter
control; C. Gross Laboratory) were provided by Marie-Agnes Petit, and
pCG179 (ptac12HrpoH+) was provided by
Philippe Bouloc (42). Plasmid pDWS2, a pBR322 derivative
carrying the recBCD region (35), was provided
by G. Smith. Cells were grown in LBT medium (Luria broth [LB]
supplemented with 25 mg of thymidine per ml). Sigma catalase from
bovine liver was used at a final concentration of 150 U per ml.
Micrographs of bacteria.
Cell samples were fixed with 4%
paraformaldehyde phenylindole (Sigma, St. Louis, Mo.), deposited on the
slides, dried, colored with 4,6-diamidino-2-phenylindole (DAPI; Sigma)
(2.5 µg/ml), and added directly to glycerol-phosphate-buffered
saline mountant (Citifluor Ltd., Canterbury, United Kingdom).
Photographs of cells were taken using an epifluorescence microscope
(Nikon) with Sensia 400 film (Fuji). The slides were scanned and
processed with the Adobe Illustrator, 7.0, program (Edinburgh, Scotland).
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RESULTS |
dnaK mutations decrease the plating efficiencies of
rep recA mutants at 30°C.
We studied the effects of
mutations of the major heat-induced chaperone protein, DnaK, in
rep, recA, and rep recA mutants. Two
dnaK mutants, TS214 (dnaK306) and CAG9271
(dnaK756), that grow at 40°C were used (45).
Either rep, recA, or both rep and recA
null mutations were introduced in these two strains as well as in
isogenic dnaK+ strains (see Material and
Methods; Table 1). Cells were grown at 30°C to exponential phase and
plated at the same temperature (Table 2).
dnaK306 and dnaK756 mutations decreased the
plating efficiency of rep mutant cells three- to fivefold in
exponential and stationary phase. The term plating efficiency is used
here as the ratio of CFU to optical density (OD). This ratio depends on
(i) the average size of the cells (OD measures cell mass per ml) and
(ii) the ability of individual cells to give rise to a colony. To
further analyze the contribution of cell filamentation to the plating
defect of these mutants, cells in exponential growth were examined by
fluorescent microscopy and DAPI staining. Microscopic observations
indicated that filamentation participates in the loss of plating
efficiency for rep dnaK756 mutant cells (Fig. 1, compare panel g with panels a and c),
whereas rep dnaK306 mutant cells were not significantly more
elongated than dnaK306 single mutants (data not shown).
recA single mutants presented a 20 to 50% plating defect
compared to isogenic recA+ cells, as expected
(3) (Table 2). In recA mutant cells, the dnaK306 mutation decreased the plating efficiency twofold
without inducing significant filamentation (Table 2; data not shown), whereas the dnaK756 mutation had little effect (Table 2;
Fig. 1e).
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FIG. 1.
Morphology of dnaK756 derivatives. Cells were
grown overnight at 30°C, diluted 100-fold, and grown for 3 h at
either 30°C (a, c, e, g) or 42°C (b, d, f, h). JJC213
(rep mutant; a, b), CAG9271 (dnaK756 mutants; c,
d), JJC546 (dnaK756 recA mutant; e, f), and JJC547
(dnaK756 rep mutant; g, h) are shown. Wild-type cells were
similar to JJC213 at 42°C and to CAG9271 at 30°C (data not shown).
Bars, 1 µm.
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The combination of
rep and
recA mutations
decreased the plating efficiency of C600 cells in exponential phase and
not in stationary
phase, as previously observed in an AB1157 background
(
43) (compare
CAG9270 and JJC636 in Table
2). The plating
efficiency of the
MC1061
rep recA mutant (JJC530) was higher
(23% of that of the
isogenic wild-type strain MC1061 [Table
2]),
suggesting that
the plating defect of
rep recA mutants grown
to exponential phase
may depend on the cellular background. The plating
efficiency
of
rep recA mutant cells was further decreased
three- to sevenfold
by
dnaK306 or
dnaK756
mutations, in part because of filamentation,
since
dnaK756
induced in
rep recA mutant cells a level of filamentation
similar to that observed in
rep mutant cells (Fig.
1g) and
dnaK306 induced a lower level (data not shown). In
rep
recA double mutants,
the defect in the ability to form colonies is
not accompanied
by filamentation (data not shown). Interestingly,
plating efficiency
was not improved in overnight cultures of the
rep recA dnaK756 mutant, suggesting that DnaK may
participate in the recovery of
plating efficiency of the
rep
recA mutant upon saturation (compare
JJC636 and JJC677 overnight
cultures in Table
2).
Taken together, these results show that (i) growth of
rep,
and to a lesser extent,
recA mutant cells is affected at
30°C by
dnaK756 or
dnaK306 mutations (Fig.
1g;
Table
2, compare
recA with
recA dnaK306 mutants)
and (ii) both
dnaK306 and
dnaK756 mutations
affect the growth of
rep recA mutant cells at 30°C.
rep recA dnaK mutants are thermosensitive for
growth.
To measure the capacity of the various strains to form
colonies at a high temperature, overnight cultures were plated at 30 and 40°C (Table 3). For all strains the
number of colonies was similar at both temperatures, with the exception
of the two rep recA dnaK mutants, which did not form
colonies at 40°C (Table 3). This shows that in the two
dnaK mutants tested, the rep recA combination of
mutations renders cells thermosensitive for growth.
The plating efficiency of
rep dnaK and
recA dnaK
mutants was not affected by temperature (Table
3). However, at 40°C
colonies
were smaller than those of single
dnaK mutants
(Fig.
2). Microscopic
observations showed
that
dnaK756 mutant cells were mildly elongated
at 42°C
(Fig.
1d). The
recA mutation increased the number and
the
length of the filaments. For unknown reasons, it also increased
the
condensation of nucleoids (Fig.
1f). The
rep dnaK756 mutant
was found to be strongly filamentous (Fig.
1h). In contrast,
dnaK306 single mutants (TS154) were more elongated at 42°C
than
dnaK756 mutants (data not shown), and when introduced
in the
dnaK306 mutant,
the
rep and
recA mutations had a mild effect on cell morphology.
In
conclusion, our results indicate that both
recA and
rep mutants
require a physiological concentration of
chaperone proteins for
normal growth and that this requirement is
additive in
rep recA mutant cells.
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FIG. 2.
Colony growth of dnaK756
derivatives. CAG9271 (dnaK756 mutant; A), JJC546
(dnaK756 recA mutant; B), JJC547 (dnaK756 rep
mutant; C), and JJC677 (dnaK756 rep recA mutant; D) are
shown. Overnight cultures grown at 30°C were streaked on LB agar
thymine (LBAT), and plates were incubated overnight at 40°C. The size
of dnaK756 colonies was not significantly different from
that of wild-type colonies at this temperature (data not shown).
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Loss of plating of the rep recA mutants at 30°C
results from oxidative damage.
The only noticeable growth defect
of the rep recA mutant in laboratory conditions is a
variable but significant defect in colony forming efficiency when cells
in exponential phase at 30°C are plated on rich medium
(43) (Table 2; Fig. 3). The
plating defect is observed for liquid cultures propagated at 30°C
regardless of the temperature at which the plates are then incubated.
The low plating efficiency of the rep recA mutants is not
observed when cells are grown at 37 or 42°C, suggesting that a higher
temperature protects rep recA mutant cells. During growth at
42°C, the steady-state level of heat shock proteins is about twice as
high as that at 30°C (12), which could be sufficient to
restore a normal plating efficiency in rep recA mutants. In
order to test whether the defect in the plating efficiency of AB1157
rep recA mutants at 30°C could be restored by an increased
concentration in heat shock proteins, plasmids overexpressing DnaK
(pNRK416) or DnaK and DnaJ (pMob-dnaKJ) were introduced in
the rep recA mutant JJC356. Plating efficiency was not
restored by the presence of either of these plasmids (data not shown).
Hence, an increased level of DnaK and DnaJ is not sufficient to restore
full viability of rep recA mutants. However, DnaK controls
the expression of several other heat shock proteins (reviewed in
reference 12). These proteins are repressed by overexpression of DnaK, and some of them may be required in rep recA mutant cells. The hypothesis that loss of colony-forming ability of rep recA mutant cells at 30°C is because of
insufficient heat shock protein expression was therefore tested by
overproduction of 
32, the rpoH gene product, which
governs the heat shock response. The
ptac12HrpoH+ plasmid was used
(42). Overexpression of 32 did not restore the plasmid
efficiency of the rep recA mutant (data not shown). In
conclusion, increasing the level of heat shock proteins did not restore
the plating efficiency of the rep recA mutant, suggesting that heat shock proteins are not at a limiting concentration at 30°C
in the rep recA mutant cells.
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FIG. 3.
Growth curves of rep recA mutant (JJC502) at
30°C. Overnight cultures grown at 37°C were diluted to an OD of
0.03, and cells were incubated in LB at 30°C with shaking. Every
hour, the OD at 650 nm was measured, and 100 µl of an appropriate
dilution was plated on LBAT plates with or without catalase to
determine the number of CFU/ml. The defect of plating was variable,
from a plateau to a 100-fold decrease in CFU, while the
recovery on catalase-containing plates was always observed. An
experiment in which the decrease was of intermediate level is shown.
Triangles, OD at 650 nm; closed squares, CFU on LBAT; open squares, CFU
on LBAT containing 150 U of catalase per ml.
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The defect in plating of
rep recA mutant cells could result
from a limiting amount of an enzymatic activity essential for
the
viability of
rep recA mutant cells other than that of heat
shock proteins. RecBCD is a good candidate because (i) it is present
in
only 10 to 20 copies per cell (
20), (ii) it is essential
in
rep mutants, and (iii) it is titrated out in cells
defective
for RecA due to the extensive DNA degradation that occurs in
these
cells (
22,
41). A decrease in the amount of
available RecBCD
enzyme in vivo can be detected with the use of T4
phages deficient
for the gene 2 product that protects linear T4
molecules against
exonuclease V-mediated degradation upon infection
(
22,
38).
As previously reported (
22), we
observed that infection by T4
2
was increased by the
presence of a
recA mutation in the recipient
cells. However,
we found that it was not further modified by inactivation
of
rep and was not significantly influenced by temperature
(data
not shown). This suggests that
rep recA mutant cells
are not more
deficient in exonuclease V activity at 30°C than at
42°C and are
not more deficient than
recA single mutants.
To check more directly
whether a decrease in the concentration of free
RecBCD complexes
could be responsible for the plating defect of
rep recA mutants,
we measured the plating efficiency of
rep recA mutants containing
the plasmid pDWS2, which carries
the
recBCD genes (
35). Overexpression
of RecBCD
from this plasmid prevented growth of T4 2
mutant phages
on
rep recA mutants as expected, while it did not
revert the
plating defect of the strain (data not shown). We conclude
that a lack
of RecBCD cannot be the reason for this plating
defect.
Interestingly, exogenous catalase has been shown to improve the
recovery of
E. coli cells under various stress conditions,
for example, heat-injured DNA-repair mutants (
27). In
addition,
bacterial catalase does not protect isolated organisms but
favors
the survival of high-density and colonial
E. coli
(
26). To test
whether the low plating efficiency of the
rep recA mutant could
result from a defect in the repair of
oxidative damage that occurs
upon plating (
26), we
measured the capability of this strain
to form colonies on
catalase-containing plates (Fig.
3). The number
of CFU was restored by
the presence of 150 U of catalase/ml in
the LB plates. This result
indicates that the defect in colony
formation results from oxidative
damage due to the presence of
compounds degraded by catalase. The
oxidative damage occurred
upon plating and not during exponential
growth in liquid medium,
as the presence of catalase in the culture did
not rescue the
cells plated on catalase-lacking plates and did not
further enhance
the plating efficiency on catalase-containing plates
(data not
shown). We conclude that the low plating efficiency of the
rep recA mutant propagated at low temperature could result
from the
additive effects of replication pauses due to the
rep mutation
and lesions due to the aerobic
environment.
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DISCUSSION |
In this work, we studied the effects of dnaK mutations
in rep, recA, and rep recA mutants and showed
that two different dnaK mutations that do not prevent the
growth of wild-type cells significantly impair the growth of
rep and rep recA mutants. We also investigated the reasons for the cryosensitivity of rep recA mutants. We
found that the presence of catalase in the plates relieves the plating defect of the strain, indicating that the defect in colony formation results from oxidative damage.
Role of DnaK in rep mutants.
The combination of
rep and dnaK mutations is sufficient to impair
growth. dnaK306 and dnaK756 mutations decrease
the plating efficiency of rep mutants three- to fivefold
independently of the growth phase (Table 2) and independently of the
temperature (Table 3). The strong filamentation induced by the
combination of rep and dnaK756 mutations is
observed at low and high temperatures (Fig. 1g and h). It can be noted
that the rep mutant cells are slightly elongated at 30°C,
which may reflect their requirement for a high level of DnaK. The
rep mutation affects the propagation of replication forks in
E. coli, and the chaperone proteins DnaK and DnaJ play a
role in the replication of several replicons. They are required for the
initiation of F, P1, and lambda (reviewed in reference 5).
In F and P1, they control the multimerization of the specific initiator
protein encoded by the replicon. In , they appear to act by
dissociating DnaB protein from the P protein, thereby allowing the
helicase to act. They are also involved in E. coli
chromosomal replication, where their role is less well understood.
Deletion of DnaK causes temperature sensitivity for cell growth,
abnormal cell division, and a reduced rate of replication at 30°C
(2). dnaK, dnaJ, and grpE mutations
cause a dramatic decrease in the level of epsilon, the proofreading subunit of DNA polymerase III, and a decrease in the apparent level of
RNase H1 (9). DnaK and DnaJ are also required for the
proper folding of UmuC, one of the subunits of DNA polymerase V (PolV)
involved in replication restart from a lesion and lesion bypass
(34, 37). The properties of the dnaK756 and
dnaK306 mutants used in this work have been characterized
(34, 45). These dnaK mutations prevent
proteolysis (measured by degradation of the pyromycyl fragment) and
mini-F plasmid replication. They differ in that the dnaK756
mutant is deficient for growth and proficient for UmuC folding,
whereas the dnaK306 mutant has the opposite properties
(34, 45). In rep mutants, chromosome
replication is slowed down, which is compensated for by more
replication forks per chromosome (6, 23). Replication
pauses due to the Rep defect may be more frequent or longer in
dnaK mutants, due to a role of chaperone proteins in the
release of the obstacles that block replication forks in rep
mutants. Alternatively, DnaK could facilitate replication restart after
restoration of a replication fork by homologous recombination. In this
case, as the defect due to the rep dnaK combination is also
observed in recA mutants, DnaK would also participate in the
restart of replication forks from arrested forks that have not
recombined. Comparison of [3H]thymidine incorporation
under various conditions did not allow us to detect a defect in
nucleotide incorporation in the rep dnaK756 double mutant
compared to the parental strains (data not shown). This suggests that
the replication defect which causes the filamentation of the rep
dnaK756 mutant is, as in rep mutants, compensated by overinitiation at the origin. Altogether, this work supports the hypothesis that chaperone proteins play a role in the normal
propagation of replication forks.
Plating defect and oxidative damage.
The plating defect of the
rep recA mutant indicates that lethal lesions occur in these
cells upon cell isolation, and the recovery of plating efficiency upon
addition of catalase in the plates shows that these lesions are caused
by oxidative compounds. This is consistent with the observation that
isolated cells are not protected against oxidative damage by their own
catalase (26). This transient inactivation of the first
line of defense, mediated by enzymes that destroy oxygen radicals,
renders essential the action of the second line of defense, mediated by
enzymes that repair lesions. Homologous recombination plays an
essential role in this process. Oxidative lesions lead to the formation
of single-strand and double-strand DNA breaks, known to be repaired by
homologous recombination (4, 16). recA
mutations also inactivate the induction of the SOS response, a set of
genes involved in DNA repair. However, inactivation of SOS induction
only in rep mutants has no effect on plating efficiency
(43), suggesting that the defect in homologous
recombination plays an essential role in the cryosensitivity of the
rep recA mutant. In contrast with cells defective in
homologous recombination, rep mutants are not more sensitive
to H2O2 than wild-type strains
(16); nevertheless, here rep mutations increase
the sensitivity of recA mutants to oxidative stress. This
suggests an additive effect of oxidative lesions and replication pauses
due to the rep mutation. The rep recA mutant
requires exonuclease V-mediated degradation for viability; however, we
show here that the plating defect of rep recA mutant cells
does not result from a lack of RecBCD. The plating defect was
suppressed at 42°C; however, it was not suppressed by overexpression of heat shock proteins. The Rep helicase is very similar to the repair helicase UvrD (11), and rep uvrD
double mutants are lethal. Interestingly, a mutation has been
described in the uvrD gene (uvrD307)
(47) that also specifically decreases the plating efficiency of certain wild-type strains of E. coli at
30°C. Whether the cryosensitivities of the uvrD307 mutant
and of rep recA mutants have a common origin remains to be determined.
DnaK and oxidative damage.
A protective effect of heat shock
proteins against oxidative damage has been previously documented in
several studies. Heat shock proteins, including DnaK, are induced by
H2O2 treatment (32; reviewed in
reference 8). A dnaK null mutation
increases the sensitivity of E. coli to
H2O2 (7). We report here that rep recA mutant cells require the following for full
viability: (i) DnaK and (ii) protection against oxidative damage.
However, no direct link between these two observations could be found. If the low level of heat shock proteins at 30°C participates in the
plating defect of rep recA mutant cells, it is not the only cause, since overproduction of heat shock proteins does not restore the
plating efficiency. Conversely, the growth defect of rep recA dnaK mutant cells at high temperature does not result only from oxidative damage: plating on catalase-containing plates of either rep recA dnaK mutant increased 10- to 100-fold the number of
colonies at 40°C (data not shown); however, all colonies tested had
acquired a suppressor mutation, suggesting that catalase only permits
some residual growth that facilitates the acquisition of suppressor mutations, without restoring viability. Therefore, the observation that
rep mutants are handicapped by either recA or
dnaK mutations and that these handicaps are additive
suggests that RecA and DnaK do different things that increase survival.
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ACKNOWLEDGMENTS |
We thank I. Matic, M. Radman, and F. Taddei for suggesting to us
the use of catalase. We are very grateful to S. Kulaskauskas and to
J. Tremblay for the bacterial micrographs. We thank M. A. Petit for careful reading of the manuscript and an anonymous referee
for insightful suggestions.
B.M. is on the CNRS staff. This work is supported in part by the
Programme de Recherche Fondamentale en Microbiologie, Maladies Infectieuses.
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FOOTNOTES |
*
Corresponding author. Mailing address:
Génétique Microbienne, INRA, 78352 Jouy en Josas Cedex,
France. Phone: (33) 1 34 65 25 14. Fax: (33) 1 34 65 25 21. E-mail:
bmichel{at}biotec.jouy.inra.fr.
Present address: Génétique Moléculaire Evolutive
et Médicale, Medical faculty-Necker Enfants Malades, E9916
INSERM, 75730 Paris Cedex 15, France.
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Journal of Bacteriology, April 2001, p. 2165-2171, Vol. 183, No. 7
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.7.2165-2171.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
