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J Bacteriol. Aug 2005; 187(15): 5367–5375.
PMCID: PMC1196036

Expression of Canonical SOS Genes Is Not under LexA Repression in Bdellovibrio bacteriovorus


The here-reported identification of the LexA-binding sequence of Bdellovibrio bacteriovorus, a bacterial predator belonging to the δ-Proteobacteria, has made possible a detailed study of its LexA regulatory network. Surprisingly, only the lexA gene and a multiple gene cassette including dinP and dnaE homologues are regulated by the LexA protein in this bacterium. In vivo expression analyses have confirmed that this gene cassette indeed forms a polycistronic unit that, like the lexA gene, is DNA damage inducible in B. bacteriovorus. Conversely, genes such as recA, uvrA, ruvCAB, and ssb, which constitute the canonical core of the Proteobacteria SOS system, are not repressed by the LexA protein in this organism, hinting at a persistent selective pressure to maintain both the lexA gene and its regulation on the reported multiple gene cassette. In turn, in vitro experiments show that the B. bacteriovorus LexA-binding sequence is not recognized by other δ-Proteobacteria LexA proteins but binds to the cyanobacterial LexA repressor. This places B. bacteriovorus LexA at the base of the δ-Proteobacteria LexA family, revealing a high degree of conservation in the LexA regulatory sequence prior to the diversification and specialization seen in deeper groups of the Proteobacteria phylum.

Bacterial cells contain several pathways targeted at the repair of DNA damage; among these, one of the most extensively studied is the SOS system. Initially described for Escherichia coli (34), the SOS response regulates in this organism the expression of up to 40 genes under direct control of the RecA and LexA proteins, which are also members of this regulon (12, 21). The products of E. coli SOS genes target a number of different cellular processes, such as inhibition of cell division, error-prone replication, or excision repair (34). The LexA protein is the repressor of the system and mediates its repression through the specific binding of its N-terminal domain to the regulatory motifs present in the promoter region of SOS genes. This regulatory motif, commonly dubbed the LexA box, has in E. coli a consensus sequence, CTGTN8ACAG (34), that has been reported also for many other members of the gamma and beta Proteobacteria (9). The RecA protein acts as sensor and inducer of the SOS system. Sensing is mediated by unspecific binding of RecA to single-stranded DNA fragments, generated by DNA damage-mediated interruption of replication (17), the enzymatic processing of broken DNA ends (29), or the inactivation of chromosome replication-involved genes (24). After binding, RecA acquires an active state that enables it to promote the autocatalytic cleavage of the LexA Ala84-Gly85 bond (20). This cleavage, carried out by LexA C-terminal residues Ser119 and Lys156, is similar to that mediated by serine proteases (19, 22) and effectively inhibits LexA from binding its target recognition sequences, thereby inducing the SOS response and activating the expression of DNA repair genes. Once DNA lesions have been repaired, RecA ceases to be activated and noncleaved LexA protein returns to its normal levels, repressing again the transcription of SOS genes. The lexA gene is widespread among bacteria and is present in most phylogenetic groups, for which different monophyletic LexA-binding motifs have been described (9, 10, 11, 35). Moreover, comparative analyses of the SOS system in different Proteobacteria classes (alpha, beta, and gamma) indicate that a common set of genes (lexA, recA, ssb, uvrA, and ruvCAB) is directly repressed by LexA in all these species and, therefore, constitutes the canonical gene composition of the SOS regulon in this phylum (9, 10).

Bdellovibrio bacteriovorus is a gram-negative, vibrio-shaped bacterium belonging to the δ-Proteobacteria class that preys on other gram-negative bacteria. Its typical life cycle consists of an obligate alternation between two distinct morphological stages: an attack and a growth phase (31). The first is initiated by a flagellated, notoriously fast free-swimming cell that is incapable of independent proliferation. In this phase, B. bacteriovorus recognizes, binds, attacks, and enters the periplasmic space of its prey. Following penetration, a variety of morphological and physiological changes take place in both B. bacteriovorus and its host, enabling B. bacteriovorus to grow efficiently into a septate filament at the expense of its host cellular material. Afterwards, the filamented B. bacteriovorus cell typically fragments into flagellated attack-phase cells, although it may form a bdellocyst that is able to linger in the ghost prey cell until harsh conditions or polluted environments die away. In spite of the fact that B. bacteriovorus wild-type strains are host-dependent predators, host-independent mutants have been isolated and have been used to study several aspects of this organism (30).

Since its parasitic lifestyle requires it to undergo regular and extensive contact with host populations, B. bacteriovorus is frequently exposed to a host of bacteriocins, microcins, and antibiotics that target DNA, and it is to be expected that such an environmental pressure should reflect on its DNA repair systems. Strict parasites such as members of the Rickettsiae, for instance, have adapted to similar conditions by adopting a constitutive expression of DNA repair genes that goes in conjunction with the loss of their lexA gene due to drastic genome reduction (2). No in vivo data are available on the B. bacteriovorus repair systems, but the sole information on this subject, coming mainly from the B. bacteriovorus genome sequence (26), indicates that B. bacteriovorus presents homologues of all the genes that constitute the canonical core of the Proteobacteria SOS system, including lexA. In this context, the LexA-binding sequence of this organism has been identified here to determine which genes constitute its LexA regulon and to analyze its response against DNA damage.


Bacterial strains and growth conditions.

The Bdellovibrio bacteriovorus HI100 host-independent strain used in this work was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), and it was grown at 30°C in PPYE medium (30). All plasmid constructions and cloning experiments were performed with E. coli DH5α by use of a pGEM-T vector (Promega). Plasmid DNA was transformed into competent E. coli cells as described previously (27).

Nucleic acid techniques.

RNA and DNA total extraction was carried out by standard methods (27). Genes and promoter fragments for electrophoretic mobility shift assays (EMSAs) were isolated by PCR from total DNA extraction, using suitable oligonucleotide primers designed in accordance with the B. bacteriovorus published sequence. Mutants in the B. bacteriovorus lexA promoter were obtained by PCR mutagenesis using oligonucleotides that carried the desired substitutions. The DNA sequence of all PCR-mutagenized fragments was determined by the dideoxy method (28) on an ALF sequencer (Pharmacia Biotech). Reverse transcriptase PCR (RT-PCR) assays were done using a Titan One Tube RT-PCR system (Roche) following the manufacturer's instructions. Real-time RT-PCR analysis of gene expression was performed for all genes as reported previously (5) and using specific internal oligonucleotide primers for each (Table (Table1)1) (26). In all cases, the absence of DNA in RNA samples was tested by PCR without reverse transcriptase addition. The specificity of primers was checked by cloning and sequencing each amplification product by the dideoxy method (28) on an ALF sequencer (Pharmacia Biotech). The RNA concentration of the gene to be analyzed was always normalized to that of the B. bacteriovorus total RNA as previously described (8). In silico identification of B. bacteriovorus LexA-regulated genes was carried out using RCGScanner, a consensus-building software for the prediction of regulatory motifs that has been previously described (9).

Oligonucleotide primers used in RT-PCR assays

Purification of LexA protein.

The B. bacteriovorus lexA gene was cloned by PCR using specific primers designed from its published sequence (26). The PCR fragment containing the B. bacteriovorus lexA gene was cloned into a pGEM-T vector and inserted into a pET15b expression vector. The pET15b derivative containing the B. bacteriovorus lexA gene was then transformed into the E. coli lexA (Def) BL21(DE3) codon plus strain (12) for overexpression of its encoding LexA protein, which was subsequently purified using a Talon metal affinity resin kit (Clontech) as reported previously (23). The purity of the B. bacteriovorus LexA protein thus obtained was above 95% as determined with Coomassie blue staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15%) polyacrylamide gels (data not shown) following standard methodology (27). The Bacillus subtilis purified LexA protein was kindly provided by Roger Woodgate. LexA protein from Anabaena PCC7120 also used in this work had been previously purified (23).

Electrophoresis mobility shift and footprinting assays.

LexA-DNA binding was analyzed for each gene promoter by electrophoresis mobility shift assays (EMSAs) using purified B. bacteriovorus LexA protein. DNA probes were prepared by PCR amplification with one of the primers labeled at its 5′ end with digoxigenin (DIG) and purifying each product in a 2% to 3% low-melting-point agarose gel. DNA-protein reaction mixtures (20 μl) typically containing 20 ng of the DIG-DNA-labeled probe and 80 nM purified LexA protein were incubated in binding buffer: 10 mM N-2-hydroxyethyl-piperazine-N′ 2-ethanesulphonic acid (HEPES), NaOH (pH 8), 10 mM Tris-HCl (pH 8), 5% glycerol, 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mg ml−1 of salmon DNA and 50 μg/ml bovine serum albumin. After 30 min at 30°C, the mixture was loaded onto a 6% nondenaturing Tris-glycine polyacrylamide gel (prerun for 30 min at 10 V/cm in 25 mM Tris-HCl [pH 8.5]-250 mM glycine-1 mM EDTA). DNA-protein complexes were separated at 150 V for 80 min, followed by transfer to a Biodine B nylon membrane (Pall Gelman Laboratory). DIG-labeled DNA-protein complexes were detected following the manufacturer's protocol (Roche). For the binding-competition experiments, a 300-fold molar excess of either specific or nonspecific unlabeled competitor DNA was also included in the mixture. All EMSAs were repeated a minimum of three times to ensure reproducibility of results. The DNase I footprinting assay carried out on the Alf sequencer (Pharmacia Biotech) was performed as described before (6). B. bacteriovorus LexA protein was added to the union reaction at a 160 nM final concentration.


Identification of the B. bacteriovorus LexA recognition sequence.

The first necessary step in the analysis of the B. bacteriovorus LexA network was the elucidation of the LexA binding sequence in this species. Taking advantage of the fact that self-regulation is to date a defining property of the lexA gene, electrophoresis mobility shift assays (EMSAs) were carried out with purified B. bacteriovorus LexA protein, using as a probe a DNA fragment extending from −201 to +73 (with respect to its translation start point) of the lexA gene. As expected, the addition of LexA protein specifically decreased the mobility of the lexA promoter fragment, while an excess of unlabeled lexA promoter abolished the delay, thereby confirming specific binding of LexA to the lexA promoter (Fig. (Fig.1).1). To further determine the position of the B. bacteriovorus LexA binding sequence, serial deletions of the lexA upstream promoter region were generated and analyzed in EMSAs with the purified LexA protein. EMSAs results suggested that the B. bacteriovorus LexA box is located between the −35 and −15 positions upstream of the lexA translation start codon (data not shown).

FIG. 1.
Electrophoretic mobility shift assay (EMSA) of the B. bacteriovorus lexA promoter in the absence (lane 1) or presence (lane 2) of 80 nM purified B. bacteriovorus LexA protein. To determine the specificity of LexA binding, a 300-fold molar excess of either ...

After honing in on the approximate location of the B. bacteriovorus LexA box, the specific sequence recognized by LexA was identified through footprinting experiments with a 172-bp fragment extending from positions −99 to +73 of the lexA promoter. The data thus obtained show that a core region of 35 nucleotides was protected by LexA binding when both lexA coding and noncoding strands were analyzed (Fig. (Fig.2),2), and a visual inspection of this protected sequence revealed the presence of the ATTTACACTGTAAGT imperfect palindrome. To further confirm that this palindrome was indeed the LexA-binding motif of B. bacteriovorus, and taking into account that the recA gene has been shown to be a robust candidate for LexA regulation, additional EMSAs were carried out with purified LexA protein and the promoter regions of the two annotated copies of recA (Bd0386 and Bd0512) in this organism.

FIG. 2.
DNase I footprinting assays with coding and noncoding Cy5-labeled strands of the DNA fragment containing the B. bacteriovorus lexA promoter in the absence or presence of 80 nM purified B. bacteriovorus LexA protein. The arrows indicate the transcriptional ...

The results revealed that only one of the two annotated recA genes (Bd0386) did bind LexA (Fig. (Fig.3),3), whereas the mobility of Bd0512 was not affected by LexA (data not shown). In this respect, it should be noted that, while the product of Bd0512 is an obvious homolog of E. coli RecA (64% identity using BLAST), the same does not hold true for the product of the Bd0386 gene (25% identity). To further elucidate whether the product of Bd0386 was a functional RecA protein, both Bd0386 and Bd0512 genes were cloned into pGEM-T vectors and used to trans-complement a recA-defective E. coli strain. Comparative analyses of survival rates following UV irradiation (data not shown) revealed that Bd0512 is able to complement a recA E. coli mutant, whereas complementation with Bd0386 does not increase survival rates compared to a noncomplemented E. coli recA strain (data not shown).

FIG. 3.
Effect of a 300-fold molar excess of B. bacteriovorus Bd0386 promoter (lane 3), lexA promoter (lane 4), or pGEM-T plasmid DNA (lane 5), used as nonspecific DNA, in the electrophoretic mobility of Bd0386 DIG-labeled promoter in presence of purified LexA ...

The above-presented data are in agreement with the distinct placement by phylogenetic inference algorithms of Bd0386 as a natural outgroup to a tree of bacterial RecA sequences (data not shown); together, both results give convincing support to the hypothesis that Bd0386 is not a recA gene. Moreover, the three open reading frames (ORFs) (Bd0385, Bd0384, Bd0383) immediately downstream of Bd0386 have been annotated, respectively, as a hypothetical protein with homology to COG0389 (dinP), a DNA polymerase III α subunit (dnaE), and a conserved hypothetical protein. Recently, a lexA-dependent DNA damage-inducible gene cassette consisting of a single polycistronic transcriptional unit that encompasses a sulA-like gene and dinP and dnaE genes has been shown to be widespread among members of the Proteobacteria (1). In the light of this, and taking into account that some internal regions of the RecA protein present a strong structural similarity to those of SulA (7), it seems likely that the product encoded by the Bd0386 ORF may be the SulA-like protein described in the aforementioned gene cassette.

In agreement with the above-described results, a close examination of the Bd0386 promoter region revealed the presence of a palindromic sequence very similar to that present in the lexA promoter (ATTTACATAGTAAGT), while no similar sequence could be found in the promoter region of its own recA gene (Bd0512). Comparison of the Bd0386 and the lexA motifs yielded the consensus sequence ATTTAC-AYW-GTAAGT, hinting at the dyad-spacer-dyad structure that is typical of LexA-binding motifs. To further elucidate which nucleotides of the observed motif were directly involved in LexA binding and thus constituted the LexA box of B. bacteriovorus, the LexA-binding motif present in the lexA promoter was analyzed through site-directed mutagenesis. Point mutations were introduced into the left (ATTTAC) and right (GTAAGT) halves of the lexA promoter motif and into the variable spacer region (ACT), and their effect on the electrophoretic mobility of the lexA promoter was analyzed through EMSAs.

EMSA results revealed that only the four internal bases of each dyad (TTAC and GTAA, respectively) were strictly required for binding and that changes in the spacer region did not affect binding (Fig. (Fig.4).4). Taken together, these data demonstrate that the presence of a TTACN3GTAA palindromic sequence is required for the binding of the B. bacteriovorus LexA protein to its own promoter, indicating that the B. bacteriovorus LexA box is substantially different from that of other members of the δ-Proteobacteria, such as Myxococcus xanthus or Geobacter sulfurreducens (6, 16).

FIG. 4.
Single-nucleotide substitutions in the ATTTACACTGTAAGT imperfect palindrome and their effect on the electrophoretic mobility of the B. bacteriovorus lexA promoter in presence of an 80 nM concentration of its own purified LexA protein. The mobility of ...

Presence of the B. bacteriovorus LexA recognition sequence upstream of additional genes.

After identification of the B. bacteriovorus LexA box, a search for its complete genome sequence was carried out using the consensus-building software RCGScanner (9) and the identified LexA-binding sequence (TTACN3GTAA) as a template. Surprisingly, besides the previously identified sequences in the promoters of lexA and Bd0386, only a low-scoring putative motif was detected in the vicinity of one of the annotated B. bacteriovorus uvrA genes (Bd2442). To analyze the binding affinity of this putative uvrA2 motif, and to determine whether the lack of additional positive results for other canonical SOS genes was due to an excessively astringent motif search, EMSA analyses were carried out on the promoter region of canonical SOS genes besides recA (i.e., uvrA1, uvrA2, ssb, and ruvCAB). The results (data not shown) revealed that none of these genes is able to bind B. bacteriovorus LexA protein and demonstrated that the low-scoring motif in the promoter of uvrA2 is not functional. Moreover, even though real-time RT-PCR experiments confirmed that both lexA and Bd0386 are induced in the presence of mitomycin C (Table (Table2),2), they also demonstrated that the expression of recA (Bd0512), uvrA1, uvrA2, and ssb is constitutive and not DNA damage inducible (Table (Table2).2). Conversely, it is worth noting that expression of the ruvCAB operon is positively triggered by DNA injuries (Table (Table2),2), even though no LexA binding motifs, or experimental LexA-binding activity, can be detected in its promoter.

Mitomycin C-mediated induction of several SOS genes in B. bacteriovorus

Regarding the Bd0386 gene, and its putative relation with the sulA-like gene of described sulA-dinP-dnaE multiple gene cassettes, RT-PCR analyses consistently demonstrated that Bd0386, Bd0385, Bd0384, and Bd0383 ORFs are encoded in a single polycistronic mRNA (Fig. (Fig.5).5). This result, together with the presence of a functional LexA-binding motif in the promoter of Bd0386, indicates that the Bd0386-Bd0385-Bd0384-Bd0383 operon is probably another instance of the aforementioned sulA-dinP-dnaE cassette and that, in accordance with that observed in other members of the Proteobacteria, expression of this gene cassette is directly regulated by LexA in B. bacteriovorus.

FIG. 5.
A) Structural arrangement of B. bacteriovorus Bd0386-dinP-dnaE genes and their surroundings. The position of primers used to determine the transcriptional organization is indicated. B) RT-PCR analysis of Bd0387-Bd0386-dinP-dnaE-Bd0383 transcripts present ...

The B. bacteriovorus LexA-binding motif binds the Cyanobacteria LexA repressor.

The here-identified LexA-binding sequence of B. bacteriovorus and the atypical conformation (lacking all the canonical SOS genes) of the B. bacteriovorus LexA regulon both raise interesting questions with regard to the particular evolution of the lexA gene in this species. In particular, and in view of the monophyletic character of LexA-binding sequences in many clades, the presence of a LexA-binding motif that is substantially divergent with respect to other previously reported δ-Proteobacteria LexA-binding sequences (of G. sulfurreducens and M. xanthus) is remarkable, as it hints at a period of heightened evolution of the LexA protein, which ultimately might have given rise to the prototypical LexA proteins and binding sequences observed in the gamma and alpha Proteobacteria subclasses (9, 10, 11). To further pin down the location of the B. bacteriovorus LexA protein in the aforementioned evolutionary thread, the relationship between the LexA protein of this organism and that of other species was explored here by an experimental cross-binding analysis of its LexA-binding sequence.

In an initial analysis, the binding capability of the LexA protein from closely related species to the B. bacteriovorus lexA promoter was analyzed through EMSAs. The results (data not shown) indicate that none of the available δ-Proteobacteria LexA proteins (G. sulfurreducens and M. xanthus) is able to bind the B. bacteriovorus lexA promoter and that the same holds true for both alpha (Rhodobacter sphaeroides) and gamma (E. coli) Proteobacteria LexA proteins, as would be expected if these proteins were the result of a further specialization from their common ancestor with B. bacteriovorus LexA.

Since the lexA gene is absent from several phylogenetic groups (e.g., Bacteroides, Green Sulfur bacteria) immediately preceding the appearance of the δ-Proteobacteria in all accepted phylogenies (13, 14), the most probable common ancestor of B. bacteriovorus LexA should be found with the members of the Cyanobacteria, whose LexA-binding sequence has recently been shown to be strongly related to the gram-positive one (23). To check whether this hypothesis held true at the binding sequence level, a cross-binding assay was carried out with Anabaena and B. subtilis LexA proteins. As shown in Fig. Fig.6,6, the B. subtilis LexA protein is unable to bind B. bacteriovorus lexA promoter but the Cyanobacteria LexA protein is manifestly able to bind it, as a LexA-DNA complex can be detected with the wild-type promoter but not with a mutant derivative unable to bind B. bacteriovorus LexA. This suggests that B. bacteriovorus LexA represents a primordial δ-Proteobacteria LexA protein, prior to the further specialization seen in the rest of species of this and other Proteobacteria classes.

FIG. 6.
EMSAs showing the binding ability of either Anabaena (lane 3) or B. subtilis (lane 4) LexA proteins to the wild-type (Wt) B. bacteriovorus lexA promoter. As a control, in lane 5 a mutant (Mut) derivative of lexA promoter, in which the A of the LexA binding ...


The results reported in this work convey conclusive evidence that the canonical SOS genes (recA, uvrA, ssb, and ruvCAB) are not repressed by LexA in B. bacteriovorus. It has also been established that the B. bacteriovorus lexA gene presents self-regulation and that its LexA protein binds and directly regulates a multiple-gene cassette consisting of four ORFs (Bd0386, Bd0385, Bd0384, Bd0383) whose homologues have been previously associated to DNA-repair activity (1). In concordance with the above-presented data, it has been shown that the lexA gene and the aforementioned multiple-gene cassette are both DNA damage inducible. Moreover, it has also been demonstrated that the expression of most Proteobacteria canonical SOS genes (recA, uvrA, and ssb) is constitutive and does not respond to DNA damage.

Taking into account the host-dependent lifestyle of B. bacteriovorus, which exposes it regularly to a collection of antagonist compounds (e.g., colicins, microcins), the fact that most canonical SOS genes are not DNA damage inducible should not constitute an unexpected result. Under such an environmental pressure, it is reasonable to expect a constitutive expression of DNA-repair pathways, such as those encompassed within the LexA regulon, to neutralize the deleterious effects of antagonist compounds. In fact, many of the obligate parasitic bacteria that thrive inside eukaryote cells, in which endogenous DNA-damaging agents abound, have lost their lexA gene and maintain most of their DNA repair genes under constitutive expression (2, 15, 32). In light of this, the here-reported DNA damage-mediated induction of the ruvCAB operon independent of LexA and the presence of a functional lexA in B. bacteriovorus constitute surprising results. In this respect, it should be noted that LexA-independent DNA damage induction of DNA repair genes has been previously reported for other bacteria (5, 6, 25) and it seems evident that, at least in some bacterial species, LexA might not be the sole regulator of the global response against DNA injuries.

In the above-described framework, however, both the presence of a functional lexA gene in B. bacteriovorus and the particular organization of its LexA regulon pose an intriguing question. If the environmental pressure stemming from the B. bacteriovorus host-dependent lifestyle is towards a progressive loss of SOS regulation, a counter-selective factor must be invoked to explain the persistence of a functional lexA gene. Apart from itself, B. bacteriovorus LexA only regulates a multiple-gene cassette with homologues in several Proteobacteria subclasses. Interestingly, in all the instances reported to date this multiple-gene cassette is explicitly regulated by LexA, either via a LexA-binding motif or through the constitution of a larger operon in which lexA is the leading gene (1, 9, 10). Taking into account that, despite the existence of a minimal core of LexA regulated genes in all bacteria studied so far which present a lexA gene, many conventional SOS genes are not under direct LexA control in some species, the fact that this multiple-gene cassette is overtly regulated by LexA in all its known occurrences suggests that regulation by LexA of one or more of its encoded products is either mandatory or extremely beneficial to the bacterial cell.

Having established that the first cassette gene (Bd0386) is not a recA homolog, we find that two complementary hypotheses arise to explain the regulation of this multiple gene cassette (typically composed of sulA, dinP, and dnaE homologues) in B. bacteriovorus and, hence, the conservation of a functional copy of LexA in this organism. On the one hand, the E. coli sulA product has been shown to be a cell division inhibitor that blocks FtsZ ring formation, leading to filamentation and, eventually, cell death (3). Hence, if Bd0386 is indeed a functional sulA homolog, its regulation by LexA should be mandatory, as has been observed in all known instances of E. coli sulA homologues (9). On the other hand, the E. coli DNA polymerase IV encoded by dinP has been shown to yield mutator phenotypes when deregulated (18), leading to a lower adaptive fitness (33). The second gene in B. bacteriovorus multiple gene cassette (Bd0385) is precisely a dinP homolog; therefore, the functional presence of LexA could be equivalently explained by the adaptive advantage of explicitly repressing the Bd0385 product until it is strictly necessary for survival (i.e., the SOS response). Following this line of thought, the third gene in the aforementioned gene cassette is a homolog of the dnaE protein. B. bacteriovorus presents two dnaE genes (Bd0384 and Bd2078), but the one included in the multiple gene cassette (Bd0384) shares the highest identity with the alpha subunit of the DnaE2 polymerase described for Mycobacterium tuberculosis, while Bd2078 is most probably the catalytic unit of its replicative polymerase. In this respect, the product of the M. tuberculosis dnaE2 gene has been shown to participate in error-prone DNA repair synthesis (4). Therefore, its is also possible that it is the presence of this dnaE2 gene in the B. bacteriovorus multiple gene cassette which has led to its explicit regulation by LexA, a fact that has been also observed in other species harboring the dnaE2 gene in the same or different genetic arrangements, such as M. tuberculosis, Pseudomonas putida, or Agrobacterium tumefaciens (1, 4).

Finally, the fact that the cyanobacterial LexA protein was able to recognize the B. bacteriovorus LexA binding box indicates that the sequence of this motif was significantly preserved during bacterial evolution, at least until the divergence process which generated more recent Proteobacteria classes took place. This line of reasoning is further reinforced by the fact that the B. bacteriovorus LexA network is markedly different from that described for members of the Cyanobacteria. The Anabaena LexA protein, for instance, has been shown to regulate the expression of canonical SOS genes such as recA, lexA, uvrA, or ssb (23) that, but for lexA, are not regulated in B. bacteriovorus. Taken together, the proximity of LexA-binding motifs and the differences in LexA regulon composition between B. bacteriovorus and the members of the Cyanobacteria support the idea that intense environmental pressure has led B. bacteriovorus to deregulate most of its canonical SOS genes in a relatively short evolutionary span and prior to the extensive diversification in LexA-binding motifs seen in ulterior Proteobacteria lineages.


This work was funded by grant BFM2004-02768/BMC from the Ministerio de Educación y Ciencia (MEC) de España and 2001SGR-206 from the Departament d'Universitats, Recerca i Societat de la Informació (DURSI) de la Generalitat de Catalunya and by the Consejo Superior de Investigaciones Científicas (CSIC). N. Salvador was recipient of a predoctoral fellowship from the DURSI, and S. Campoy is recipient of a postdoctoral contract from INIA-IRTA.

We are deeply grateful to Roger Woodgate for generously providing us with B. subtilis LexA. We acknowledge Joan Ruiz for his excellent technical assistance.


1. Abella, M., I. Erill, M. Jara, G. Mazón, S. Campoy, and J. Barbé. 2004. Widespread distribution of a lexA-regulated DNA damage-inducible multiple gene cassette in the Proteobacterium phylum. Mol. Microbiol. 54:212-222. [PubMed]
2. Andersson, S. G., A. Zomorodipour, J. O. Andersson, T. Sicheritz-Ponten, U. C. Alsmark, R. M. Podowski, A. K. Naslund, A. S. Eriksson, H. H. Winkler, and C. G. Kurland. 1998. The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396:133-140. [PubMed]
3. Bi, E., and J. Lutkenhaus. 1993. Cell division inhibitors SulA and MinC prevent formation of the FtsZ ring. J. Bacteriol. 175:1118-1125. [PMC free article] [PubMed]
4. Boshoff, H. I. M., M. B. Reed, C. E. Barry III, and V. Mizrahi. 2003. DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113:183-193. [PubMed]
5. Campoy, S., G. Mazón, A. R. Fernández de Henestrosa, M. Llagostera, P. Brant-Monteiro, and J. Barbé. 2002. A new regulatory DNA motif of the gamma subclass Proteobacteria: identification of the LexA protein binding site of the plant pathogen Xylella fastidiosa. Microbiology 148:3583-3597. [PubMed]
6. Campoy, S., M. Fontes, S. Padmanabhan, P. Cortes, M. Llagostera, and J. Barbé. 2003. LexA-independent DNA damage-mediated induction of gene expression in Myxococcus xanthus. Mol. Microbiol. 49:769-781. [PubMed]
7. Cordell, S. C., E. J. H. Robinson, and J. Löwe. 2003. Crystal structure of the SOS cell division inhibitor SulA and in complex with FtsZ. Proc. Natl. Acad. Sci. USA 100:7889-7894. [PMC free article] [PubMed]
8. Dinamarca, M. A., I. Aranda-Olmedo, A. Puyet, and F. Rojo. 2003. Expression of the Pseudomonas putida OCT plasmid alkane degradation pathway is modulated by two different global control signals: evidence from continuous cultures. J. Bacteriol. 185:4772-4778. [PMC free article] [PubMed]
9. Erill, I., M. Escribano, S. Campoy, and J. Barbé. 2003. In silico analysis reveals substantial variability in the gene contents of the gamma Proteobacteria LexA-regulon. Bioinformatics 19:2225-2236. [PubMed]
10. Erill, I., M. Jara, N. Salvador, M. Escribano, S. Campoy, and J. Barbé. 2004. Differences in LexA regulon structure among Proteobacteria through in vivo assisted comparative genomics. Nucleic Acids Res. 32:6617-6626. [PMC free article] [PubMed]
11. Fernández de Henestrosa, A. R., E. Rivera, A. Tapias, and J. Barbé. 1998. Identification of the Rhodobacter sphaeroides SOS box. Mol. Microbiol. 28:991-1003. [PubMed]
12. Fernández de Henestrosa, A. R., T. Ogi, S. Aoyagi, D. Chafin, J. J. Hayes, H. Ohmori, and R. Woodgate. 2000. Identification of additional genes belonging to the LexA regulon in Escherichia coli. Mol. Microbiol. 35:1560-1572. [PubMed]
13. Griffiths, E., and R. S. Gupta. 2001. The use of signature sequences in different proteins to determine the relative branching order of bacterial divisions: evidence that Fibrobacter diverged at a similar time to Chlamydia and the Cytophaga-Flavobacterium-Bacteroides division. Microbiology 147:2611-2622. [PubMed]
14. Gupta, R. S., and E. Griffiths. 2002. Critical issues in bacterial phylogeny. Theor. Popul. Biol. 61:423-434. [PubMed]
15. Himmelreich, R., H. Hilbert, H. Plagens, E. Pirkl, B. C. Li, and R. Herrmann. 1996. Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res. 24:4420-4449. [PMC free article] [PubMed]
16. Jara, M., C. Nuñez, S. Campoy, A. R. Fernández de Henestrosa, D. R. Lovley, and J. Barbé. 2003. Geobacter sulfurreducens has two autoregulated lexA genes whose products do not bind the recA promoter: differing responses of lexA and recA to DNA damage. J. Bacteriol. 185:2493-2502. [PMC free article] [PubMed]
17. Kenyon, C. J., and G. C. Walker. 1980. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc. Natl. Acad. Sci. USA 77:2809-2815. [PMC free article] [PubMed]
18. Kim, S. R., G. Maenhaut-Michel, M. Yamada, Y. Yamamoto, K. Matsui, T. Sofuni, T. Nohmi, and H. Ohmori. 1997. Multiple pathways for SOS-induced mutagenesis in Escherichia coli: an overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA. Proc. Natl. Acad. Sci. USA 94:13792-13797. [PMC free article] [PubMed]
19. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. [PubMed]
20. Little, J. W. 1991. Mechanism of specific LexA cleavage: autodigestion and the role of RecA coprotease. Biochimie 73:411-422. [PubMed]
21. Little, J. W., D. Mount, and C. R. Yanish-Perron. 1981. Purified LexA protein is a repressor of the recA and lexA genes. Proc. Natl. Acad. Sci. USA 78:4199-4203. [PMC free article] [PubMed]
22. Luo, Y., R. A. Pfuetzner, S. Mosimann, M. Paetzel, E. A. Frey, M. Cherney, B. Kim, J. W. Little, and C. J. Strynadka. 2001. Crystal structure of LexA: a conformational switch for regulation of self-cleavage. Cell 106:585-594. [PubMed]
23. Mazón, G., J. M. Lucena, S. Campoy, A. R. Fernández de Henestrosa, P. Candau, and J. Barbé. 2004. LexA-binding sequences in Gram-positive and cyanobacteria are closely related. Mol. Gen. Genomics 271:40-49. [PubMed]
24. O'Reilly, E. K., and K. N. Kreuzer. 2004. Isolation of SOS constitutive mutants of Escherichia coli. J. Bacteriol. 186:7149-7160. [PMC free article] [PubMed]
25. Pérez-Capilla, T., M.-R. Baquero, J.-M. Gómez-Gómez, A. Ionel, S. Martín, and J. Blázquez. 2005. SOS-independent induction of dinB transcription by β-lactam-mediated inhibition of cell wall synthesis in Escherichia coli. J. Bacteriol. 187:1515-1518. [PMC free article] [PubMed]
26. Rendulic, S., P. Jagtap, A. Rosinus, M. Eppinger, C. Barr, C. Lanz, H. Keller, C. Lambert, K. J. Evans, A. Goesmann, F. Meyer, R. E. Sockett, and S. C. Schuster. 2004. A predator unmasked: life cycle of Bdellovibrio bacteriovorus from a genomic perspective. Science 303:689-692. [PubMed]
27. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
28. Sanger, F., S. Nicklen, and S. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. [PMC free article] [PubMed]
29. Sassanfar, M., and J. W. Roberts. 1990. Nature of SOS-inducing signal in Escherichia coli. The involvement of DNA replication. J. Mol. Biol. 212:79-96. [PubMed]
30. Seidler, R. J., and M. P. Starr. 1969. Isolation and characterization of host-independent bdellovibrios. J. Bacteriol. 100:769-785. [PMC free article] [PubMed]
31. Starr, M. P., and R. J. Seidler. 1971. The bdellovibrios. Annu. Rev. Microbiol. 25:649-678. [PubMed]
32. Stephens, R. S., S. Kalman, C. Lammel, J. Fan, R. Marathe, L. Aravind, W. Mitchell, L. Olinger, R. L. Tatusov, Q. Zhao, E. V. Koonin, and R. W. Davis. 1998. Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282:754-759. [PubMed]
33. Viguera, E., M. Petranovic, D. Zaharadka, K. Germain, D. S. Ehrlich, and B. Michel. 2003. Lethality of bypass polymerases in Escherichia coli cells with a defective clamp loader complex of DNA polymerase III. Mol. Microbiol. 50:193-204. [PubMed]
34. Walker, G. C.,. 1984. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol. Rev. 48:60-93. [PMC free article] [PubMed]
35. Winterling, K. W., D. Chafin, J. J. Hayes, J. Sun, A. S. Levine, R. E. Yasbin, and R. Woodgate. 1998. The Bacillus subtilis DinR binding site: redefinition of the consensus sequence. J. Bacteriol. 180:2201-2211. [PMC free article] [PubMed]

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