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1 Centre de Recerca en Sanitat Animal (CReSA), Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
2 Biomedical Applications Group, Centro Nacional de Microelectrónica, 08193 Bellaterra, Spain
3 Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
Correspondence
Jordi Barbé
jordi.barbe{at}uab.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In Escherichia coli, the SOS regulon consists of more than 40 genes, whose functions are involved in DNA replication, DNA repair, mutagenesis and control of the cell cycle (Fernández de Henestrosa et al., 2000
; Courcelle et al., 2001). Under normal conditions, the SOS network remains repressed by the product of the lexA gene (Walker, 1984). The E. coli LexA protein binds specifically to a 16 bp palindrome (CTGTN8ACAG) named the LexA box, located in the promoter region of SOS genes, whose expression is normally down-regulated to a basal level (Little et al., 1981). After DNA-damage-mediated stalling of the replication fork, RecA acquires an active conformation (RecA*) by binding to ssDNA (Sassanfar & Roberts, 1990). RecA* mediates the autohydrolysis of the E. coli LexA repressor between residues Ala84 and Gly85, resulting in the expression of the SOS genes (Little, 1991; Little et al., 1980). Once DNA damage is effectively repaired, RecA* concentration decreases, LexA ceases to be hydrolysed, and the non-cleaved LexA synthesized de novo once again inhibits the transcriptional expression of the SOS network.
The evolutionary history of the SOS system across the domain Bacteria is complex. First of all, and with very few exceptions (such as Cytophaga, Flavobacterium, Bacteroides and Epsilonproteobacteria), the lexA gene seems to be present in all bacterial families in which drastic genetic reduction (such as that observed in Buchnera, Rickettsia, Aquifex and Mycoplasma, among others) has not taken place. Furthermore, a clear relationship seems to exist between the LexA-binding sequence and the branching order of several bacterial phylogenetic groups from their common ancestor (Mazón et al., 2004b). Therefore, and in agreement with the bacterial genome sequences known so far, several groups can be established. The first comprises all the Gram-positive bacteria and closely related phyla, such as the Cyanobacteria and the green non-sulfur bacteria. All these phyla possess the GAACN4GTTC sequence or a related motif as their specific LexA-binding sequence (Winterling et al., 1998; Fernández de Henestrosa et al., 2002; Mazón et al., 2004a). On the other hand, Alphaproteobacteria have a GTTCN7GTTC direct repeat as their LexA box (Fernández de Henestrosa et al., 1998; Tapias & Barbé, 1999), whereas Beta- and Gammaproteobacteria (Erill et al., 2003) share the same LexA-binding sequence as that found in E. coli (CTGTN8ACAG). However, an intermediate phylum (Fibrobacter) between Proteobacteria and Cyanobacteria possesses a LexA box that seems to represent a transition from the Gram-positive one to that seen in E. coli (Mazón et al., 2004b).
Several DNA repair genes, including some belonging to the canonical LexA regulon (recA and uvrA), are usually involved in the resistance of bacteria to acidic pH (Raja et al., 1991; Thompson & Blaser, 1995; Hanna et al., 2001). However, some bacterial species whose environmental growth conditions are very acidic show a constitutive expression of the recA gene that is mediated either by the absence of a LexA box on the recA promoter (e.g. Acidobacillus thioferrooxidans; Ramesar et al., 1989) or by the lack of a lexA regulatory gene (e.g. Helicobacter pylori; Tomb et al., 1997).
Acidobacterium capsulatum is a Gram-negative, acidophilic, chemo-organotrophic bacterium, containing menaquinone, of which very few isolates have been cultured (Kishimoto et al., 1991; Hiraishi et al., 1995). This micro-organism has been proposed to belong to a new bacterial phylum that branched approximately at the same time as the phylum Fibrobacter, and significantly earlier than the Proteobacteria (Ludwig et al., 1997; Quaiser et al., 2003). Despite its extreme growth conditions, no data exist on the DNA repair system of A. capsulatum. For this reason, and taking advantage of the fact that the A. capsulatum genome is being sequenced (http://www.tigr.org), here we report the cloning of the A. capsulatum lexA gene, the purification of its product, and the characterization of its binding site, in an effort to unravel both the composition of its LexA regulon and the evolution of this gene network across the domain Bacteria.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. E. coli was grown in LB medium at 37 °C and antibiotics were added to the cultures at the concentrations reported in Sambrook et al. (1992). A. capsulatum ATCC 51196 was grown in M-269 medium with 0·1 g l–1 yeast extract (pH 3·5) at 30 °C (Hiraishi et al., 1995). E. coli cells were transformed with plasmid DNA as described by Sambrook et al. (1992). Oligonucleotide primers for PCR, restriction enzymes, T4 DNA ligase and polymerase, and the ‘DIG DNA labelling and detection kit’, were from Roche. Genomic DNA of A. capsulatum was obtained by standard procedures (Sambrook et al., 1992) from a 10 ml culture grown at 30 °C in M-269. The DNA sequence of all PCR-mutagenized fragments was determined by the dideoxy method (Sanger et al., 1977) on an ALF sequencer (Amersham-Pharmacia).
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).
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). Rhodobacter sphaeroides LexA protein, also used in this work, had been previously purified (Tapias et al., 2002).
Mobility shift assays.
LexA–DNA complexes were detected by electrophoretic mobility shift assays (EMSAs), using purified LexA proteins. Typical 20 µl reactions containing 10 ng DIG-DNA labelled probe and 20 nM final concentration of either pure A. capsulatum or pure R. sphaeroides LexA were incubated in binding buffer (5 mM Tris/HCl, 5 mM MgCl2, 25 mM NaCl, 5 %, v/v, glycerol, 1 µg bulk carrier DNA and 50 µg BSA ml–1). After 30 min incubation, the reaction mixture was loaded onto a 6 % non-denaturing Tris/glycine/polyacrylamide gel. DNA–protein complexes were separated at 100 V for 1 h, and then transferred to a Biodine B membrane (Pall Gelman Laboratory), followed by the detection of DIG-labelled DNA–protein complexes, following the manufacturer's protocol (Roche).
RT-PCR analysis.
Mitomycin C-mediated induction of several genes was analysed by real-time quantitative RT-PCR of total A. capsulatum RNA with LightCycler (Roche), as described previously (Campoy et al., 2002; Bustin, 2002), and using the appropriate primers. The Titan One Tube RT-PCR system (Roche) was used, as described by Campoy et al. (2002), to perform RT-PCR experiments and to determine the transcriptional organization of the imuA-imuB-dnaE gene cassette.
Phylogenetic analysis.
Sequence data of F. succinogenes, Myxococcus xanthus and A. capsulatum were obtained from TIGR through their website at http://www.tigr.org. Protein sequences for all other organisms were acquired from the Microbial Genome Database for Comparative Analysis website (http://mbgd.genome.ad.jp/) and the US Department of Energy Joint Genome Institute website (http://genome.jgi-psf.org/mic_home.html). All proteins used for phylogenetic analyses are listed in Supplementary Table S1, where their locus, accession numbers and micro-organism abbreviations are also given. Alignments of protein sequences were carried out using a combined procedure to improve alignment quality. Protein sequences were first aligned through CLUSTALW version 1.83 (Thompson et al., 1994), using default (10), 25 and 5 gap-opening penalties (GOPs) for the multiple alignment stage, and generating three different alignments. These three alignments, together with a local alignment generated by the T-COFFEE lalign method, were integrated as libraries into T-COFFEE version 1.37 (Notredame et al., 2000) for optimization. The optimized alignment was then visually inspected with BioEdit version 5.0.9 (Hall, 1999) and submitted to Gblocks version 0.91b (Castresana, 2000) with the half-gaps setting and, otherwise, default parameters, in order to select conserved positions and discard poorly aligned and phylogenetically unreliable information.
Phylogenetic analyses of the refined alignments were carried out using MrBayes version 3.1.1 (Ronquist & Huelsenbeck, 2003), applying a mixed four-category Gamma distributed rate model plus proportion of invariable sites model (invgamma). MrBayes Metropolis-Coupled Markov Chain Monte Carlo runs were carried out with four independent chains for 106 generations. The resulting phylogenetic trees, which are the product of four independent MrBayes runs, were plotted with TREEVIEW version 1.6.6 (Page, 1996). In all cases, only branch support values over 0·85 are shown.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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b). To locate more precisely the LexA-binding region, the A. capsulatum lexA promoter was divided into two fragments (designated LexA1 and LexA2), which were obtained by PCR amplification with suitable oligonucleotides. The two fragments were DIG end-labelled and then used as probes in an EMSA (Fig. 2a). A stable DNA–protein complex was observed when both fragments were incubated in the presence of purified A. capsulatum LexA (Fig. 2c). These data indicate that the region to which the LexA protein binds was located downstream of position –35 of the lexA promoter.
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). To test whether this sequence was actually the A. capsulatum LexA box, an EMSA experiment with a LexA2-derivative fragment, in which the first GTTC motif was substituted by an AGGT tetranucleotide, was performed. Fig. 2(d) shows that the GTTC motif was needed by A. capsulatum LexA to successfully bind its promoter. Furthermore, R. sphaeroides LexA protein was also able to bind the wild-type LexA2 fragment, but not its mutant derivative (Fig. 3a). Likewise, the A. capsulatum LexA protein was able to bind the wild-type R. sphaeroides recA promoter but not a mutant derivative with a modified LexA Box (Fig. 3b). These experiments clearly demonstrate that R. sphaeroides and A. capsulatum recognize the same LexA-binding sequence. In this respect, it is worth noting that the same LexA box (GTTCN7GTTC) is present in the lexA gene promoter region of Solibacter usitatus sp. Ellin6076, another member of the phylum Acidobacteria whose sequence is being completed by the Joint Genome Institute (http://www.jgi.doe.gov).
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, 2004). Some of these genes (lexA, recA and ruvA) are also regulated by LexA in Bacillus subtilis (Dubnau & Lovett, 2002). Furthermore, it has recently been reported that a gene (dnaE2) that encodes a second alpha subunit of DNA polymerase III, and which is found either isolated in the chromosome (Mycobacterium tuberculosis) or belonging to a multiple gene cassette (many Beta- and Gammaproteobacteria species), is also under LexA control (Boshoff et al., 2003; Abella et al., 2004; Galhardo et al., 2005). To analyse the constitution of the A. capsulatum LexA regulon, TBLASTN was employed to search the A. capsulatum genomic database for recA, dinP, uvrA, ruvAB, ssb and dnaE2, using their respective homologues in the Alphaproteobacteria. It is worth noting that, besides all the searched proteobacterial or B. subtilis lexA-regulated genes (recA, dinP, uvrA, ruvAB and ssb), two independent copies (iid1 and iid2) of the multiple gene cassette constituted by the imuA-imuB-dnaE2 genes were also detected. Furthermore, the iid1 and iid2 cassettes both constitute single transcriptional units when analysed by RT-PCR (Fig. 4).
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shows that only iid1, iid2 and dinP upstream regions were able to bind the A. capsulatum LexA protein. It must be noted that a sequence search revealed that there are no ORFs immediately upstream of these genes, indicating that each gene is the first one of a transcriptional unit (data not shown). In accordance with these results, A. capsulatum LexA-binding sites were only found in iid1, iid2 and dinP promoter regions (data not shown).
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shows that, as expected, lexA, iid1 and iid2 transcriptional units are induced by DNA damage. It is worth noting also that, among the canonical proteobacterial LexA-regulated genes present in A. capsulatum, only recA is DNA-damage inducible in this organism, despite the fact LexA does not bind to its promoter region (Fig. 5).
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), and the same holds true for the phylum Fibrobacter (Griffiths & Gupta, 2001). Nevertheless, F. succinogenes possesses a LexA-binding sequence that seems to represent a transition between that of Gram-positive bacteria and that of Beta- and Gammaproteobacteria (Mazón et al., 2004b). In contrast, the A. capsulatum LexA box seems clearly related to that of the Alphaproteobacteria, which branched later than Deltaproteobacteria, a subclass in which F. succinogenes-related LexA recognition motifs can be observed (Campoy et al., 2003; Mazón et al., 2004b). This fact strongly suggested the possibility of a lateral gene transfer (LGT) event concerning the lexA gene between Acidobacteria and Alphaproteobacteria.
Accordingly, phylogenetic analyses using the LexA protein reveal a strong relationship between Acidobacteria and Alphaproteobacteria. As can be seen in Fig. 6, both types of LexA protein cluster together with a high support value. Conversely, when other proteins belonging to the Alphaproteobacteria SOS regulon (such as RecA, UvrA, Ssb and UvrD) are used for phylogenetic analysis, the placement of the Alphaproteobacteria is in concordance with the established phylogenetic location of that phylum (Ludwig et al., 1997), and is also supported by high support values (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), and this motif, with slight variations, extends also to green non-sulfur bacteria (Fernández de Henestrosa et al., 2002) and Cyanobacteria (Mazón et al., 2004a). The LexA-binding motif is overspecified in terms of information content (LexA boxes are apparently too long for the number of LexA sites in the genome), and this may be the reason behind the vast diversification observed in LexA-binding motifs, which has led to the proliferation of seemingly dissimilar LexA boxes, such as the aforementioned Gram-positive one and that observed in E. coli (CTGTN8ACAG). In spite of this, several major features can be identified among these divergent motifs, and we have previously provided experimental support for a basic evolutionary history of LexA-binding motifs based on a vertical inheritance model (Mazón et al., 2004b).
Among the different LexA motifs described so far, the Alphaproteobacteria LexA box is perhaps the most divergent and the most intriguing. First described in R. sphaeroides (Fernández de Henestrosa et al., 1998), the Alphaproteobacteria LexA box is monophyletic for this proteobacterial subclass (Erill et al., 2004), and presents a direct-repeat structure that, up to now, had not been described for any other bacterial clade. This fundamental departure from the standard palindromic structure of all other known LexA-binding motifs, together with a highly divergent protein sequence, have made it difficult to reconstruct the precise evolutionary history of the Alphaproteobacteria LexA, but we have previously shown that its most probable origin lies in an evolutionary pathway different from the one that gave rise to the Gamma- and Betaproteobacteria LexA proteins (Mazón et al., 2004b).
The identification here of an Alphaproteobacteria-like LexA box in the Acidobacteria, a lineage that is supposed to branch deeply from either the Chlamydia/Planctomyces or the Gram-positive lines (Hiraishi et al., 1995), thus represents a key piece of evidence in this respect and gives rise to several questions. On the one hand, the identification of the A. capsulatum LexA box demonstrates that the GTTCN7GTTC LexA-binding motif existed well before the Alphaproteobacteria emerged. Based on the tendency towards diversification observed in the LexA-binding sequence, it seems very unlikely that the same motif, and precisely such a divergent motif, should have arisen independently in two different phyla. Moreover, the ability reported here of Alphaproteobacteria and Acidobacterium LexA proteins to effectively bind one another's promoters is further strong evidence of a common origin for both proteins.
Taking into account that no motif similar to that of the Alphaproteobacteria has been identified so far in any of the studied phylogenetic groups that are intermediate between the Alphaproteobacteria and Acidobacteria, the most plausible explanation for the experimental evidence reported here seems to be an LGT event. In agreement with this line of reasoning, the incongruent placement of the Alphaproteobacteria in the LexA tree, clustering precisely with the Acidobacteria at the natural branching point of the latter, gives further credence to the hypothesis that an LGT event took place between the two clades. Furthermore, the fact that studies using RecA, UvrA, Ssb and UvrD proteins locate both Acidobacterium and the Alphaproteobacteria at their accepted branching points, in accordance with observations using well-established phylogenetic markers such as the 16s RNA, suggests that the transfer proposed here occurred from Acidobacteria, or an immediate ancestor presenting the GTTCN7GTTC motif as its LexA box, to the Alphaproteobacteria. In accordance with this line of reasoning, an alignment of several LexA proteins that are representative of different bacterial groups (Fig. 1) reveals that the Alphaproteobacteria LexA protein possesses a 33 amino acid insertion downstream of the predicted third helix (
3) of its binding domain that is not seen in any other bacterial group. This gives further credence to the direction of the LGT deduced from the LexA tree, and also supports the hypothesis that this insert does not affect the DNA binding domain (DBD) (Knegtel et al., 1995).
The data reported in this work also demonstrate that the A. capsulatum recA gene is DNA-damage inducible (Table 2), although the A. capsulatum LexA protein is unable to bind to the recA gene promoter (Fig. 5). A similar result has also been described for other bacterial species, such as M. tuberculosis, Xylella fastidiosa and Myx. xanthus, in which several DNA-repair-related genes (such as uvrA and ssb) are DNA-damage inducible, even though their expression is not directly under LexA protein control (Brooks et al., 2001; Campoy et al., 2002, 2003). However, to date, the mechanism controlling the LexA-independent DNA-damage-mediated induction of these genes is not known. Likewise, it is also not known if these bacterial species (M. tuberculosis, X. fastidiosa and A. capsulatum) possess a similar control mechanism to regulate the expression of DNA-damage-inducible genes that lack a LexA box in their promoters. Further work is necessary to understand how this lexA-independent gene induction takes place, as well as to determine its relevance in the bacterial response systems against DNA damage.
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ACKNOWLEDGEMENTS |
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Received 26 July 2005;
revised 16 December 2005;
accepted 19 December 2005.
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X174 used as a molecular weight (MW) marker is also shown.
