1 Unité de Recherches Laitières et Génétique Appliquée, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas, France
2 Laboratoire de Microbiologie et Génétique Moléculaires UMR 5100, CNRS Université Paul Sabatier, 118, route de Narbonne, 31062 Toulouse cedex, France
Correspondence
Meriem El Karoui
meriem{at}diamant.jouy.inra.fr
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The extent of genetic plasticity displayed by S. pneumoniae raises the question of whether cellular processes other than natural transformation are involved. One process known to generate plasticity is double-strand DNA break (DSB) repair by homologous recombination. This process sometimes leads to chromosomal rearrangements and is thus a potential cause of genomic plasticity (Gruss & Michel, 2001). In Gram-positive bacteria, the exonuclease/helicase RexAB is a major component of the homologous recombination process and is essential for DSB repair (Chedin & Kowalczykowski, 2002
; Quiberoni et al., 2001b
). It has been studied in Bacillus subtilis (where it is called AddAB; Doly et al., 1974
; Kooistra & Venema, 1991
) and in Lactococcus lactis (El Karoui et al., 1998
), and is the functional homologue of the RecBCD enzyme of Escherichia coli. Unlike RecBCD, which has a single nuclease (and two helicases) to degrade both strands, the RexAB enzyme comprises two distinct nuclease activities that appear to each degrade one of the two strands (Quiberoni et al., 2001a
). Nuclease activities have been mapped to motifs present at the C-terminal extremities of both RexA and RexB subunits (Quiberoni et al., 2001a
), which are conserved in all RexAB homologues thus far examined (Quiberoni et al., 2001b
). RexAB exonuclease activity degrades DNA from a double-strand end, but is attenuated when it encounters a short DNA sequence called Chi (Chedin et al., 1998
; El Karoui et al., 1998
). After a Chi encounter, the helicase activity remains (Chedin et al., 2000
), leading to formation of a 3'-OH single-strand extremity (the preferential substrate for RecA-mediated homologous pairing reaction) and subsequent repair of the molecule by homologous recombination. The sequence comprising Chi has been shown to vary according to the species (E. coli, L. lactis, Haemophilus influenzae and B. subtilis; for review see El Karoui et al., 1999
). It is interesting to note that despite the importance of exonuclease/helicase-Chi couples in genome integrity via DSB repair, the structure of their components is only poorly conserved.
To characterize the role of RexAB in S. pneumoniae, we constructed rexAB insertional mutants and examined the phenotypes of these mutants with respect to growth, DNA repair capacity and recombination in gene conversion. AddAB has been proposed in B. subtilis to have an additional role in recombination during natural transformation (Kooistra et al., 1988), but this observation seems to be strain-dependent (Fernandez et al., 2000
). To clarify this issue we also tested the natural transformation ability of our rexAB mutants.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains were grown in THY medium (30 g dehydrated ToddHewitt broth l1, 5 g yeast extract l1; Difco). Antibiotic concentrations used for selection of transformants were: chloramphenicol, 5 µg ml1; erythromycin, 0·2 µg ml1; spectinomycin (Spc), 100 µg ml1; and streptomycin (Str), 200 µg ml1. For transformation, pre-competent cells were prepared as described by Martin et al. (2000) and incubated with synthetic Competence Stimulating Peptide (25 ng ml1) for 10 min before addition of chromosomal or plasmid DNA.
In vitro mariner mutagenesis.
Mutagenesis was carried out essentially as described previously (Martin et al., 2000). A 7·3 kb DNA fragment encoding rexAB was PCR-amplified using primers ODH9 and ODH17 (ODH9, 5'-ATTCGGACTTCTTTGACAGAA-3' at the beginning of rexB; ODH17, 5'-ATTCTTTGGTAGCTTGTTCCC-3' at the end of rexA), and strain R800 chromosomal DNA as template. Plasmid pR412 (Martin et al., 2000
) was used as a source of the SpcR mariner minitransposon to mutagenize the rexAB fragment in vitro, which was then transformed into R800. Ten SpcR transformants were checked by PCR and shown to carry a mariner insertion. Two of them (strains DHP14 and DHP15) were further analysed; the precise insertion position was determined by PCR and DNA sequencing using primers ODH9, ODH17, MP127 and MP128 (MP127, 5'-CCGGGGACTTATCAGCCAACC-3'; MP128, 5'-TACTAGCGACGCCATCTATGTG-3'; Martin et al., 2000
).
UV irradiation.
Cells were grown in liquid culture to exponential phase to an OD550 of approximately 0·15. Dilutions were then plated and cells were UV-irradiated at 5, 10 and 25 J m2 using a UV stratalinker (Stratagene; the wavelength was 254 nm and the energy was calculated according to the manufacturer's protocol see www.stratagene.com/manuals/7003406.pdf). Survival was assessed after 24 h incubation at 37 °C.
High molecular weight (HMW) plasmid detection.
HMW multimer accumulation was detected as described by Sourice et al. (1998). Total DNA was prepared using cells grown to an OD550 of approximately 0·2 with the Qiagen Genomic-tip 20/G kit (according to the manufacturer's protocol, except that 10 mg lysozyme ml1 and 50 U mutanolysin ml1 were added to facilitate cell lysis). HMW multimers were detected by Southern blot hybridization using a plasmid-specific probe. The Southern blot was scanned and the ratio of HMW versus total plasmid DNA content was quantified using ImageQuant v5 software.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Inactivation of rexAB affects growth and DNA repair
RexAB is a major component of the DSB repair machinery. DSB occurs during the course of normal cellular processes like chromosome replication (Gruss & Michel, 2001) or because of exposure to DNA-damaging agents (Thoms & Wackernagel, 1998
). As the accumulation of non-repaired DSB during growth usually leads to cell death, rexAB mutants are expected to display reduced growth capacity and reduced survival after exposure to DNA-damaging agents.
Both rexA (strain DHP15) and rexB (strain DHP14) were severely impaired for growth, as monitored by OD550 measurements (Fig. 2, filled symbols). This may suggest that only a fraction of the cell population is viable. Indeed, for a given OD550, the numbers of colony-forming centres (CFC) of both rexAB mutants were lower than those obtained with the wild-type (wt) strain (Fig. 2
, open symbols). rexA and rexB mutant viability (CFC per OD550 unit) was around 20 % of that of the wt strain (calculated from data in Fig. 2
). These results are in keeping with viabilities reported for E. coli recBCD or B. subtilis addAB mutant strains (Capaldo et al., 1974
; Kooistra et al., 1988
). The observed lower ratio of CFC to OD550 in the rexAB strains could possibly reflect the presence of elongated cells due to the mutation. However, examination of wt and mutant strains by microscopy revealed no differences in cell shape or size: the ratio of diplococci to tetracocci and longer chains was identical (data not shown). Cell length measurements further confirmed that the rexAB mutation had no effect on S. pneumoniae cell size; the mean cell length was 1·01±0·2 µm for the wt strain and 0·98±0·18 µm for both rexAB mutants. Note that both wt and rexAB S. pneumoniae cells died quickly after reaching stationary phase (as deduced from the rapid decrease in CFC) while OD550 remained constant.
|
|
|
RexAB inactivation has a slight effect on gene conversion
As a component of a homologous recombination pathway, RexAB might be involved in recombination events occurring during gene conversion. We tested whether rexAB mutants are affected in gene conversion using a previously developed test system (Sung et al., 2001). The assay was performed in the R960 strain, which carries two alleles of the rpsL gene, the dominant StrS rpsL wt allele (linked to a kanamycin resistance gene) and the StrR rpsL41 allele. Thus R960 is normally StrS, but StrR clones arise at significant levels. It was previously shown that these clones are the result of a homologous recombination event between rpsL41 and the rpsL wt allele (Sung et al., 2001
). We introduced the rexA and rexB mutations into the R960 background; the gene conversion frequency was determined as the proportion of StrR cells among the total number of viable cells in an exponentially grown culture. The conversion frequency was slightly reduced in rexAB mutants (
three- and fivefold for rexB and rexA, respectively; Table 2
) compared to the
40- to 200-fold reduction observed in a recA mutant (Sung et al., 2001
). While the reduction was slight, we consider it significant, as the standard deviations observed between independent measurements were very small. These results may suggest that 5080 % of the conversion events are RexAB-dependent. Nevertheless, other tests would be required to confirm the biological importance of RexAB in gene conversion events. We conclude that RexAB is not a major player in gene conversion in S. pneumoniae, and at least another RexAB-independent pathway is likely to exist to initiate conversion. This is not surprising in light of the multiple pathways identified in other bacteria that generate single-strand substrates for RecA annealing. For example, gene conversion in E. coli is mediated by the RecBCD pathway (Zieg & Kushner, 1977
), but can also be mediated by the RecF or RecE pathway (Kobayashi, 1992
).
|
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ballester, S., Lopez, P., Alonso, J. C., Espinosa, M. & Lacks, S. A. (1986). Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids. Gene 41, 153163.[CrossRef][Medline]
Berka, R. M., Hahn, J., Albano, M., Draskovic, I., Persuh, M., Cui, X., Sloma, A., Widner, W. & Dubnau, D. (2002). Microarray analysis of the Bacillus subtilis K-state: genome-wide expression changes dependent on ComK. Mol Microbiol 43, 13311345.[CrossRef][Medline]
Biswas, I., Maguin, E., Ehrlich, S. D. & Gruss, A. (1995). A 7-base-pair sequence protects DNA from exonucleolytic degradation in Lactococcus lactis. Proc Natl Acad Sci U S A 92, 22442248.[Abstract]
Capaldo, F. N., Ramsey, G. & Barbour, S. D. (1974). Analysis of the growth of recombination-deficient strains of Escherichia coli K-12. J Bacteriol 118, 242249.[Medline]
Chedin, F. & Kowalczykowski, S. C. (2002). A novel family of regulated helicases/nucleases from Gram-positive bacteria: insights into the initiation of DNA recombination. Mol Microbiol 43, 823834.[CrossRef][Medline]
Chedin, F., Noirot, P., Biaudet, V. & Ehrlich, S. D. (1998). A five-nucleotide sequence protects DNA from exonucleolytic degradation by AddAB, the RecBCD analogue of Bacillus subtilis. Mol Microbiol 29, 13691377.[CrossRef][Medline]
Chedin, F., Ehrlich, S. D. & Kowalczykowski, S. C. (2000). The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro. J Mol Biol 298, 720.[CrossRef][Medline]
Claverys, J. P., Prudhomme, M., Mortier-Barriere, I. & Martin, B. (2000). Adaptation to the environment: Streptococcus pneumoniae, a paradigm for recombination-mediated genetic plasticity? Mol Microbiol 35, 251259.[CrossRef][Medline]
Dabert, P., Ehrlich, S. D. & Gruss, A. (1992). Chi sequence protects against RecBCD degradation of DNA in vivo. Proc Natl Acad Sci U S A 89, 1207312077.[Abstract]
Doly, J., Sasarman, E. & Anagnostopoulos, C. (1974). ATP-dependent deoxyribonuclease in Bacillus subtilis and a mutant deficient in this activity. Mutat Res 22, 1523.[Medline]
Dopazo, J., Mendoza, A., Herrero, J. & 13 other authors (2001). Annotated draft genomic sequence from a Streptococcus pneumoniae type 19F clinical isolate. Microb Drug Resist 7, 99125.[CrossRef][Medline]
Dubnau, D. (1999). DNA uptake in bacteria. Annu Rev Microbiol 53, 217244.[CrossRef][Medline]
El Karoui, M., Ehrlich, D. & Gruss, A. (1998). Identification of the lactococcal exonuclease/recombinase and its modulation by the putative Chi sequence. Proc Natl Acad Sci U S A 95, 626631.
El Karoui, M., Biaudet, V., Schbath, S. & Gruss, A. (1999). Characteristics of Chi distribution on different bacterial genomes. Res Microbiol 150, 579587.[CrossRef][Medline]
Fernandez, S., Ayora, S. & Alonso, J. C. (2000). Bacillus subtilis homologous recombination: genes and products. Res Microbiol 151, 481486.[CrossRef][Medline]
Garcia, E., Ronda, C., Garcia, J. & Lopez, R. (1985). a rapid procedure to detect the autolysin phenotype in Streptococcus pneumoniae. FEMS Microbiol Lett 29, 7781.[CrossRef]
Gruss, A. & Michel, B. (2001). The replication-recombination connection: insights from genomics. Curr Opin Microbiol 4, 595601.[CrossRef][Medline]
Haijema, B. J., Hamoen, L. W., Kooistra, J., Venema, G. & van Sinderen, D. (1995). Expression of the ATP-dependent deoxyribonuclease of Bacillus subtilis is under competence-mediated control. Mol Microbiol 15, 203211.[Medline]
Haijema, B. J., Noback, M., Hesseling, A., Kooistra, J., Venema, G. & Meima, R. (1996). Replacement of the lysine residue in the consensus ATP-binding sequence of the AddA subunit of AddAB drastically affects chromosomal recombination in transformation and transduction of Bacillus subtilis. Mol Microbiol 21, 989999.[CrossRef][Medline]
Hamoen, L. W., Smits, W. K., Jong Ad, A., Holsappel, S. & Kuipers, O. P. (2002). Improving the predictive value of the competence transcription factor (ComK) binding site in Bacillus subtilis using a genomic approach. Nucleic Acids Res 30, 55175528.
Kobayashi, I. (1992). Mechanisms for gene conversion and homologous recombination: the double-strand break repair model and the successive half crossing-over model. Adv Biophys 28, 81133.[CrossRef][Medline]
Kooistra, J. & Venema, G. (1991). Cloning, sequencing, and expression of Bacillus subtilis genes involved in ATP-dependent nuclease synthesis. J Bacteriol 173, 36443655.[Medline]
Kooistra, J., Vosman, B. & Venema, G. (1988). Cloning and characterization of a Bacillus subtilis transcription unit involved in ATP-dependent DNase synthesis. J Bacteriol 170, 47914797.[Medline]
Kuzminov, A. (1995). Collapse and repair of replication forks in Escherichia coli. Mol Microbiol 16, 373384.[Medline]
Kuzminov, A. (2001). Single-strand interruptions in replicating chromosomes cause double-strand breaks. Proc Natl Acad Sci U S A 98, 82418246.
Lopez, R., Sanchez-Puelles, J. M., Garcia, E., Garcia, J. L., Ronda, C. & Garcia, P. (1986). Isolation, characterization and physiological properties of an autolytic-deficient mutant of Streptococcus pneumoniae. Mol Gen Genet 204, 237242.[Medline]
Martin, B., Garcia, P., Castanie, M. P. & Claverys, J. P. (1995). The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction. Mol Microbiol 15, 367379.[Medline]
Martin, B., Prudhomme, M., Alloing, G., Granadel, C. & Claverys, J. P. (2000). Cross-regulation of competence pheromone production and export in the early control of transformation in Streptococcus pneumoniae. Mol Microbiol 38, 867878.[CrossRef][Medline]
Oggioni, M. R. & Claverys, J. P. (1999). Repeated extragenic sequences in prokaryotic genomes: a proposal for the origin and dynamics of the RUP element in Streptococcus pneumoniae. Microbiology 145, 26472653.
Ogura, M., Yamaguchi, H., Kobayashi, K., Ogasawara, N., Fujita, Y. & Tanaka, T. (2002). Whole-genome analysis of genes regulated by the Bacillus subtilis competence transcription factor ComK. J Bacteriol 184, 23442351.
Quiberoni, A., Biswas, I., El Karoui, M., Rezaiki, L., Tailliez, P. & Gruss, A. (2001a). In vivo evidence for two active nuclease motifs in the double-strand break repair enzyme RexAB of Lactococcus lactis. J Bacteriol 183, 40714078.
Quiberoni, A., Rezaiki, L., El Karoui, M., Biswas, I., Tailliez, P. & Gruss, A. (2001b). Distinctive features of homologous recombination in an old microorganism, Lactococcus lactis. Res Microbiol 152, 131139.[CrossRef][Medline]
Sourice, S., Biaudet, V., El Karoui, M., Ehrlich, S. D. & Gruss, A. (1998). Identification of the Chi site of Haemophilus influenzae as several sequences related to the Escherichia coli Chi site. Mol Microbiol 27, 10211029.[CrossRef][Medline]
Sung, C. K., Li, H., Claverys, J. P. & Morrison, D. A. (2001). An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae. Appl Environ Microbiol 67, 51905196.
Tettelin, H., Nelson, K. E., Paulsen, I. T. & 36 other authors (2001). Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293, 498506.
Thoms, B. & Wackernagel, W. (1998). Interaction of RecBCD enzyme with DNA at double-strand breaks produced in UV-irradiated Escherichia coli: requirement for DNA end processing. J Bacteriol 180, 56395645.
Tiraby, G., Fox, M. S. & Bernheimer, H. (1975). Marker discrimination in deoxyribonucleic acid-mediated transformation of various Pneumococcus strains. J Bacteriol 121, 608618.[Medline]
Zieg, J. & Kushner, S. R. (1977). Analysis of genetic recombination between two partially deleted lactose operons of Escherichia coli K-12. J Bacteriol 131, 123132.[Medline]
Received 18 February 2004;
revised 23 April 2004;
accepted 27 April 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |