Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, 48149 Münster, Germany
Correspondence
Friedhelm Meinhardt
meinhar{at}uni-muenster.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ515540 (recA1) and AJ515541 (recA2).
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INTRODUCTION |
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Although the basic regulatory principles of the SOS network composed of LexA and RecA are considered to be a universal phenomenon in prokaryotes, recent studies of analogous systems in species from other bacterial taxa have revealed major variations from the E. coli model. While the SOS box of E. coli and most other members of the -subclass of Proteobacteria consists of the 16 bp palindrome CTGTN8ACAG (Walker, 1984
), the consensus sequence of the LexA-binding site in Xanthomonadaceae clearly differs (Campoy et al., 2002
). Deviating SOS boxes were also identified in the
- and
-subdivisions of Proteobacteria (Tapias et al., 2002
; Jara et al., 2003
; Campoy et al., 2003
). Furthermore, such differences appear not to be restricted to various repressor-binding sequences, but also include the number of din genes constituting the SOS regulon. While analyses of available genomic sequences revealed recA being present throughout the bacteria, lexA homologous genes, however, were found to be absent in some species (Liveris et al., 2004
). Furthermore, for other bacteria two copies of lexA were reported to exist (Jara et al., 2003
).
In the well-studied Gram-positive bacterium Bacillus subtilis, the transcriptional repressor of din genes is the LexA homologue DinR, which binds to a palindrome (termed the DinR box) with the consensus sequence CGAACRNRYGTTCG (Winterling et al., 1998). Furthermore, by analogy with E. coli, RecA functions as the activator of SOS induction (Yasbin et al., 1991
). So far, six loci [recA, dinR, uvrBA (formerly dinA), dinB, tagC (formerly dinC) and yneABynzC] have been shown to be under DinR control (Marrero & Yasbin, 1988
; Raymond-Denise & Guillen, 1991
; Cheo et al., 1991
; Kawai et al., 2003
). Compared to the multitude of din genes found in E. coli, the number of as yet identified and proven din loci in B. subtilis is low, suggesting that many din genes remain to be discovered. Expression of the B. subtilis recA gene is not only derepressed under SOS conditions as in E. coli, but is also activated during development of natural competence (Haijema et al., 1996
; Hamoen et al., 2001
). Such SOS-independent activation is mediated by the competence transcription factor ComK, which binds specifically to sequence motifs consisting of two AT boxes (consensus sequence AAAA-N5-TTTT) located upstream of recA and other late competence genes (Van Sinderen et al., 1995
). Activation of recA during competence development seems reasonable since uptake of exogenous DNA is followed by recombinational integration into the chromosome. Thus, expression of the two global regulons, i.e. SOS response and competence development, is linked in B. subtilis (Yasbin et al., 1991
, 1992
).
Taking into account the divergence concerning different SOS regulons among several Gram-negative bacterial groups, variations of the common pattern can be assumed within Gram-positive bacteria, too. However, except for the recA gene of Bacillus anthracis, which contains a Group I self-splicing intron (Ko et al., 2002), essentially nothing is known about recA expression in representatives of the genus other than B. subtilis. Thus, we looked for recA and its function in DNA repair in Bacillus megaterium. Interestingly, this biotechnologically relevant species (Vary, 1994
) has been reported to resist DNA damage much more efficiently than B. subtilis (English & Vary, 1986
). As a result of our work, B. megaterium unlike several other Bacillus species was found to harbour two recA genes, both being damage-inducible and functional in DNA repair.
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METHODS |
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Transcript and primer extension analysis of the B. megaterium recA genes.
Cells of B. megaterium were grown in minimal medium to an OD546 of 1·8. Cultures were then divided into two samples, one of which was supplemented with 0·2 µg MMC ml1. During further cultivation 5 ml samples were taken at hourly intervals, from which total RNA was isolated using hot phenol as previously described (Nahrstedt & Meinhardt, 2004). For analysis of heterologous recA transcription in E. coli, cells were cultivated in LB supplemented with 75 µg ampicillin ml1, and RNA was isolated by applying the RNeasy Midi Kit (Qiagen) according to the manufacturer's recommendations. In each case, RNA samples were additionally supplemented with 40 U RNasin Plus (Promega), and their concentration was determined spectrophotometrically. For Northern blotting, equal amounts of total RNA (20 µg for B. megaterium and 3 µg for E. coli) were separated by electrophoresis through 1·5 % (w/v) formaldehyde agarose gels with MOPS as the running buffer and subsequently transferred onto nylon membranes by vacuum blotting. After UV cross-linking for 5 min and prehybridization for 1 h, hybridization was carried out overnight at 55 °C; signals were subsequently detected with CDP-Star. In this manner, recA2 transcripts were analysed in RNA isolated from mutant MS991 by a recA2 RNA probe, whereas RNA from wild-type cells was analysed using a probe specific for recA1. These probes were generated by applying the DIG RNA labelling kit (Roche Diagnostics). The following PCR products were used as templates for in vitro transcription: a fragment ranging from nucleotide 1812 to 2369 (produced by A3 and A6) for recA1, and a fragment from nucleotide 897 to 1564 (produced by M9 and M8) for recA2 (nucleotide positions correspond to the respective sequence submitted to the EMBL database). The binding positions of these probes are depicted in Figs 1 and 3(a)
. In primer extension reactions total RNA (20 µg) isolated from cells treated with 0·2 µg MMC ml1 for 4 h was used. Synthesis of cDNA was performed with Superscript II RNase H Reverse Transcriptase (Invitrogen) according to the manufacturer's recommendations in combination with the IRD800 fluorescent-labelled primers A-P2-IRD (for recA1) and M10-IRD (for recA2), respectively. After heating the samples at 65 °C for 10 min, 200 U transcriptase was added in a total volume of 20 µl and incubated at 42 °C for 70 min. Samples were then kept at 95 °C for 5 min, and after digestion with RNase A for 15 min at 37 °C, cDNA was precipitated using 2-propanol and finally dissolved in sequencing-stopping solution. As templates in sequence reactions with the same primers, the following DNA fragments were used: a recA1 PCR product ranging from nucleotide 731 to 1219 (produced by A-P1 and A-P2) and a recA2 HindIII fragment comprising nucleotides 1 to 1298 (positions correspond to the respective sequence submitted to the EMBL database). Both sequencing and extension products were then analysed in parallel on a LI-COR sequencer, model 4000L.
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Construction of recA disruption vectors and gene knockout experiments.
For targeted gene disruption, derivatives of the temperature-sensitive shuttle vectors pUCTV2 (Wittchen & Meinhardt, 1995) and pUCTV3 were applied. The latter carries the bgl gene of Paenibacillus macerans encoding an extracellular (1,3-1,4)-
-glucanase as an additional marker which can be screened by using LB plates containing 0·02 % (w/v) lichenin and subsequently overlaying grown colonies with Congo red solution (0·2 %, w/v). A 1086 bp deletion spanning the complete ORF of recA1 was generated by applying flank A (amplified with A11/A12) and flank B (A8/A13) (see also Fig. 1
). For construction of pDRECA1, the respective deletion cassette was first cloned in pUCBM20, and subsequently ligated into the single EcoRI site of pUCTV3. Similarly, two different cassettes for the disruption of recA2 were developed: in pDRECMcat1, the disruption cassette consists of flank A (amplified with M1/M2) and flank B (M3/M4), creating a deletion of 1237 bp covering nearly the complete recA2 ORF (see also Fig. 3a
). pDRECMcat2 carries flank C (M7/M8) and flank B (mentioned above), causing a 337 bp deletion within the 3'-end of recA2. In both cases, the cat (chloramphenicol acetyltransferase) gene of Staphylococcus aureus was inserted in between the respective flanks. In pDRECMcat3, cat was directly integrated into the single HincII site within the centre of flank C, which was subsequently ligated into pUCTV3. Vector-carrying clones of B. megaterium DSM 319 or MS991 were selected on tetracycline-containing plates at 30 °C. Integrants carrying the entire vector inside the chromosomal locus were selected by growth on plates containing 1·25 µg tetracycline ml1 at 42 °C. Single colonies of such candidates were further cultivated on plates without tetracycline at 42 °C. Plasmid-cured clones were identified by loss of tetracycline resistance (and of
-glucanase activity if pUCTV3 derivatives were used).
Construction of recA expression vectors for complementation experiments in E. coli.
A 1719 bp fragment carrying the recA1 ORF including its promoter and terminator was amplified via PCR with primers A-P1 and A19 using Vent DNA polymerase. Similarly, a 1888 bp segment carrying the complete recA2 was isolated by applying the primers M-P1 and M6. Both fragments were each ligated into the single SmaI site of pACYC177 (in the same orientation relative to the vector's backbone), resulting in vectors pACYCrecA1 and pACYCrecA2. For construction of pACYCrecA2-P1, upstream of a promoterless recA2 (1510 bp; amplified with M9-SmaI and M6) which was integrated in pACYC177, a fragment including the recA1 promoter (489 bp; amplified with A-P1 and A-P2) was inserted. The sequences of all products were verified by DNA sequencing.
UV and MMC survival measurements.
Strains were cultivated to mid-exponential phase (B. megaterium in minimal medium and recombinant E. coli strains in LB with 75 µg ampicillin ml1). Cells were then washed in 15 mM NaCl and serially diluted to produce titres suitable for viable counts. For UV survival measurements, cells were plated on LB and irradiated with increasing doses of UV light using a UV-C lamp with an intensity of 1 J m2 s1. In the case of MMC survival measurements, cells were plated onto LB medium with increasing amounts of MMC. Plates were then incubated overnight in the dark, and relative survival was calculated by relating the number of colonies to those of control plates that had not been irradiated.
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RESULTS AND DISCUSSION |
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recA2 cannot be disrupted in B. megaterium
The UV survival of recA1 mutant MS991 indicates RecA2 to be functional in complementing loss of RecA1. In contrast, RecA2 does not appear to be able to fully complement the function of RecA1 in repair of DNA damage caused by MMC. In this context, it is of note that UV and MMC affect different RecA functions. Repair of UV damage primarily reflects NER, and thus the role of RecA in SOS induction. The bifunctional alkylating agent MMC predominantly generates interstrand cross-links through reaction with guanine residues. For B. subtilis it has been shown that the repair of such two-strand damage involves the concerted action of both RR and NER (Friedman & Yasbin, 1983). However, since RR is the more crucial component in interstrand repair, MMC is routinely used for monitoring RecA as a recombinase. Anyhow, our present survival studies do not provide evidence for different functions of the two proteins (e.g. RecA1 only functioning as a recombinase and RecA2 exclusively being involved in SOS induction). In fact, it appears plausible that the double gene dose present in B. megaterium might have a greater impact on survival when RecA acts as a recombinase rather than fulfilling its regulatory function. To further investigate the functionality of RecA2, we tried to inactivate the respective gene via targeted gene disruption. In addition to a deletion approach, the cat (chloramphenicol acetyltransferase) gene from S. aureus was introduced as an additional marker to enhance screening efficiencies. The general procedure of such a gene replacement using a replicating vector can be divided into two steps. Firstly, the entire vector is integrated into the desired chromosomal locus via one of the two homologous flanks; if the respective flank is entirely located within the ORF this first integration step already results in a gene disruption, while a flank situated outside the ORF leads to its restoration. Subsequent excision of the vector via the same flank restores the wild-type, while recombination via the other flank eventually results in the desired mutant. However, although three different disruption vectors displaying various combinations of flanks were used, all attempts to inactivate recA2 (either in wild-type DSM 319 or in recA1 mutant MS991) failed. In vector pDRECMcat1, constructed for a complete recA2 deletion, the respective flanks A and B were situated in such a way that either type of integration resulted in restoration of recA2, and indeed, both recombinational events (i.e. via flank A and B, respectively) were identified (data not shown); however, from further curings of such integrants only the wild-type lacking resistance to chloramphenicol was obtained. Following a second approach, vector pDRECMcat2 was applied to create a disruption at the 3'-end of recA2. Here, only integration via flank B was found, and again, further curings of these strains resulted exclusively in wild-type cells. In a third disruption strategy applying pDRECMcat3, integration via both flanks would already cause inactivation of the gene, and in fact, neither type of integrants was obtained. Altogether, these results clearly demonstrated that in our hands recA2 cannot be inactivated via targeted gene disruption, although recA1 and a number of other genes (Wittchen & Meinhardt, 1995
; Strey et al., 1999
; Lee et al., 2001
; Nahrstedt & Meinhardt, 2004
) have been successfully disrupted in B. megaterium by applying similar approaches. Apart from the fact that in several instances recA mutants were reported to show decreased viability as well as an irregular nucleoid morphology (Sciochetti et al., 2001
), in many bacteria such mutants can be obtained. In fact, we previously performed a knockout of the single recA gene from Bacillus licheniformis via a similar deletion strategy (H. Nahrstedt & F. Meinhardt, unpublished results). Hence, in general recA is not considered to be essential for cell viability. To our knowledge, the only other recA gene for which a disruption was achieved only under special circumstances is that of Streptomyces lividans (Muth et al., 1997
), in which viable mutant strains were found to lack only short extensions of the RecA C-terminus, and displayed residual RecA activity. Recent studies in S. lividans suggested that an additional mutation (resulting in a sporulation defect) is required to tolerate total recA deficency, although its role remains obscure (Vierling et al., 2001
). Although the observed inability to inactivate recA2 demands further investigation, our present data indicate that this locus might be required for sustaining cell viability in B. megaterium.
recA1 and recA2 complement recA defects in E. coli
It has been previously reported that expression of recA genes may have lethal effects on their cloning hosts, at least on multi-copy vectors; for example, cloning of recA from B. subtilis was found to be critical not only in E. coli, but also in B. subtilis itself (Marrero & Yasbin, 1988). Consistent with such findings, our initial attempts to clone the B. megaterium recA genes faced similar problems (not shown). Since no low-copy plasmids for Bacillus were readily available, we made use of appropriate E. coli vectors. Eventually, low-copy plasmid pACYC177 in combination with the native B. megaterium recA promoters facilitated cloning in a RecA null mutant of E. coli. Due to the high conservation of RecA proteins, such interspecies complementation approaches have been successfully applied not only for several recA genes of Gram-negative bacteria but also for some from Gram-positive species such as Clostridium perfringens (Johnston et al., 1997
) and B. anthracis (Ko et al., 2002
). The resulting vectors pACYCrecA1 and pACYCrecA2 (for cloning strategies see Methods) were used in parallel with pACYC177 (as the control) for transformation of the RecA null mutant E. coli JM109. Subsequently, recA transcription was confirmed by Northern analyses with the specific RNA probes (see also Figs 1 and 3a
), yielding transcripts with sizes of 1·3 kb (recA1) and 1·2 kb (recA2), which demonstrated that both promoters are functional in E. coli. However, although equal amounts of RNA were applied, the recA2 signals always appeared to be stronger compared to the ones of recA1, suggesting a higher expression level for recA2 (not shown). The two plasmid-carrying strains were then analysed with respect to single-filament growth (in the presence of 0·1 µg MMC ml1 for SOS induction) and by measuring survival either following UV irradiation or in the presence of MMC. Single-filament growth is a typical SOS phenomenon resulting from inhibition of cell division in the event of DNA damage (Huisman & D'Ari, 1981
) since SulA, a component of the SOS response in E. coli, prevents formation of the FtsZ ring. As induction of sulA depends on RecA, the phenotype of single-filament growth should not be visible in a RecA null mutant unless it is complemented. As shown in Fig. 6(a)
, complementation with plasmid pACYCrecA2 resulted in filamentation, while for the growth of the pACYCrecA1-harbouring strain this phenotype was hardly observable, with only minor deviations in cell morphology compared to the control strain. Thus, either the gene product of recA1 is less active in SOS induction, or the amount of heterologous protein is not sufficient for eliciting such a response in E. coli. Besides single-filament growth, RecA complementation should also increase survival of a RecA null mutant after UV irradiation. The data in Fig. 6(b)
show that UV survival of both recA strains was indeed enhanced. However, survivability of the pACYCrecA2 strain was significantly higher than that of the pACYCrecA1 strain. Analogous studies performed with MMC yielded similar results (Fig. 6c
); both genes enhanced survival in the presence of MMC, but the effect of recA2 was more significant than that of recA1. Altogether, the results demonstrate that both B. megaterium RecAs can mediate DNA repair in E. coli, as shown by the use of UV light as well as MMC. Strikingly, however, the complementation effects of the two RecAs were quite different in strength. RecA2 turned out to be much more effective, while the impact of RecA1 was rather poor. This discrepancy becomes particularly evident when filamentous growth is examined. One possible explanation for these results might be the fact that the transcription levels of the two genes are different. Similar to the results in B. megaterium, the Northern analysis in E. coli suggested a higher expression level of recA2 compared to recA1. When the effect of recA2 under control of the recA1 promoter was subsequently checked by the use of vector pACYCrecA2-P1 (see also Methods), the respective strain displayed the same phenotypes as obtained for the pACYCrecA1-carrying strain, i.e. hardly any filamentation was observable, and survival after UV irradiation or in the presence of MMC was clearly lower compared to the pACYCrecA2 strain (data not shown). These results indicate that the different complementation effects of the two B. megaterium RecAs are most likely due to different expression levels of the respective genes in E. coli rather than to different functionalities of the respective gene products.
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Conclusions
B. megaterium contains two functional recA copies, designated recA1 and recA2. Involvement of recA1 in DNA repair was evidenced by a recA1 mutant which displayed increased sensitivity to MMC. The lack of UV sensitivity of that particular mutant, however, indicated that recA2 could complement loss of recA1 with respect to UV-mediated DNA damage. Since all efforts to disrupt recA2 were unsuccessful, further functional studies were performed by complementation experiments in E. coli. Both genes were able to complement recA defects to a certain degree, with recA1 appearing less effective than recA2. Consistently, single-filament formation was induced by recA2 only. Such differences are most likely due to higher expression of recA2 in E. coli. Accordingly, similar expression patterns were observed in B. megaterium, in which transcription of both genes was inducible by DNA damage. This finding accords well with the presence of potential DinR boxes located within the promoters, suggesting negative transcriptional control by a DinR homologue as for the uvrBA locus of B. megaterium, upstream of which such a DinR-box-like motif was found as well (Nahrstedt & Meinhardt, 2004). Since the gene encoding the SOS repressor DinR was recently identified (H. Nahrstedt & F. Meinhardt, unpublished results), a regulon similar to the SOS systems in B. subtilis is present in B. megaterium. However, in marked contrast, B. megaterium possesses two functional recA genes, which presumably enhances efficiency of this highly conserved DNA repair mechanism.
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ACKNOWLEDGEMENTS |
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Received 15 September 2004;
revised 27 October 2004;
accepted 15 November 2004.
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