| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
2 Biomedical Applications Group, Centro Nacional de Microelectrónica, 08193 Bellaterra, Spain
3 Centre de Recerca en Sanitat Animal (CReSA), 08193 Bellaterra, Spain
4 Unité de Microbiologie, INRA, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle, France
Correspondence
Jordi Barbé
jordi.barbe{at}uab.es
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In E. coli, the LexA protein controls the expression of some 40 genes (Fernández de Henestrosa et al., 2000; Courcelle et al., 2001), including both the lexA and recA genes, which are, respectively, the negative and positive regulators of the SOS response (Walker, 1984). The E. coli LexA protein specifically recognizes and binds to an imperfect 16 bp palindrome with consensus sequence CTGTN8ACAG, designated the E. coli SOS or LexA box (Walker, 1984). Both in vitro and in vivo experiments have shown that binding to single-stranded DNA fragments, generated by DNA damage-mediated inhibition of replication, activates the RecA protein (Sassanfar & Roberts, 1990). Once in its active state, RecA promotes the autocatalytic cleavage of LexA, resulting in the expression of the genes regulated by this repressor (Little, 1991). Hydrolysis of the E. coli LexA protein is mediated by its Ser119 and Lys156 residues, in a mechanism similar to that of proteolysis by serine proteases (Luo et al., 2001). After DNA repair, the RecA protein ceases to be activated and, consequently, non-cleaved LexA protein returns to its usual levels, repressing again the genes that are under its direct negative control.
Even though some notable exceptions have been reported, the increasing availability of microbial genome sequences has revealed that LexA is present in many bacterial species and in most phyla. So far, all the identified and characterized LexA proteins display two conserved domains that are clearly differentiated. The N-domain, ending at the Ala-Gly bond where the protein is cleaved after DNA damage activation of RecA (Little, 1991), has three 
helices that are necessary for the recognition and binding of LexA to the SOS box (Fogh et al., 1994; Knegtel et al., 1995). Conversely, the C-domain contains amino acids that are essential for the serine-protease-mediated auto-cleavage and for the dimerization process necessary for repression (Luo et al., 2001).
The sequence of the LexA box is strongly conserved among related bacterial species. In fact, the LexA box has been shown to be monophyletic for several bacterial phyla, and this feature has been successfully exploited in phylogenetic analyses (Erill et al., 2003). Thus, in the Gram-positive phylum the LexA-binding motif presents a CGAACRNRYGTTYC consensus sequence (Winterling et al., 1998) that, with slight variations (Davis et al., 2002), is conserved among all its members and is also found in the phylogenetically close green non-sulfur bacteria that, nonetheless, are Gram-negative bacteria (Fernández de Henestrosa et al., 2002). Apart from the ‘Gammaproteobacteria’, in which the consensus sequence CTGTN8ACAG is monophyletic and seems to extend to those ‘Betaproteobacteria’ that possess a lexA gene (Erill et al., 2003), alternative LexA-binding sequences with a high degree of conservation have also been described in other groups. For instance, the direct repeat GTTCN7GTTC is the LexA-binding sequence of the ‘Alphaproteobacteria’ harbouring a lexA gene, a group that includes the Rhodobacter, Sinorhizobium, Agrobacterium, Caulobacter and Brucella genera (Fernández de Henestrosa et al., 1998; Tapias & Barbé, 1999). Still, in other phyla where the LexA-binding motif has been identified, more data are required to gauge the conservation of the LexA box. Such is the case of the ‘Deltaproteobacteria’, for which a CTRHAMRYBYGTTCAGS consensus motif has been identified in one of its members, the fruiting body forming Myxococcus xanthus (Campoy et al., 2003).
The existence of different LexA recognition motifs and the monophyletic or paraphyletic nature of those studied so far indicate that the appearance of new LexA-binding motifs marks turning points in the evolutionary history of both this protein and its respective host species. Previous work has demonstrated that the cyanobacteria LexA box (RGTACNNNDGTWCB) derives directly from that of Gram-positive bacteria (Mazón et al., 2004). Nevertheless, a huge gap is still apparent in the further evolutionary pathway of the LexA box that leads from the Cyanobacteria up to other bacterial phyla of later appearance, such as the Proteobacteria. Protein signature analyses have established that Fibrobacter succinogenes branched from a common bacterial ancestor immediately before the Proteobacteria phylum (Griffiths & Gupta, 2001). F. succinogenes is an anaerobic Gram-negative bacterium that inhabits the rumen and caecum of herbivores and, for a long time, this organism was included in the Bacteroides genus. Recent 16S rRNA analyses, however, have granted Fibrobacter a new bacterial phylum of its own (Maidak et al., 1999; Ludwig & Schleifer, 1999).
In an effort to recreate the evolutionary history of the LexA protein through the changes in its recognition sequence, and taking advantage of the fact that the F. succinogenes genome is now partially sequenced, the lexA gene of this bacterial species has been isolated and its encoded product has been purified to determine its DNA recognition sequence. The results obtained here are in accordance with the newly established branching point of F. succinogenes, and introduce a novel element that allows a finer drawing of the evolutionary path of the LexA recognition sequence from Gram-positive bacteria to ‘Gammaproteobacteria’.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
. E. coli and F. succinogenes ATCC 19169 strains were grown in LB (Sambrook et al., 1992) and in a chemically defined medium (Gaudet et al., 1992) with 3 g cellobiose l–1, respectively. Antibiotics were added to the cultures at reported concentrations (Sambrook et al., 1992). E. coli cells were transformed with plasmid DNA as described by Sambrook et al. (1992). All restriction enzymes, PCR-oligonucleotide primers, T4 DNA ligase and polymerase, and the DIG-DNA labelling and detection kit were from Roche. DNA from F. succinogenes cells was extracted as described by Forano et al. (1994).
|
. To facilitate subcloning of some PCR DNA fragments, specific restriction sites were incorporated into the oligonucleotide primers. These restriction sites are identified in Table 2. Mutants in the F. succinogenes lexA promoter were obtained by PCR mutagenesis, using oligonucleotides carrying designed substitutions (Table 2). The DNA sequence of all PCR-mutagenized fragments was determined by the dideoxy method (Sanger et al., 1977) on an ALF Sequencer (Amersham Pharmacia). In all cases the entire nucleotide sequence was determined for both DNA strands.
|
), corresponding to nucleotides –276 to –249 and +653 to +678, with respect to its proposed translational starting point. The 954 bp PCR fragment obtained was cloned into the pGEM-T vector (Promega), obtaining the pUA1033 plasmid. To confirm that no mutation was introduced during the amplification reaction, the sequence of the fragment was determined. The plasmid pUA1038 was constructed in order to create and express a glutathione S-transferase (GST)–F. succinogenes LexA fusion protein. The first step in the construction of this plasmid was to amplify the F. succinogenes lexA gene from plasmid pUA1033, using the primers LexAEcoRI and LexADw. The resulting DNA fragment was cloned into pGEM-T, to give pUA1037. Following excision with EcoRI and SalI, the lexA gene was inserted into the pGEX4T1 expression vector (Amersham Pharmacia) immediately downstream of the GST-encoding gene that is under the T7 promoter control. The initiation codon of the LexA protein was placed immediately downstream of the EcoRI sites in the LexAEcoRI primer, such that the lexA gene could be fused to GST in-frame. The insert of pUA1037 was sequenced in order to ensure that no mutations were introduced during amplification.
To overproduce the LexA–GST fusion protein, the pUA1037 plasmid was transformed into the E. coli BL21(
DE3) codon-plus strain (Stratagene). Cells of the resulting BL21 codon-plus strain were diluted in 0·5 l LB medium and incubated at 37 °C until they reached an OD600 of 0·8. Fusion-protein expression was induced at this time by the addition of IPTG to a final concentration of 1 mM. Following incubation for an additional 3 h at 37 °C, cells were collected by centrifugation for 15 min at 3000 g. The bacterial pellet was resuspended in PBS buffer (10 mM Na2HPO4, 1·7 mM KH2PO4, 140 mM NaCl, 2·7 mM KCl, pH 7·4) containing Complete Mini protease inhibitors cocktail (Roche). The resulting cell suspensions were lysed by sonication. Unbroken cells and debris were removed by centrifugation for 20 min at 14 000 g. The supernatant containing the GST–LexA fusion protein was incubated with PBS/Glutathione Sepharose 4B beads (Amersham Pharmacia) for 2 h at 4 °C, in order to affinity purify the fusion protein. The beads were then washed twice with PBS containing 0·1 % Triton and three times with PBS without detergent.
The sequence Leu-Val-Pro-Arg-Gly-Ser is located immediately downstream of the GST coding sequence in the pGEX4T vector series, and serves as a linker between the LexA and GST moieties of the fusion proteins. This hexapeptide is recognized by the protease thrombin, which cleaves at the Arg-Gly bond. It was therefore possible to release the F. succinogenes LexA protein from the Sepharose beads by incubating a 700 µl bed volume of beads with 25 units of thrombin (Amersham Pharmacia) in 1 ml PBS. The supernatants containing the F. succinogenes LexA protein with an additional 5 aa tail at their N-terminal (Gly-Ser-Pro-Glu-Phe), was visualized in a Coomassie blue-stained 13 % SDS-PAGE gel (Laemmli, 1970). Their purity was greater than 98 % (data not shown).
LexA proteins from Bacillus subtilis, E. coli, Anabaena PCC 7120, M. xanthus and Rhodobacter sphaeroides also used in this work had been previously purified (Winterling et al., 1998; Tapias et al., 2002; Campoy et al., 2003; Mazón et al., 2004).
Mobility-shift assays and DNase I footprinting.
LexA–DNA complexes were detected by electrophoresis mobility-shift assays (EMSAs) using purified LexA proteins. DNA probes were prepared by PCR amplification using one of the primers labelled at its 5' end with DIG (Roche) (Table 2), purifying each product in a 2–3 % low-melting-point agarose gel depending on DNA size. DNA–protein reactions (20 µl), typically containing 10 ng DIG-labelled DNA probe and 40 nM of the desired purified LexA protein, were incubated in binding buffer: 10 mM HEPES/NaOH (pH 8), 10 mM Tris/HCl (pH 8), 5 % glycerol, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 2 µg poly(dG-dC) and 50 µg BSA ml–1. After 30 min at 30 °C, the mixture was loaded onto a 5 % non-denaturing Tris/glycine polyacrylamide gel (pre-run for 30 min at 10 V cm–1 in 25 mM Tris/HCl, pH 8·5, 250 mM glycine, 1 mM EDTA). DNA–protein complexes were separated at 150 V for 1 h, followed by transfer to a Biodine B nylon membrane (Pall Gelman Laboratory). DIG-labelled DNA–protein complexes were detected by following the manufacturer's protocol (Roche). For the binding-competition experiments, a 300-fold molar excess of either specific or non-specific unlabelled competitor DNA was also included in the mixture. Protein concentrations were determined as described by Bradford (1976). All EMSAs were repeated a minimum of three times to ensure reproducibility of the results. DNase I footprinting assays were performed using the ALF Sequencer (Amersham Biosciences) as described previously (Patzer & Hantke, 2001; Campoy et al., 2003).
In silico phylogenetic analysis.
Preliminary sequence data of F. succinogenes unfinished genome was obtained from The Institute for Genomic Research (TIGR) through their website at http://www.tigr.org, and protein sequences for all other organisms were obtained from the Microbial Genome Database for Comparative Analysis website (http://mbgd.genome.ad.jp/) and the TIGR Comprehensive Microbial Resource (CMR). Identification of additional LexA-binding genes was carried out using the RCGScanner software (Erill et al., 2003), using known E. coli LexA-governed genes (Fernández de Henestrosa et al., 2000; Erill et al., 2003) and the here-reported LexA box of F. succinogenes to scan and then filter through the consensus method putative LexA-binding sites across the F. succinogenes genome.
For phylogenetic analyses, protein sequences for each gene under study were aligned using the CLUSTALW program (Higgins et al., 1994). Multiple alignments were then used to infer phylogenetic trees with the SEQBOOT, PROML and CONSENSE programs of the PHYLIP 3.6 software package (Felsenstein, 1989), applying the maximum-likelihood method on 100 bootstrap replicates. The resulting phylogeny trees were plotted using TreeView (Page, 1996).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
, the addition of increasing concentrations of LexA to a fragment extending from –154 to +169 of the F. succinogenes lexA gene promoter (with respect to its proposed translational starting point) produces one retardation band whose intensity is directly related to the amount of protein used. The formation of this DNA–LexA complex is specific, since it is sensitive to competition by an excess of unlabelled lexA promoter, but not to competition by non-specific DNA (Fig. 1b). Moreover, EMSAs performed using different-sized fragments containing the lexA promoter as a probe demonstrated that the LexA recognition sequence must lie between positions –72 and –57 of this promoter (data not shown).
|
). A visual inspection of this DNA sequence revealed the presence of the imperfect palindrome TGCCCAGTTGTGCA in its central region. To determine whether this motif was really involved in LexA binding, the effect of single substitutions in each nucleotide of this palindrome on the formation of the LexA protein–lexA promoter complex was analysed. Results (Fig. 3) indicate that a single substitution in any position of the TGC trinucleotide, as well as in the last C of the TGCCC motif, abolishes LexA binding. Likewise, mutagenesis of any position of the GTGCA motif also inhibits DNA–LexA complex formation. In contrast, the substitution of single nucleotides immediately surrounding either the TGCCC or the GTGCAT motifs does not affect LexA binding. Taken together, these results show that the TGCNCNNNNGTGCA imperfect palindrome is the F. succinogenes LexA-binding sequence of the lexA gene, since it is required for the binding of the LexA protein to the lexA promoter. Additional single substitutions generating two different perfect palindromes were carried out. The binding ratio of the LexA protein to both the TGCAC-N4-GTGCA palindrome and the wild-type sequence was practically the same. In contrast, there was a substantial reduction (about 75 %) in the LexA-binding ratio to the TGCCC-N4-GGGCA palindrome. This demonstrates that a TGCAC-N4-GTGCA perfect palindrome is the most likely consensus for the LexA box of F. succinogenes.
|
|
cI protein) (Little, 1984; Burckhardt et al., 1988; Nohmi et al., 1988). Nevertheless, of these two proteins only the prophage repressors are able to bind DNA-specific sequences. To eliminate the possibility that the F. succinogenes LexA was in fact a residual prophage repressor, a phylogenetic analysis of LexA proteins from several bacterial groups was performed. The results obtained (Fig. 4), together with phylogenetic trees including the cI repressor as an outgroup, indicate that the F. succinogenes LexA protein identified here is most probably a descendant of a Gram-positive LexA protein, and rule out the possibility of lateral gene transfer (LGT) from such an unspecified source as a residual prophage. To further validate this hypothesis, an in silico analysis of the F. succinogenes genome sequence was carried out using the RCGScanner program (Erill et al., 2003) in search of other genes with significant TGCNCNNNNGTGCA-like palindrome motifs upstream of their coding regions. Imperfect palindrome motifs were found upstream of the recA (TGCACAAAAGTTCA), uvrA (TATTCAAATGTTCA), ssb (TGCCTCCTCGAGCA) and ruvAB (AGCTCAAAGGCGCA) genes, and competitive EMSA experiments demonstrated that their promoters also bind F. succinogenes LexA (Fig. 5). Since these genes are under the control of the LexA protein in many bacterial species, the possibility that the F. succinogenes lexA gene identified here was the result of convergent evolution from a residual prophage repressor was definitively eliminated.
|
|
was constructed from a multiple alignment of available LexA protein sequences from relevant members of the Gram-positive and Cyanobacteria phyla and the ‘Alphaproteobacteria’, ‘Betaproteobacteria’ and ‘Gammaproteobacteria’ classes, and those of both F. succinogenes and M. xanthus. As expected, the resulting tree reveals that all these LexA proteins derive from the LexA of Gram-positive bacteria. However, closer examination of the phylogenetic tree indicates that at least two divergent paths originated from the Gram-positive LexA protein: one leading to the ‘Deltaproteobacteria’, ‘Betaproteobacteria’ and ‘Gammaproteobacteria’ LexA, with the F. succinogenes LexA as an intermediate step, and the other giving rise to the Cyanobacteria LexA and, unexpectedly, the ‘Alphaproteobacteria’ LexA.
To analyse whether the relationships between the different LexA proteins displayed in the phylogenetic tree were also reflected in their respective binding sites, a sequence comparison between the aforementioned LexA-binding sequences and that of F. succinogenes was carried out. This comparison reveals the presence of marked resemblances among several nucleotide positions (Fig. 6) that are consistent with a common phylogenetic origin. Moreover, and in accordance with the dual-branching hypothesis prompted by LexA protein phylogeny, on close inspection these resemblances again suggest two putative evolutionary lanes emerging from the Gram-positive LexA box: one giving rise to the Cyanobacteria and ‘Alphaproteobacteria’ LexA box and the other leading to both the F. succinogenes and M. xanthus LexA boxes and, ultimately, resulting in the ‘Betaproteobacteria’ and ‘Gammaproteobacteria’ LexA box.
|
), the B. subtilis LexA protein is able to bind the F. succinogenes LexA box with the introduction of only five substitutions (the T as well as the two internal Cs of the TGCCC motif, plus the internal G and A of GTGCAT), a number that, considering the evolutionary distance between the species, is remarkably low. Similarly, the M. xanthus LexA protein is able to bind a F. succinogenes lexA-derivative promoter in which only the flanking bases at each end of the TGCCCAGTTGTGCA palindrome have been substituted for a C and a G, respectively, and the G of the internal GTGCA motif has been replaced by a T. Finally, the E. coli LexA protein can effectively bind to the lexA promoter recognized by M. xanthus LexA if only three additional changes to the mutant promoter are made: substitution of the CC duet for TA on the TGCC tetranucleotide and a change from T to A in the TTC trinucleotide. The fact that both these generated motifs are very close to experimentally validated LexA-binding motifs of B. subtilis and E. coli (Fig. 7a) indicates that a mutational transition similar to the one proposed here could certainly have taken place between the LexA-binding sequences of these species.
|
, it was found that the simple addition of three nucleotides (chosen in accordance with the ‘Alphaproteobacteria’ LexA-box consensus sequence) between the AGTAC and GTTC motifs of the cyanobacterial LexA box was sufficient to enable the binding of the R. sphaeroides LexA protein to the mutant LexA box in the Anabaena lexA gene promoter (Fig. 7b). Furthermore, and although significant binding of the R. sphaeroides LexA protein to the Anabaena lexA promoter could be easily accomplished with the single insertion event described above, the introduction of an additional single-point mutation (substitution of T for A in the GTAC tetranucleotide) to the mutant Anabaena lexA promoter dramatically increased the recognition ability of the R. sphaeroides LexA repressor (Fig. 7b). Again, the fact that experimentally confirmed LexA-binding motifs closely resembling the motifs generated here are present in R. sphaeroides (Fig. 7b) gives further support to the evolutionary pathway here proposed. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
helices involved in DNA binding (Fig. 8). A straight comparison of the N-terminal domain of F. succinogenes and M. xanthus LexA protein sequences with the consensus sequences of this region for Gram-positive, Cyanobacteria and ‘Alphaproteobacteria’ LexA proteins reveals no amino acid insertions in the residues that, in E. coli, have been shown to participate directly in DNA-binding activity, nor in their immediate neighbours (Fig. 8).
|
helices that are involved in DNA binding. This suggests that, since their respective LexA boxes are markedly different, these amino acids must be required for the maintenance of the overall DNA recognition complex instead of being used for specific binding. This is the case for T5, Q8, E10, P26, S39, L50, G54 and R64, following the numbering of the E. coli LexA protein. Likewise, other residues present a low degree of substitution that, in addition, correspond to amino acids of the same family: L4, I15, E30, L47, K53, I56 and I66. This fact suggests that these residues must also be related to structural functions of the LexA helix–turn–helix (HTH) complex rather than to the specific recognition of the DNA-binding sequence. It has been suggested that, in E. coli, the third helix of the LexA HTH complex plays the leading role in specific DNA recognition (Knegtel et al., 1995). However, other residues in the remaining helices or between them must also play a significant part in specific DNA recognition, since a F. succinogenes LexA protein derivative in which the sequence of the third helix has been replaced through directed mutagenesis with that of E. coli LexA can not bind the E. coli-like CTGTN8ACAG motif (data not shown).
Furthermore, we have also demonstrated that a functional ‘Alphaproteobacteria’ LexA-binding sequence may be easily generated from the cyanobacterial one through a single insertion event while, in turn, the cyanobacterial LexA box derives directly from the Gram-positive one (Mazón et al., 2004). The use of DNA recognition motifs in combination with other phylogenetic evidence has been proposed earlier as a measure of divergence to refine phylogenetic analyses and as a milestone to highlight branching points in evolution (Rodionov et al., 2001; Rajewsky et al., 2002; Erill et al., 2003). Therefore, the experimental evidence of relatedness between Alpha and Cyanobacteria LexA boxes takes on a new relevance when combined with the fact that these two groups do also cluster together in the phylogenetic tree of LexA proteins (Fig. 4). This close relationship between ‘Alphaproteobacteria’ and Cyanobacteria is clearly at odds with the traditional positioning of the ‘Alphaproteobacteria’ class in the bacterial evolutionary tree, as prompted by the RecA protein (Fig. 9; Eisen, 1995) and 16S rRNA and signature protein phylogenies (Woese et al., 1984; Gupta & Griffiths, 2002), since these three phylogenetic techniques place the ‘Alphaproteobacteria’ very close to the ‘Betaproteobacteria’ and far removed from either Cyanobacteria or Gram-positive bacteria. The best explanation for this divergence from conventional phylogenetic data is to suppose that, after branching from other Proteobacteria classes, ‘Alphaproteobacteria’ lost their vertically transmitted lexA gene, but incorporated later a novel lexA copy through LGT from either a cyanobacterium or a bacterial species closely related to this phylum. This LGT event, however, must have occurred very early in the evolutionary history of the ‘Alphaproteobacteria’, since the same protein is present in all ‘Alphaproteobacteria’ that have not suffered major reductions in chromosome size (e.g. Rickettsia), and GC content and codon usage of the extant lexA genes are in perfect agreement with the mean values for each of the ‘Alphaproteobacteria’ hosting them. In this context, it should be stressed that the loss of the lexA gene does not seem to be a very unusual event in bacterial evolution, as it has already been described in several genera (including Aquifex, Borrelia, Campylobacter, Chlamydia, Helicobacter, Mycoplasma and Rickettsia). Up to now, a common characteristic of those bacteria for which the lack of a lexA gene had been described was that they had undergone a major reduction in chromosome size, suggesting that massive genome reduction was a convergent evolutionary cause for the loss of the lexA gene. However, given that the ‘Alphaproteobacteria’ species analysed here do not show significant reductions in genetic material, our data concerning their LexA protein breaks with this traditional assumption and hints at the possible existence of losses and lateral acquisitions of the lexA gene among bacteria. Although further work is still necessary to elucidate whether similar LGT events have taken place in other bacterial phyla, the evidence reported here of lateral transfer of the lexA gene sheds new light on the evolutionary history of complex regulatory networks like the LexA-governed SOS response and validates the previously reported use of regulatory motifs, in combination with phylogenetic and protein signature studies, as reliable indicators of phylogenetic history.
|
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Burckhardt, S. E., Woodgate, R., Scheuermann, H. R. & Echols, H. (1988). UmuD mutagenesis protein of Escherichia coli: overproduction, purification and cleavage by RecA. Proc Natl Acad Sci U S A 85, 1811–1815.
Campoy, S., Fontes, M., Padmanabhan, S., Cortes, P., Llagostera, M. & Barbe, J. (2003). LexA-independent DNA damage-mediated induction of gene expression in Myxococcus xanthus. Mol Microbiol 49, 769–781.[CrossRef][Medline]
Combet, C., Blanchet, C., Geourjon, C. & Deléage, G. (2000). NPS@: network protein sequence analysis. Trends Biochem Sci 25, 147–150.[CrossRef][Medline]
Courcelle, J., Khodursky, A., Peter, B., Brown, P. O. & Hanawalt, P. C. (2001). Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics 158, 41–64.
Davis, E. O., Dullaghan, E. M. & Rand, L. (2002). Definition of the mycobacterial SOS box and use to identify LexA-regulated genes in Mycobacterium tuberculosis. J Bacteriol 184, 3287–3295.
Eisen, J. A. (1995). The RecA protein as a model molecule for molecular systematic studies of bacteria: comparison of trees of RecAs and 16S rRNAs from the same species. J Mol Evol 41, 1105–1123.[Medline]
Erill, I., Escribano, M., Campoy, S. & Barbé, J. (2003). In silico analysis reveals substantial variability in the gene contents of the Gamma Proteobacteria LexA-regulon. Bioinformatics 19, 2225–2236.
Felsenstein, J. (1989). PHYLIP: phylogeny inference package (version 3.2). Cladistics 5, 164–166.
Fernández de Henestrosa, A. R., Rivera, E., Tapias, A. & Barbé, J. (1998). Identification of the Rhodobacter sphaeroides SOS box. Mol Microbiol 28, 991–1003.[CrossRef][Medline]
Fernández de Henestrosa, A. R., Ogi, T., Aoyagi, S., Chafin, D., Hayes, J. J., Ohmori, H. & Woodgate, R. (2000). Identification of additional genes belonging to the LexA regulon in Escherichia coli. Mol Microbiol 35, 1560–1572.[CrossRef][Medline]
Fernández de Henestrosa, A. R., Cuñé, J., Erill, I., Magnuson, J. K. & Barbé, J. (2002). A green nonsulfur bacterium, Dehalococcoides ethenogenes, with the LexA binding sequence found in gram-positive organisms. J Bacteriol 184, 6073–6080.
Fogh, R. H., Ottleben, G., Rüterjans, H., Schnarr, M., Boelens, R. & Kaptein, R. (1994). Solution structure of the LexA repressor DNA binding domain determined by 1H NMR spectroscopy. EMBO J 13, 3936–3944.[Medline]
Forano, E., Broussolle, V., Gaudet, G. & Bryant, J. A. (1994). Molecular cloning, expression and characterization of a new endoglucanase gene from Fibrobacter succinogenes S85. Current Microbiol 28, 7–14.
Gaudet, G., Forano, E., Dauphin, G. & Delort, A. M. (1992). Futile cycling of glycogen in Fibrobacter succinogenes as shown by in situ 1H-NMR and 13C-NMR investigation. Eur J Biochem 207, 155–162.[Medline]
Griffiths, E. & Gupta, R. S. (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.
Gupta, R. S. & Griffiths, E. (2002). Critical issues in bacterial phylogeny. Theor Popul Biol 61, 423–434.[CrossRef][Medline]
Higgins, D., Thompson, J., Gibson, T., Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Knegtel, R. M. A., Fogh, R. H., Ottleben, G., Rüterjans, H., Dumoulin, P., Schnarr, M., Boelens, R. & Kaptein, R. (1995). A model for the LexA repressor DNA complex. Proteins 21, 226–236.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Little, J. W. (1984). Autodigestion of lexA and phage lambda repressors. Proc Natl Acad Sci U S A 81, 1375–1379.
Little, J. W. (1991). Mechanism of specific LexA cleavage: autodigestion and the role of RecA coprotease. Biochimie 73, 411–422.[Medline]
Ludwig, W. & Schleifer, K. H. (1999). Phylogeny of Bacteria beyond the 16S rRNA standard. ASM News 65, 752–757.
Luo, Y., Pfuetzner, R. A., Mosimann, S., Paetzel, M., Frey, E. A., Cherney, M., Kim, B., Little, J. W. & Strynadka, C. J. (2001). Crystal structure of LexA: a conformational switch for regulation of self-cleavage. Cell 106, 585–594.[CrossRef][Medline]
Maidak, B. L., Cole, J. R., Parker, C. T., Jr & 11 other authors (1999). A new version of the RDP (Ribosomal Database Project). Nucleic Acids Res 27, 171–173.
Mazón, G., Lucena, J. M., Campoy, S., Fernández de Henestrosa, A. R., Candau, P. & Barbé, J. (2004). LexA-binding sequences in Gram-positive and cyanobacteria are closely related. Mol Genet Genomics 271, 40–49.[CrossRef][Medline]
Nohmi, T., Battista, J. R., Dodson, L. A. & Walker, G. C. (1988). RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci U S A 85, 1816–1820.
Norioka, N., Hsu, M. Y., Inouye, S. & Inouye, M. (1995). Two recA genes in Myxococcus xanthus. J Bacteriol 177, 4179–4182.
Page, R. D. M. (1996). TreeView: an application to display phylogenetic trees on personal computers. CABIOS 12, 357–358.
Patzer, S. I. & Hantke, K. (2001). Dual repression by Fe2+-Fur and Mn2+-MntR of the mntH gene, encoding an NRAMP-like Mn2+ transporter in Escherichia coli. J Bacteriol 183, 4806–4813.
Rajewsky, N., Socci, N., Zapotocky, M. & Siggia, E. D. (2002). The evolution of DNA regulatory regions for proteo-gamma bacteria by interspecies comparisons. Genome Res 12, 298–308.
Rodionov, D. A., Mironov, A. A. & Gelfand, M. S. (2001). Transcriptional regulation of pentose utilisation systems in the Bacillus/Clostridium group of bacteria. FEMS Microbiol Lett 205, 305–314.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1992). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, S. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74, 5463–5467.
Sassanfar, M. & Roberts, J. W. (1990). Nature of SOS-inducing signal in Escherichia coli. The involvement of DNA replication. J Mol Biol 212, 79–96.[CrossRef][Medline]
Tapias, A. & Barbé, J. (1999). Regulation of divergent transcription from the uvrA-ssb promoters in Sinorhizobium meliloti. Mol Gen Genet 262, 121–130.[CrossRef][Medline]
Tapias, A., Fernández, S., Alonso, J. C. & Barbé, J. (2002). Rhodobacter sphaeroides LexA has dual activity: optimising and repressing recA gene transcription. Nucleic Acids Res 30, 1539–1546.
Walker, G. C. (1984). Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev 48, 60–93.
Winterling, K. W., Chafin, D., Hayes, J. J., Sun, J., Levine, A. S., Yasbin, R. E. & Woodgate, R. (1998). The Bacillus subtilis DinR binding site: redefinition of the consensus sequence. J Bacteriol 180, 2201–2211.
Woese, C. R., Stackebrandt, E., Weisburg, W. C. & 8 other authors (1984). The phylogeny of purple bacteria: the alpha subdivision. Syst Appl Microbiol 5, 315–326.[Medline]
Received 10 May 2004;
revised 14 July 2004;
accepted 15 July 2004.
This article has been cited by other articles:
|
|
 |
|
  N. Jochmann, A.-K. Kurze, L. F. Czaja, K. Brinkrolf, I. Brune, A. T. Huser, N. Hansmeier, A. Puhler, I. Borovok, and A. Tauch Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays Microbiology, May 1, 2009; 155(5): 1459 - 1477. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Baumbach, A. Tauch, and S. Rahmann Towards the integrated analysis, visualization and reconstruction of microbial gene regulatory networks Brief Bioinform, January 1, 2009; 10(1): 75 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Abella, S. Campoy, I. Erill, F. Rojo, and J. Barbe Cohabitation of Two Different lexA Regulons in Pseudomonas putida J. Bacteriol., December 15, 2007; 189(24): 8855 - 8862. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Mazon, S. Campoy, I. Erill, and J. Barbe Identification of the Acidobacterium capsulatum LexA box reveals a lateral acquisition of the Alphaproteobacteria lexA gene. Microbiology, April 1, 2006; 152(Pt 4): 1109 - 1118. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Erill, S. Campoy, G. Mazon, and J. Barbe Dispersal and regulation of an adaptive mutagenesis cassette in the bacteria domain Nucleic Acids Res., January 10, 2006; 34(1): 66 - 77. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
