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Vol. 180, Issue 13, 3405-3409, July 1, 1998
Genetic Recombination in Bacillus
subtilis 168: Effects of recU and recS
Mutations on DNA Repair and Homologous Recombination
Silvia
Fernández1,
Alexei
Sorokin2, and
Juan C.
Alonso1*
1 Centro Nacional de Biotecnología,
Consejo Superior de Investigaciones Científicas, Campus
Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid,
Spain,1 and
2 Institut National de la
Recherche Agronomique
Génétique Microbienne, 78352 Jouy-en-Josas Cedex, France2
 |
ABSTRACT |
Bacillus subtilis recombination-deficient mutants were
constructed by inserting a selectable marker (cat gene)
into the yppB and ypbC coding regions. The
yppB:cat and ypbC:cat
null alleles rendered cells sensitive to DNA-damaging agents, impaired
plasmid transformation (25- and 100-fold), and moderately affected
chromosomal transformation when present in an otherwise
Rec+ B. subtilis strain. The yppB
gene complemented the defect of the recG40 strain.
yppB and ypbC and their respective null alleles were termed "recU" and "recU1"
(recU:cat) and "recS" and
"recS1" (recS:cat),
respectively. The recU and recS mutations were
introduced into rec-deficient strains representative of the
(recF), 
(addA5 addB72), 
(recH342), and 
(recG40) epistatic groups.
The recU mutation did not modify the sensitivity of
recH cells to DNA-damaging agents, but it did affect inter-
and intramolecular recombination in recH cells. The
recS mutation did not modify the sensitivity of
addAB cells to DNA-damaging agents, and it marginally
affected recF, recH, and recU
cells. The recS mutation markedly reduced (about 250-fold)
intermolecular recombination in recH cells, and there were
reductions of 10- to 20-fold in recF, addAB,
and recU cells. Intramolecular recombination was blocked in
recS recF, recS addAB, and recS
recU cells. RecU and RecS have no functional counterparts in
Escherichia coli. Altogether, these data indicate that the
recU and recS proteins are required for DNA
repair and intramolecular recombination and that the recF
( epistatic group), addAB (), recH (),
recU (), and recS genes provide overlapping activities that compensate for the effects of single mutation. We
tentatively placed recS within a new group, termed
"
."
| |
INTRODUCTION |
In Bacillus subtilis,
several recombination-deficient mutants with increased sensitivities to
DNA-damaging agents have been isolated. Genetic analysis of chromosomal
and plasmid transformations has demonstrated that to be efficient,
homologous recombination requires the products of the recA,
recB, recD, recF, recU
(formerly termed "recG"), recH,
recL, recN, recP, recR,
addA, addB, and mfd genes (reviewed in
reference 6), in addition to the enzymes involved in
general DNA metabolism, such as DNA polymerases, SSB, Hbsu, DNA ligase,
and topoisomerases, among others (15, 20).
Three qualitatively distinct situations may arise when the frequency of
genetic exchange of a double-mutant strain is analyzed. (i) The
frequency may be equal to that of the more deficient single-mutant parent. (ii) The frequency may be equal to the sum of each of the
single-mutant parents (additive effect). (iii) The frequency may be
greater than the sum of each of the single-mutant parents (synergistic
effect) (11). Considering these possibilities, we have
classified the B. subtilis double-Rec
strains
into different epistatic groups (, , , and groups) (3). The epistatic group activity requires the
recF, recL, recR, and recN
genes (2). Recently, the RecF and RecR proteins have been
biochemically characterized (7-9). The epistatic group
activity is dependent on the addA and addB genes
(3). The addA and addB genes encode
different subunits of the multifunctional enzyme AddAB (also termed
exonuclease V [Exo V] or RecBCD in Escherichia coli
[6, 19]). The epistatic group activity requires
the recP and recH genes (6). The epistatic group activity may require the recB,
recD, and recU genes (6). The and
epistatic groups, which are poorly characterized, do not seem to
have a counterpart in E. coli. No double-mutant strains
impaired in the functions classified within groups and have
been described.
In this report, we genetically characterize the products of the
B. subtilis recU (which codes for an unknown activity) and recS (which codes for a putative DNA helicase) genes that
are involved in DNA repair and homologous recombination. The phenotype associated with recU cells is consistent with our previous
grouping, but we were unable to classify recS cells within
any previously recognized epistatic group (see reference
6). Unless otherwise stated, the indicated genes and
products are of B. subtilis origin.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
The E. coli JM103
strain was used (23). All B. subtilis strains
listed in Table 1 are isogenic with
strain YB886 (rec+ control). The ypbC
(National Biomedical Research Foundation-Protein Identification
Resources [NBRF-PIR] and Swiss-Prot accession no. .Gb_Ba) and
yppB (.Gb_Ba) genes were PCR amplified with specific
oligonucleotides and cloned (Table 1). The transfer of the
recU:cat deletion/insertion allele (termed recU1) and recS:cat deletion/insertion
allele (termed recS1) from pCB182 and pCB176, respectively,
into the B. subtilis chromosome by a double-crossover event
was performed as previously described (5).
Chemical treatment.
Methyl methanesulfonate (MMS) was
purchased from Eastman Kodak, Rochester, N.Y. 4-Nitroquinoline-1-oxide
(4NQO) was purchased from Sigma Chemical Co., St. Louis, Mo. The
chemical treatment of the mutant strains was performed essentially as
previously described (1).
Transformation of bacteria.
Competent cells were prepared as
described by Rottländer and Trautner (25) and Maniatis
et al. (23) for B. subtilis and E. coli, respectively. Plasmid transformants were selected on tryptone-yeast agar medium (25) containing chloramphenicol
or erythromycin at 5 µg/ml. Met+ recombinants were
selected by plating on minimal agar containing all nutritional
requirements except methionine (25).
Recombination frequencies.
Relative transformation
frequencies were used as a measure of recombination. B. subtilis competent cells (about 5.0 × 107
cells/ml) were transformed according to the method of Rottländer and Trautner (25). The yield of Met+ or plasmid
transformants was corrected for DNA uptake and cell viability as
described by Alonso et al. (1). DNA uptake, which is taken
as a measurement of competence, was monitored as described previously
(25).
| |
RESULTS AND DISCUSSION |
Replacement of B. subtilis recU and recS
genes by use of a selectable marker.
Recently by molecular
cloning, DNA sequencing, and computer homology comparisons, a B. subtilis open reading frame (ORF), ypbC, was identified
as coding for a putative recombinational helicase, the gene for which
is "recQ-like" (coordinates 4291 to 5781 in the sequence
with accession no. .Gb_Ba) (27). Furthermore, a
B. subtilis region (plasmid pMP29-42) able to rescue the
recG40 mutation (24) has been sequenced. The
pMP29-42-borne DNA segment bears two truncated ORFs, termed
"yppB" and "yppC" (27)
(accession no. .Gb_Ba).
To identify and clone the
recG gene of
B. subtilis, a PCR-amplified DNA with coding capacity for
yppB or
yppC was used to
transform the
recG40 strain. Upon transformation of the MMS-sensitive
(MMS
s)
recG40 strain with purified DNA
fragments, we learned that only
the
yppB ORF fully
complements the
recG40 defect, since
yppB encodes
a protein unrelated to the
E. coli recG product. To avoid
confusion
in the nomenclature and for the sake of simplicity, we have
renamed
this ORF as "
recU" and the mutant allele as
"
recU40."
To learn whether the putative
ypbC gene codes for a product
involved in homologous recombination and recombinational repair
and
to challenge the grouping of the
B. subtilis rec genes
(described
above), we cloned the chloramphenicol acetyltransferase
gene (
cat)
239 and 415 bp downstream of the ATG codons of
the
ypbC and
recU genes, respectively. The
resulting alleles were used to replace
the
ypbC and
recU genes.
Strains containing the expected gene substitution were obtained by
natural transformation of the
B. subtilis rec+
strain and its isogenic
rec-deficient derivatives with
linearized
plasmid-borne
recU:
cat
(
recU1) and
ypbC:
cat (Table
1). The
presence
of the desired replacement was confirmed by PCR amplification
and nucleotide sequence analysis (data not shown). In all cases,
integration had occurred by a double-crossover event, as predicted
for
transformation of competent cells with linear plasmid DNA
(reviewed in
reference
13).
To analyze if the constructs were impaired in recombinational repair
processes, we exposed the mutant strains to the lethal
effect of MMS.
Both
recU1 and
ypbC:
cat strains were
unable to
form colonies on plates containing 300 (2.7 mM) and 100 (0.9 mM)
µg of MMS per ml, respectively, whereas the wild-type strain
formed
colonies even in plates containing 450 µg (4 mM) of MMS per
ml.
The
recU1 and
ypbC:
cat strains,
exposed to the killing action
of 10 mM MMS, were more sensitive than
the otherwise
rec+ strain (Fig.
1A and Fig.
2). The same results were
obtained when
the cells were challenged with 0.1 mM 4NQO (data not
shown). It
is likely, therefore, that the
ypbC gene product
is involved in
DNA repair. We have renamed the
recS gene and
its mutant allele
"
recS1"
(
ypbC:
cat). As revealed in Fig.
1A, the cells
carrying
the
recU1 null allele are slightly more sensitive
to the killing
action of MMS than those of the
recU40
strain, and the defect
can be complemented by a plasmid-borne
recU gene (coordinates
2536 to 2874 in the sequence with
accession no. .Gb_Ba).
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Fig. 1.
Survival of B. subtilis strains following
exposure to 10 mM MMS. (A) Survival of the rec+
strain (solid circles), the recU40 strain (open circles),
the recU40 strain carrying a plasmid-borne recU
gene (solid triangles), and the recU1 strain (solid
squares). (B) Survival of rec+ (solid circles),
recH342 (open squares), recU1 (solid
squares), and recH342 recU1 (solid triangles) strains.
The chemical treatment of DNA repair-deficient mutant strains was
performed essentially as previously described (1).
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|
Effect of DNA-damaging agents in recU and
recH mutant strains.
To date in B. subtilis, 13 genes involved in homologous recombination
(recA, addA, addB, recF,
recL, recR, recN, recH,
recP, recB, recD, recU, and
mfd) have been identified (reviewed in reference 6). Genetic analysis of double-rec
mutants with chromosomal and plasmid transformation has led to the
classification of the rec genes, other than recA,
into four epistatic groups (3). The genes involved in the
(recF, recL, recR, and
recN) and (addA and addB) groups
are well characterized (7, 8, 19), but those comprising the
(recH and recP) and (recB,
recD, and recU) groups are poorly defined.
Previously, we have shown that the
recU40 (
group) gene
in combination with mutations in the
addA addB (
epistatic group)
genes was more sensitive to the killing action of MMS
and 4NQO
than the single-mutant parent, whereas with a mutation in the
recF (
group) gene, it was as sensitive as the single
parent
(
3,
5). However, no data are available about the
effect
of double mutations in the
and
groups. The
recH342 strain
was selected as a representative of the
epistatic group. We
have constructed a
recU recH double
mutant and challenged these
cells with the killing action of 10 mM MMS.
As revealed in Fig.
1B, the
recH mutation did not affect the
MMS sensitivity of the
recU cells.
Effects of recS, recF, recH,
recU, and addAB mutations on MMS sensitivity.
B. subtilis rec+ and its isogenic
rec-deficient derivative strains (BG431, BG435, BG429, and
BG433 [listed in Table 1]) were exposed to the killing action of 10 mM MMS. The recF, addAB, recH, and
recU genes were selected as representatives of the , , and groups, respectively. As revealed in Fig. 2, various
degrees of increased sensitivity were observed. The recS,
addAB, and recH strains are moderately affected,
whereas the recF and recU strains are very
sensitive to the killing action of MMS compared to the rec+ control (Fig. 2). The recS
mutation partially suppressed the sensitivity of addAB cells
to MMS (Fig. 2A). The recS
mutation moderately increased the MMS sensitivity of recH,
recU, and recF cells (Fig. 2B, C, and D).
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Fig. 2.
Survival of B. subtilis strains following
exposure to 10 mM MMS. (A) Survival of rec+
(solid circles), recS1 (open circles), addA addB
(solid squares), and addA addB recS1 (solid triangles)
strains. (B) Survival of rec+ (solid circles),
recS1 (open circles), recH342 (solid squares),
and recH342 recS1 (solid triangles) strains. (C) Survival of
rec+ (solid circles), recS1 (open
circles), recF15 (solid squares), and recF15
recS1 (solid triangles) strains. (D) Survival of
rec+ (solid circles), recS1 (open
circles), recU40 (solid squares), and recU40
recS1 (solid triangles) strains.
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Together, these results indicate that (i) the
recS product
is needed for the removal of DNA damage; (ii) the
recS genes
contribute
to different extents to the removal of DNA damage in
recH,
recU,
and
recF cells; and (iii)
recS partially suppresses the defect
of the
addAB
cells.
Effects of recF, addAB, recU,
recH, and recS mutations on genetic
recombination.
To study the effects of the recF,
addAB, recH, recU, and recS
mutations on genetic recombination, we analyzed the requirement for
these functions in natural transformational recombination. We measured
the frequency of transformation by using plasmid and chromosomal DNA.
Transformation in B. subtilis involves the transfer of naked
double-stranded DNA from the media to the recipient competent uninucleated cell (22, 26). During DNA uptake, a set of
competence (com) gene products degrades one of the DNA
strands and takes up the other one in a linear single-stranded form
(reviewed in reference 13). Therefore, by the
activity of the com genes, the donor DNA is presented to the
recombinational machinery in a form such that it is ready for homology
searching (synaptic stage) on the parental molecule (13,
22). Hence, in our analysis, we are studying the synaptic and
postsynaptic stages but neglect the involvement of rec
functions in the presentation of the substrate (presynaptic stage).
B. subtilis transformation with chromosomal DNA (chromosomal
transformation) does not require replication for integration
of donor
markers (
13,
22). In contrast, both replication and
recombination functions are required for the establishment of
plasmid
DNA (
13,
22). Canosi et al. (
12) and de Vos et
al.
(
12a) have proposed that after uptake of the oligomeric
plasmid
DNA molecule (a monomer is inactive in transformation) and
synthesis
of the complementary strand, pairing of one of the incoming
single-stranded
DNA ends with the newly replicated strand results in
plasmid establishment.
By measuring both chromosomal (intermolecular
recombination) and
plasmid (intramolecular recombination)
transformation, we can
examine different types of events.
B. subtilis competent cells
were transformed with 1 µg of
homologous chromosomal DNA or plasmid
DNA per ml to determine the
transformation frequency of the Rec
mutant strains. The
frequency of appearance of
met+ transformants in
the single-Rec
strain and in certain
double-Rec
strains has been previously reported (
1,
3). Here, the
experiments were performed in parallel for
comparison with other
strains (Table
2).
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Table 2
Effect of RecU and RecS on homologous recombination as
measured by transformation of chromosomal and
plasmid DNAa
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|
Except for
recA, the frequency of chromosomal transformation
(intermolecular recombination) in the singly
rec-deficient
strain
did not change more than fourfold relative to the
rec+ value (
3) (Table
2). The same
results were found for plasmid
transformation.
The
recU null allele (
recU1) and
recU40 mutations reduce the chromosomal transformation less
than threefold (
1) (Table
2). As previously reported,
plasmid transformation is marginally
affected by the
recU40
mutation, but it is reduced 25-fold and
100-fold in the
recU1 and
recS1 cells, respectively (Table
2).
Since (i) the
recH (epistatic group
) mutation blocked
(>5,000-fold reduction) chromosomal transformation of
recF
(group
) and
addAB (group
) cells (
3,
5)
and reduced about 250-fold
that of
recS cells (Table
2) but
did not affect chromosomal transformation
of
recU cells
(group
), and (ii) the
recH mutation blocks plasmid
transformation of
recF addAB cells (
5) and
reduced more than
1,000-fold that of
recS cells (Table
2)
but did not affect (2-fold)
plasmid transformation of
recU
cells (Table
2), we can classify
recH and
recS
into two different epistatic groups by using transformational
recombination, and confirmed our previous classification of
recH and
recU within different epistatic groups
(
3). The classification
of
recH within group
and
recU within
was based on the following
facts. (i)
recH cells are only moderately sensitive to DNA-damaging
agents, whereas
recU cells are very sensitive to these
agents
(
1,
3). (ii) Double-mutant strains impaired in the
plus
or
plus
epistatic groups are blocked in
chromosomal recombination,
whereas the
plus
or
plus
groups are moderately affected
(
1,
3). (iii) Transductional
intramolecular recombination
is modestly affected in the double
plus
mutants but is highly
impaired in the
plus
mutants
(
4). (iv) Chromosomal transformation
is blocked in
hbs
recH cells but is reduced only 11-fold in
hbs recU40
cells (
15). To reconcile these differences with the data
presented in this report, we have to assume that certain gene
products
of the
and
epistatic groups cooperate and that
recH and
recU operate at different stages in the recombination
pathway.
Both the
and
groupings will tentatively be maintained
until
more information becomes available.
The frequency of chromosomal transformation of the
recS null
allele was reduced about 4-fold, whereas plasmid transformation
was
reduced about 100-fold (Table
2). The
recS mutation reduced
chromosomal transformation in
recF,
recU, and
addAB cells about
10- to 20-fold and that in
recH
cells about 250-fold (Table
2).
Plasmid transformation was reduced by
the
recS mutation more than
1,000-fold in strains
representative of the
,
, and
epistatic
groups and was
reduced about 200-fold in the
epistatic group.
On the basis of these and previous results, we conclude that no
interaction exists between the RecU protein and the
addAB and
recF gene products, whereas a significant interaction
may
exist between the RecU and RecH proteins. No interaction was
observed
between the
recS and the
addAB,
recH,
recU, and
recF functions.
We
tentatively place the
recS mutation within a different
epistatic
group, termed "
."
RecU and RecS proteins have no functional counterparts in E. coli.
A homology search of the RecU and RecS proteins was
performed with the protein sequences available in the NBRF-PIR (release 50) and Swiss-Prot (release 34) databases. Significant identity (about
50%) was observed between the 23.9-kDa RecU protein (database accession no. ) and an equivalent 23.1-kDa product of
Streptococcus pneumoniae (27) (accession no.
ypoa_strpn, ypoa_stror), and 34% identity was observed with a 19.3-kDa
protein of Mycoplasma genitalium (17) (accession
no. ). A protein similar to RecU has not been detected so far in
gram-negative bacteria. Both E. coli and Haemophilus
influenzae seem to lack this protein (10, 16).
The 56.5-kDa RecS protein (accession no. .GB_Ba) shares 34 to
36% identity with
E. coli RecQ (
27) (accession
no. BVECRQ) and the putative
H. influenzae RecQ
(accession no.
U32756_D) and
B. subtilis "RecQ"
(accession no.
AF027868.Gb_Ba)
proteins. The degree of identity of RecS
with both
E. coli RecQ
and the putative
B. subtilis "RecQ" protein could be enhanced
up to 43 and 40%,
respectively, if only the regions containing
the seven-amino-acid
motifs of DExH-box DNA helicases (first 330
residues) were used for the
alignment. The putative
B. subtilis "RecQ" protein
(
yocI) shares 40% identity with the
E. coli RecQ
protein.
Unlike
E. coli containing only one
recQ gene
product with DNA helicase activity (
28),
B. subtilis could possess more than
one such putative helicase
(
21). This is consistent with the
fact that human cells
contain genes that code for three related
putative helicases (RecQL
[human RecQL], accession no.
A55311),
HumBS [Bloom's syndrome,
accession no.
A57570], and HumWS [Werner's
syndrome, accession no.
L76937]) (see reference
14). Although
the
B. subtilis "
recQ" gene has not yet been tested, the
gene
seems to be nonessential for cell viability (reviewed in
references
14 and
20).
It is generally accepted that the role of
E. coli RecQ (and
probably
B. subtilis "RecQ") protein, alone or in
combination
with the
E. coli RecJ protein, is to generate a
3'-terminal single-stranded
DNA to which RecA could bind in a step
proceeding to the homologous
pairing. With chromosomal and plasmid
transformation, we are measuring
the synaptic and postsynaptic stages
of recombination (
13,
22).
In this report, we show that the
RecS protein is required in either
the synaptic stage, postsynaptic
stage, or both stages. It remains
to be documented if RecS is a bona
fide DNA helicase and if the
helicase activity participates directly in
recombination. We also
report here that deletion of the
recS
gene partially suppressed
the defect of
addAB cells. The
AddAB protein is a multifunctional
enzyme associated with nuclease and
helicase activities (
19,
20); however, the defects generated
by the
addA5 and
addB71 mutations in this genetic
background remain to be characterized.
| |
ACKNOWLEDGMENTS |
This research was partially supported by grant PB 96-0817 from
DGCICYT and grant 06G/004/96 from the Consejería de
Educación y Cultura de la Comunidad de Madrid to J.C.A. and
grants from GREG (décision 21) and ECC (BIO2-CT93-0272 and
BIO2-CT94-2011) to S. D. Ehrlich. S.F. is supported by a Comunidad
de Madrid training grant.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centro Nacional
de Biotecnología, CSIC, Campus Universidad Autónoma de
Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: (34) 91 585 4546. Fax:
(34) 91 585 4506. E-mail: jcalonso{at}cnb.uam.es.
| |
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Abstract
Introduction
