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Research Paper |
Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain1
Fundo de Defesa da Citricultura (Fundecitrus), 14807-040, VI. Melhado- C. P. 391, Araraquara, Sao Paulo, Brazil2
Centre de Recerca en Sanitat Animal (CReSA), Universitat Autònoma de Barcelona-Institut de Recerca i Tecnologia Agroalimentària (UAB-IRTA), Bellaterra, 08193 Barcelona, Spain3
Author for correspondence: Jordi Barbé. Tel: +34 93 581 1837. Fax: +34 93 581 2387. e-mail: jordi.barbe{at}uab.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Keywords: DNA damage, gene expression, SOS system
Abbreviations: DIG, digoxigenin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). In Escherichia coli, the SOS response has been widely studied, with at least 40 genes constituting this regulon (Fernandez de Henestrosa et al., 2000 ; Courcelle et al., 2001 ). The recA and lexA gene products control the expression of the E. coli SOS network to which both belong, LexA protein being the negative regulator (Walker, 1984 ). It has been demonstrated that the RecA protein is converted into an active conformation after binding to single-stranded DNA regions generated by DNA damage-mediated inhibition of replication or by enzymic processing of broken DNA ends (Sassanfar & Roberts, 1990 ). After activation, RecA promotes autocatalytic cleavage of LexA, inducing the SOS genes (Walker, 1984 ). E. coli LexA cleavage occurs in the Ala84-Gly85 bond and is mediated by its Ser119 and Lys156 residues by a similar mechanism to that of serine proteases (Little, 1991 ; Luo et al., 2001 ). Once DNA damage has been repaired, RecA is no longer activated and the LexA protein level increases again, inhibiting expression of SOS genes. Recently, it has been suggested that the product of dinI is involved in the arrest of RecA protein activation (Voloshin et al., 2001 ). Likewise, it has also been shown that some E. coli genes are DNA damage-inducible in a lexA-independent pathway, suggesting that other regulatory pathways could be involved in the E. coli DNA damage mediated response (Courcelle et al., 2001 ; Khil & Camerini-Otero, 2002 ).
The E. coli LexA repressor specifically binds regions placed upstream of SOS genes. These LexA binding sites are imperfect 16-bp palindromes whose consensus sequence is CTGTN8ACAG (Walker, 1984 ), which has been designated as the E. coli SOS box. The presence of a similar LexA regulon has been described in other bacterial groups, although the LexA-binding sequence is not conserved. Thus, the imperfect palindrome CGAACRNRYGTTYC and the GTTCN7GTTC direct repeat are the LexA boxes of Gram-positive bacteria and the alpha subclass Proteobacteria, respectively (Winterling et al., 1998 ; Fernandez de Henestrosa et al., 1998 ; Tapias & Barbé, 1999 ). While the recA gene has been found in all sequenced eubacterial genomes, lexA appears to be absent in some of them. In this way, a lexA-like gene does not seem to be present in Aquifex aeolicus, Borrelia burgdorferi, Chlamydia pneumoniae, Mycoplasma pneumoniae, Campylobacter jejuni, Helicobacter pylori and Porphyromonas gingivalis. Moreover, and with the exception of the plant-pathogenic bacterium Xylella fastidiosa, all members of the gamma subclass Proteobacteria whose genome sequences have been published (E. coli, Pseudomonas aeruginosa, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae) present an E. coli-like LexA box upstream of the promoter genes belonging to the SOS system. Nevertheless, and despite the absence of this E. coli-like LexA box, a lexA homologue gene is present in the genome of X. fastidiosa (Simpson et al., 2000 ). This fact indicates that, unlike what happens in both Gram-positive and alpha subclass Proteobacteria (Winterling et al., 1998 ; Tapias & Barbé, 1999 ), a significant heterogeneity exists in the very compact phylogenetic gamma subclass, with respect to the sequence of the control region of the common LexA regulon.
Differences among the LexA networks of several bacteria seems to be not only limited to the LexA box, but also to the regulation mechanism. So, whereas the E. coli LexA represses gene transcription by precluding RNA polymerase binding to the promoter region (Brent & Ptashne, 1981 ; Little et al., 1981 ; Bertrand-Burggraf et al., 1987 ), Rhodobacter sphaeroides LexA interferes with the clearance process besides also being able to act as a transcriptional activator (Tapias et al., 2002 ). All together indicate that it is not suitable to postulate a general model to explain how the LexA regulon functions in the Domain Bacteria, but this aspect must be studied in each one of their several phylogenetic groups. Likewise, the number of evolutionary analyses of regulatory sequences as well as of the constitution of different gene networks in the several bacterial phyla is increasing (Eisen & Hanawalt, 1999 ; Tan et al., 2001 ; Makarova et al., 2001 ; Panina et al., 2001 ; Rodionov et al., 2001 ; Roy et al., 2002 ). Nevertheless, and for this kind of studies to be carried out, a previous identification of new regulatory sequences, as well as of the genes which are under their control, is required.
In this context, and also to further characterize the differences existing among the LexA regulon of several groups of the gamma subclass Proteobacteria, the X. fastidiosa lexA gene has been cloned, overexpressed in E. coli and its product purified to determine the sequence to which it binds. Furthermore, the effect of DNA damage on the expression of X. fastidiosa genes whose homologues are under LexA regulation in E. coli has also been analysed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. E. coli and X. fastidiosa strains were grown at either 37 °C or 29 °C in LB or PW medium (Davis et al., 1981 ; Sambrook et al., 1992 ), 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 X. fastidiosa cells was extracted as described by Monteiro et al. (2001) .
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. To facilitate subcloning of some PCR-amplified DNA fragments, specific restriction sites were incorporated into the oligonucleotide primers. These restriction sites are shown in Table 2. Mutants in the X. fastidiosa 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 (Pharmacia Biotech). In all cases the entire nucleotide sequence was determined for both DNA strands.
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) corresponding to nucleotides -273 to -252 and +706 to +726, with respect to its proposed translational starting point (Simpson et al., 2000 ). The 999 bp PCR fragment obtained was cloned into the pGEM-T vector giving plasmid pUA945. To confirm that no mutation was introduced during the amplification reaction, the sequence of the fragment was determined. pUA945 was used as a template in PCR amplifications with LexANdeI and LexABamHI primers (Table 2), and the fragment obtained was again cloned in pGEM-T. The putative ATG initial triplet of LexA is part of the NdeI restriction site of the LexANdeI primer which enables IPTG-mediated overexpression of the X. fastidiosa LexA protein. After digestion with NdeI and BamHI, the lexA gene was cloned downstream of the T7 promoter of the pET15b expression vector (Novagen) carrying a N-terminal tag containing six histidine residues according to standard procedures (Sambrook et al., 1992 ), and the ligation mix was transformed into DH5
competent cells. Once confirmed that no mutation had been introduced in the lexA gene contained in the pUA973 plasmid by DNA sequencing, this was transformed into the E. coli BL21(DE3) strain for overexpression of the LexA protein.
Purification of the X. fastidiosa LexA protein.
His-tag fusion protein was purified by using the Talon Metal Affinity Resin Kit (Clontech) as described by the manufacturer. To carry out this, E. coli BL21(DE3) cells containing pUA973 were grown in 250 ml LB broth with shaking until OD550 0·8. Protein expression was induced by adding 10 mM IPTG to the culture, which was incubated at 37 °C for an additional 3 h. Cells were harvested by centrifugation at 5000 g for 10 min at 4 °C, resuspended in 10 ml washing solution (50 mM NaH2PO4, 300 mM NaCl, pH 7) to which protease inhibitor cocktail (Complete Mini, EDTA free; Roche) was added to the concentration indicated by the supplier. After sonication of the cells, the lysate was centrifuged at 14000 g for 20 min, and the supernatant was collected. Metal Affinity Resin (Clontech; 2 ml), previously equilibrated with washing buffer, was added to the supernatant and the mix was agitated for 20 min at room temperature to allow binding of the tagged LexA protein to the resin. Afterwards, it was centrifuged and washed several times with washing buffer. The resin was then transferred to a gravity-flow column and washed again with 10 ml washing buffer. Tagged LexA protein was eluted by adding 10 ml elution buffer (50 mM NaH2PO4, 300 mM NaCl, 150 mM imidazole) and the eluate was collected in 500 µl fractions. To determine the fraction containing the majority of the tagged LexA protein, SDS-PAGE in 15% polyacrylamide gels was performed according to standard procedures (Laemmli, 1970 ). The selected fraction was more than 98% pure as determined by Coomassie blue staining as shown in Fig. 1.
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). DNA probes were prepared by PCR amplification using one of the primers labelled at its 5' end with digoxigenin (DIG) (Table 2) and 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 ng purified X. fastidiosa 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. 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. When used, the protein amount of crude extract of IPTG-induced E. coli BL21(DE3) cells carrying either the pUA973 plasmid or the pET15b vector alone added to the binding mixture was about 500 ng. Protein concentrations were determined as described by Bradford (1976) . All mobility shift assays were repeated a minimum of three times to assure reproducibility of the results.
RT-PCR analysis of X. fastidiosa gene expression.
To determine the transcriptional organization of X. fastidiosa lexA and recA genes, reverse transcriptase (Roche) was used to generate cDNA by RT-PCR using total RNA from X. fastidiosa as a template and the pair of primers indicated in Table 2 and designed to amplify a PCR product of 355 bp if the lexA and recA genes constituted a single transcription unit. Total RNA from X. fastidiosa was prepared with Trizol as described by the supplier (Gibco) and treated with RNase-free DNase I (Roche) to assure the absence of contaminating DNA. RNA concentration and its integrity were determined by A260 measurements and 1% formaldehyde-agarose gel electrophoresis, respectively (Sambrook et al., 1992 ). In all RT-PCR experiments, the absence of contaminating DNA in RNA samples after RNase-free DNase I (Roche) treatment was confirmed by processing a duplicate of them in the same way but without reverse transcriptase addition.
Mitomycin C-mediated induction of several genes studied in this work was carried out by real-time quantitative RT-PCR analysis of total X. fastidiosa RNA with the LightCycler apparatus (Roche), using the LC-RNA master SYBR green I kit (Roche) and primers indicated in Table 2, following supplier’s instructions. The concentration of total RNA of both treated and untreated cultures was adjusted to the same value. The amount of mRNA of each gene was determined by plotting it against a standard curve generated by the amplification of an internal fragment of the X. fastidiosa 16S rRNA with the appropriate primers indicated in Table 2. In all determinations, the amount of mRNA of X. fastidiosa XF1375 and XF2223 genes, encoding tryptophan synthase and threonine synthase, respectively, was also determined. These two genes were used as negative controls because their expression is not DNA-damage-inducible in E. coli (Courcelle et al., 2001 ).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). To more exactly locate the binding region, the X. fastidiosa lexA promoter was divided into four fragments (designated as LexA1, LexA2, LexA3 and LexA4) obtained by PCR amplification with the oligonucleotides listed in Table 2, DIG end-labelled and then used as probes in gel retardation assays (Fig. 3a). A stable DNA–protein complex was observed when fragments LexA1, LexA2 and LexA3 were incubated in the presence of purified X. fastidiosa LexA, but there was no change in the mobility of the LexA4 fragment under the same conditions (Fig. 3b). These data indicate that the region to which the LexA protein binds, or at least a portion of it, must lie between positions -25 and -10 of the lexA promoter. No DNA–protein complex was detected when a 355 bp fragment comprising 295 bp of the upstream region of recA gene was used as a probe (Fig. 3b). This is in agreement with the close proximity existing between the X. fastidiosa lexA and recA genes, which is only 184 bp (Fig. 3a) and with the fact that both constitute a single transcriptional unit in this organism (Fig. 4) as shown by RT-PCR analysis of the X. fastidiosa RNA with RecAup and RecAdw primers beginning 111 bp upstream and 60 bp downstream of the translational stop and starting codons of the lexA and recA genes, respectively (Fig. 3a). It is worth noting that this is the first bacterial species described so far in which lexA and recA genes constitute a single transcriptional unit.
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, lanes 3 and 4, respectively). The same DNA–LexA complex was obtained when, instead of purified LexA repressor, crude extracts from IPTG-induced E. coli BL21(DE3) cells overexpressing the X. fastidiosa LexA (Fig. 1, lane 2) were used (Fig. 5a, lane 5). Moreover, no DNA–LexA complex was detected when crude extracts of E. coli BL21(DE3) cells carrying the pET15b plasmid alone were employed in gel retardation assays with the LexA3 fragment (Fig. 5a, lane 6). These results unequivocally demonstrate that the complex was indeed formed by the specific binding of the X. fastidiosa LexA protein to the lexA promoter.
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ACCA) and the right (CTAAAAGG) halves of this palindrome, and their effects on the mobility of the LexA3 fragment were analysed.
Results obtained indicated that no DNA–LexA complex was formed when probes containing substitutions in either the left or right halves of the palindrome were employed (Fig. 5b, lanes 3 and 5). Furthermore, a three-base insertion between the TTAG and CTAA motifs also abolished the gel retardation of the band (Fig. 5b, lane 4).
To determine which bases of this TTAGN8CTAA palindrome were directly involved in the LexA interaction with the DNA, the effect of a single substitution in each one of the nucleotides of the palindrome and its immediately surrounding bases was tested. Results obtained indicated that a single nucleotide substitution in every position of the TTAG motif, as well as in any of the first three nucleotides (CTA) of the second motif of the palindrome, eliminates the DNA–LexA complex formation (Fig. 6). Additionally, mutagenesis of either T or A placed immediately upstream of the CTA trinucleotide also prevents DNA–LexA complex formation (Fig. 6). In accordance with these data, neither of the mutant fragments that do not form the DNA–LexA complex was able to abolish the mobility shift of the wild-type fragment originated in the presence of purified X. fastidiosa LexA protein when used in competition experiments (data not shown). These results demonstrate that binding of the X. fastidiosa LexA protein to the lexA promoter requires the TTAGN6TACTA imperfect palindrome. In agreement with the fact that lexA and recA genes are co-transcribed (Fig. 3b and Fig. 4), no motif presenting a sequence related with this TTAGN6TACTA palindrome is found immediately upstream of the X. fastidiosa recA gene.
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).
LexA protein binds different gene promoters in X. fastidiosa and E. coli
As mentioned above, at least 40 genes are directly under the control of the LexA repressor in E. coli (Fernandez de Henestrosa et al., 2000 ; Courcelle et al., 2001 ). A TBLASTX search using each one of the encoded products of these E. coli LexA-regulated genes as a query has enabled us to demonstrate that, besides lexA and recA, only eight of them (uvrA, uvrB, ssb, recN, ruvAB, dinG, ftsK and yigN) are present in the genome of X. fastidiosa. Sequence analysis of the upstream regions of these eight X. fastidiosa genes revealed that none has a sequence related to the TTAGN6TACTA palindrome. In agreement with this fact, fragments containing 400 bp of the upstream region of these eight genes were not able to inhibit the DNA–X. fastidiosa LexA complex formation when used as unlabelled competitors in gel retardation experiments with the DIG-labelled LexA3 fragment as a probe (data not shown). Moreover, a search in the X. fastidiosa genome database using the Findpatterns Program of the Genetics Computer Group package (Devereux et al., 1984 ) has revealed the presence of the TTAGN6TACTA palindrome upstream of six ORFs: lexA–recA, XF1271, XF1417, XF1614, XF1823 and XF2313. It was further verified by competitive gel retardation assays (Fig. 7) that the LexA repressor only binds to the promoter of XF2313 encoding a putative DNA-modification methylase (Simpson et al., 2000 ). A similar case in which a putative LexA-binding sequence has been identified by in silico analysis, but which afterwards does not bind the LexA protein in vitro, has also been for the E. coli dinJ gene (Fernández de Henestrosa et al., 2000). The reasons for this lack of binding have not been established although they could be attributed to additional factors such as the structure of the region or the secondary role of the neighbouring bases of the regulatory sequence, as has been demonstrated for several transcriptional regulators (Winterling et al., 1998 ; Griffith & Wolf, 2001 ; Davis et al., 2002 ). In agreement with this, the sequence between both motifs of the imperfect palindrome TTAGN6TACTA is very different in XF1271, XF1417, XF1614 and XF1823 than in XF2313 and lexA (Fig. 7). It must be noted that in the X. fastidiosa genome there is another ORF (called XF2297) which is almost identical to XF2313 although it is slightly shorter probably due to a frameshift near its N terminus, suggesting that XF2297 derives from XF2313. Nevertheless, a 600 bp fragment containing the upstream region of XF2297 neither presents a copy of the TTAGN6TACTA motif nor binds the X. fastidiosa LexA protein (data not shown). The LexA-binding sequence was perhaps lost in the genetic rearrangement that produced XF2297 from XF2313.
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, 1997 ; Brooks et al., 2001 ).
Moreover, the TTAGN6TACTA palindrome is also present in an upstream region 169 nt long in the Xanthomonas campestris lexA gene, in which it has been shown that its own LexA protein binds, although the precise binding sequence has not been established (Yang et al., 2001 ). It must be noted that Xyl. fastidiosa and Xan. campestris LexA proteins present a high degree of identity in their N-terminal regions. According to this fact, the DNA binding sequence of both bacteria are expected to be very similar. Furthermore, the fact that the TTAGN6TACTA palindrome is perfectly conserved in the lexA promoters of Xanthomonas oryzae, Xyl. fastidiosa pv. oleander and Xyl. fastidiosa pv. almonder, as well as in the promoter of the XF2313-like genes of these two former species (Fig. 8), leads us to propose this sequence as the LexA binding site of the Order Xanthomonadales. The TTAGN6TACTA palindrome is the first LexA-binding sequence different from the E. coli-like one identified in the gamma subclass Proteobacteria.
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indicates that both transcriptional units are induced by DNA damage, whereas expression of two non-DNA-damage-related genes (those encoding tryptophan and threonine synthase) is not significantly stimulated by mitomycin C. It is worth noting that all LexA-regulated E. coli genes which are also present in X. fastidiosa, with the exception of ssb, are also DNA damage-inducible in this organism despite the fact they cannot bind to their own LexA protein (Table 3). A similar result has also been described for M. tuberculosis in which uvrA and uvrB are DNA damage-inducible but do not bind the LexA protein, whereas recN, dinP and dinG have their transcriptions neither stimulated by DNA injuries nor bind LexA (Brooks et al., 2001 ). The DNA damage-mediated transcription of genes that do not bind LexA protein in either M. tuberculosis or X. fastidiosa could be explained by two different mechanisms: i) the existence of a lexA-independent pathway of induction, or ii) the presence of an additional transcriptional regulatory factor which is under LexA control. Nevertheless, this last possibility seems to be unlikely since a gene encoding a putative transcriptional regulator, aside from lexA, presenting its own LexA-binding sequence has not been detected in either the M. tuberculosis or X. fastidiosa genomes, (Simpson et al., 2000 ; Cole et al., 1998 ). Additional investigations are necessary to better understand the molecular basis of this behaviour. Moreover, the fact that bacterial species belonging to phylogenetic groups as different as Gram-positive and the gamma subclass of Proteobacteria have DNA damage-inducible genes which are unable to bind the LexA repressor suggests that this phenomenon may be widespread in the bacterial world.
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ACKNOWLEDGEMENTS |
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254.[Medline]
Brent, R. & Ptashne, M. (1981). Mechanism of action of the lexA gene product. Proc Natl Acad Sci USA 78, 4204-4208.
Brooks, P. C., Movahedzadeh, F. & Davis, E. O. (2001). Identification of some DNA damage-inducible genes of Mycobacterium tuberculosis: apparent lack of correlation with LexA binding. J Bacteriol 183, 4459-4467.
Cole, S. T., Brosch, R., Parkhill, J. & 36 other authors. (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[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, J. M., French, W. J. & Schaad, N. (1981). Axenic culture of the bacteria associated with phony disease of peach and plum leaf scald. Curr Microbiol 6, 309-314.
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.
Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387-395.
Eisen, J. A. & Hanawalt, P. C. (1999). A phylogenomic study of DNA repair genes, proteins, and processes. Mutat Res 435, 171-213.[Medline]
Fernandez de Henestrosa, A. R., Rivera, E., Tapias, A. & Barbé, J. (1998). Identification of the Rhodobacter sphaeroides SOS box. Mol Microbiol 28, 991-1003.[Medline]
Fernandez 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.[Medline]
Griffith, K. L. & Wolf, R. E. (2001). Systematic mutagenesis of the DNA binding sites for SoxS in the Escherichia coli zwf and fpr promoters: identifying nucleotides required for DNA binding and transcription activation. Mol Microbiol 40, 1141-1154.[Medline]
Khil, P. P. & Camerini-Otero, R. D. (2002). Over 1000 genes are involved in the DNA damage response of Escherichia coli. Mol Microbiol 44, 89-105.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Little, J. W. (1991). Mechanism of specific LexA cleavage: autodigestion and the role of RecA coprotease. Biochimie 73, 411-422.[Medline]
Little, J. W., Mount, D. & Yanisch-Perron, C. R. (1981). Purified LexA protein is a repressor of the recA and lexA genes. Proc Natl Acad Sci USA 78, 4199-4203.
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.[Medline]
Makarova, K. S., Mironov, A. A. & Gelfand, M. S. (2001). Conservation of the binding site for the arginine repressor in all bacterial lineages. Genome Biol 2, 131-138.
Monteiro, P. B., Teixeira, D. C., Palma, R. R., Garnier, M., Bové, J. M. & Renaudin, J. (2001). Stable transformation of the Xylella fastidiosa citrus variegated chlorosis strain with oriC plasmids. Appl Environ Microbiol 67, 2263-2269.
Panina, E. M., Mironov, A. A. & Gelfand, M. S. (2001). Comparative analysis of Fur regulons in Gamma-proteobacteria. Nucleic Acids Res 29, 5195-5206.
Rivera, E., Vila, L. & Barbé, J. (1996). The uvrB gene of Pseudomonas aeruginosa is not DNA damage inducible. J Bacteriol 178, 5550-5554.
Rivera, E., Vila, L. & Barbé, J. (1997). Expression of the Pseudomonas aeruginosa uvrA gene is constitutive. Mutat Res 377, 149-155.[Medline]
Rodionov, D. A., Mironov, A. M. & Gelfand, M. S. (2001). Transcriptional regulation of pentose utilisation systems in the Bacillus/Clostridium group of bacteria. FEMS Microbiol Lett 205, 305-314.[Medline]
Roy, S., Sahu, A. & Adhya, S. (2002). Evolution of DNA binding motifs and operators. Gene 285, 169-173.[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 USA 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.[Medline]
Silhavy, T. J., Berman, M. L. & Enquist, L. W. (1984). Experiments With Gene Fusions. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Simpson, A. J. G., Reinach, F. C., Arruda, P. & 113 other authors (2000). The genome sequence of the plant pathogen Xylella fastidiosa. Nature 406, 151–159.[Medline]
Tan, K., Moreno-Hagelsieb, G., Collado-Vives, J. & Stormo, G. D. (2001). A comparative genomics approach to prediction of new members of regulons. Genome Res 11, 566-584.
Tapias, A. & Barbé, J. (1999). Regulation of divergent transcription from the uvrA-ssb promoters in Sinorhizobium meliloti. Mol Gen Genet 262, 121-130.[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.
Voloshin, O. N., Ramirez, B. E., Bax, A. & Camerini-Otero, R. D. (2001). A model for the abrogation of the SOS response by an SOS protein: a negatively charged helix in DinI mimics DNA in its interaction with RecA. Genes Dev 15, 415-427.
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.
Yang, Y. C., Yang, M. K., Kuo, T. T. & Tu, J. (2001). Structural and functional characterization of the lexA gene of Xanthomonas campestris pathovar citri. Mol Gen Genet 265, 316-326.
Received 4 June 2002;
accepted 26 July 2002.
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 J. M. Hare, S. N. Perkins, and L. A. Gregg-Jolly A Constitutively Expressed, Truncated umuDC Operon Regulates the recA-Dependent DNA Damage Induction of a Gene in Acinetobacter baylyi Strain ADP1. Appl. Envir. Microbiol., June 1, 2006; 72(6): 4036 - 4043. [Abstract] [Full Text] [PDF]
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 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]
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 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]
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S. Campoy, N. Salvador, P. Cortes, I. Erill, and J. Barbe Expression of Canonical SOS Genes Is Not under LexA Repression in Bdellovibrio bacteriovorus J. Bacteriol., August 1, 2005; 187(15): 5367 - 5375. [Abstract] [Full Text] [PDF]
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H. Nahrstedt, C. Schroder, and F. Meinhardt Evidence for two recA genes mediating DNA repair in Bacillus megaterium Microbiology, March 1, 2005; 151(3): 775 - 787. [Abstract] [Full Text] [PDF]
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A. R. Fernandez de Henestrosa, J. Cune, G. Mazon, B. L. Dubbels, D. A. Bazylinski, and J. Barbe Characterization of a New LexA Binding Motif in the Marine Magnetotactic Bacterium Strain MC-1 J. Bacteriol., August 1, 2003; 185(15): 4471 - 4482. [Abstract] [Full Text] [PDF]
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M. Jara, C. Nunuz, S. Campoy, A. R. Fernandez de Henestrosa, D. R. Lovley, and J. Barbe 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., April 15, 2003; 185(8): 2493 - 2502. [Abstract] [Full Text] [PDF]
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