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Jordi Barbé - One of the best experts on this subject based on the ideXlab platform.

  • An SOS Regulon under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria
    Journal of bacteriology, 2015
    Co-Authors: Neus Sanchez-alberola, Jordi Barbé, Susana Campoy, David Emerson, Ivan Erill
    Abstract:

    The SOS response is a transcriptional regulatory network governed by the LexA repressor that activates in response to DNA damage. In the Betaproteobacteria, LexA is known to target a palindromic sequence with the consensus sequence CTGT-N8-ACAG. We report the characterization of a LexA regulon in the iron-oxidizing betaproteobacterium Sideroxydans lithotrophicus. In silico and in vitro analyses show that LexA targets six genes by recognizing a binding motif with the consensus sequence GAACGaaCGTTC, which is strongly reminiscent of the Bacillus subtilis LexA-binding motif. We confirm that the closely related Gallionella capsiferriformans shares the same LexA-binding motif, and in silico analyses indicate that this motif is also conserved in the Nitrosomonadales and the Methylophilales. Phylogenetic analysis of LexA and the alpha subunit of DNA polymerase III (DnaE) reveal that the organisms harboring this noncanonical LexA form a compact taxonomic cluster within the Betaproteobacteria. However, their LexA gene is unrelated to the standard Betaproteobacteria LexA, and there is evidence of its spread through lateral gene transfer. In contrast to other reported cases of noncanonical LexA-binding motifs, the regulon of S. lithotrophicus is comparable in size and function to that of many other Betaproteobacteria, suggesting that a convergent SOS regulon has reevolved under the control of a new LexA Protein. Analysis of the DNA-binding domain of S. lithotrophicus LexA reveals little sequence similarity with that of other LexA Proteins targeting similar binding motifs, suggesting that network structure may limit site evolution or that structural constrains make the B. subtilis-type motif an optimal interface for multiple LexA sequences. IMPORTANCE Understanding the evolution of transcriptional systems enables us to address important questions in microbiology, such as the emergence and transfer potential of different regulatory systems to regulate virulence or mediate responses to stress. The results reported here constitute the first characterization of a noncanonical LexA Protein regulating a standard SOS regulon. This is significant because it illustrates how a complex transcriptional program can be put under the control of a novel transcriptional regulator. Our results also reveal a substantial degree of plasticity in the LexA recognition domain, raising intriguing questions about the space of Protein-DNA interfaces and the specific evolutionary constrains faced by these elements.

  • Cohabitation of Two Different LexA Regulons in Pseudomonas putida
    Journal of Bacteriology, 2007
    Co-Authors: Marc Abella, Ivan Erill, Susana Campoy, Fernando Rojo, Jordi Barbé
    Abstract:

    In contrast to the vast majority of the members of the domain Bacteria, several Pseudomonas and Xanthomonas species have two LexA genes, whose products have been shown to recognize different LexA binding motifs, making them an interesting target for studying the interplay between cohabiting LexA regulons in a single species. Here we report an analysis of the genetic composition of the two LexA regulons of Pseudomonas putida KT2440 performed with a genomic microarray. The data obtained indicate that one of the two LexA Proteins (LexA1) seems to be in control of the conventional Escherichia coli-like SOS response, while the other LexA Protein (LexA2) regulates only its own transcriptional unit, which includes the imuA, imuB, and dnaE2 genes, and a gene (PP_3901) from a resident P. putida prophage. Furthermore, PP_3901 is also regulated by LexA1 and is required for DNA damage-mediated induction of several P. putida resident prophage genes. In silico searches suggested that this marked asymmetry in regulon contents also occurs in other Pseudomonas species with two LexA genes, and the implications of this asymmetry in the evolution of the SOS network are discussed.

  • Insights into the LexA regulon of Thermotogales
    Antonie van Leeuwenhoek, 2006
    Co-Authors: Gerard Mazón, Susana Campoy, Antonio R. Fernández De Henestrosa, Jordi Barbé
    Abstract:

    The LexA genes of Thermotoga maritima and Petrotoga miotherma , both members of the Order Thermotogales , have been cloned and their transcriptional organization, as well as the functional characteristics of their encoded products, analyzed. In both bacterial species, the LexA gene was found to be co-transcribed together with another four ( T. maritima ) or three ( P. miotherma ) upstream open-reading frames. The P. miotherma LexA was able to bind promoters of both the cognate LexA encoding operon and the uvrA gene but not to that of the recA . Conversely, LexA Protein and crude cell extracts from T. maritima were unable to bind promoters governing the expression of either its LexA or recA genes. In agreement with these observations, no functional copy of the P. miotherma LexA box, corresponding to the GANTN_6GANNAC motif, seems to be present in the T. maritima genome. Giving support to the proposal that the evolutionary branching order of the Order Thermotogales is very close to that of Gram-positive bacteria, the P. miotherma LexA Protein was still able to recognize the previously described LexA-binding sequence for Gram-positive bacteria.

  • Identification of the Acidobacterium capsulatum LexA box reveals a lateral acquisition of the Alphaproteobacteria LexA gene.
    Microbiology (Reading England), 2006
    Co-Authors: Gerard Mazón, Ivan Erill, Susana Campoy, Jordi Barbé
    Abstract:

    Acidobacterium capsulatum is the most thoroughly studied species of a new bacterial phylogenetic group designated the phylum Acidobacteria. Through a tblastn search, the A. capsulatum LexA gene has been identified, and its product purified. Electrophoretic mobility shift assays have shown that A. capsulatum LexA Protein binds specifically to the direct repeat GTTCN(7)GTTC motif. Strikingly, this is also the LexA box of the Alphaproteobacteria, but had not previously been described outside this subclass of the Proteobacteria. In addition, a phylogenetic analysis of the LexA Protein clusters together Acidobacterium and the Alphaproteobacteria, moving the latter away from their established phylogenetic position as a subclass of the Proteobacteria, and pointing to a lateral gene transfer of the LexA gene from the phylum Acidobacteria, or an immediate ancestor, to the Alphaproteobacteria. Lastly, in vivo experiments demonstrate that the A. capsulatum recA gene is DNA-damage inducible, despite the fact that a LexA-binding sequence is not present in its promoter region.

  • Expression of Canonical SOS Genes Is Not under LexA Repression in Bdellovibrio bacteriovorus
    Journal of bacteriology, 2005
    Co-Authors: Susana Campoy, Ivan Erill, Noelia Salvador, Pilar Cortés, Jordi Barbé
    Abstract:

    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.

Mei-kwei Yang - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of Xanthomonas axonopodis pv. citri LexA: recognition of the LexA binding site.
    Molecular genetics and genomics : MGG, 2002
    Co-Authors: Mei-kwei Yang, Yen-chun Yang, Chien-hsiu Hsu
    Abstract:

    Levels of LexA transcripts are markedly increased upon exposure of Xanthomonas axonopodis pathovar citri (X. a. pv. citri) to the DNA-damaging agent mitomycin C. Preliminary electrophoretic mobility-shift data led us to propose that binding of LexA Protein to the sequence upstream of the LexA coding region is responsible for low promoter activity in the uniduced state. We determined that the LexA Protein binds to the region located between the transcription start site and the translation initiation codon of the LexA gene of X. a. pv. citri. Using a DNase I footprinting technique, we identified a 19-bp palindromic sequence, TTAGTAGTAATACTACTAA (TTAGN11CTAA), located in this region as the binding sequence for the LexA Protein of X. a. pv. citri, and showed that the two halves of the palindrome have to be in the inverted repeat orientation to permit binding of LexA. We also showed that almost any mutation in this sequence, including changes in the length of the spacer region of the palindrome, destroyed its ability to bind LexA both in vitro and in vivo.

  • Genetic organization of the LexA, recA and recX genes in Xanthomonas campestris.
    FEMS microbiology letters, 2002
    Co-Authors: Yen-chun Yang, Chien-hsiu Hsu, Chun-ping Chou, Mei-kwei Yang
    Abstract:

    A gene cluster containing LexA, recA and recX genes was previously identified and characterized in Xanthomonas campestris pathovar citri (X. c. pv. citri). We have now cloned and sequenced the corresponding regions in the Xanthomonas campestris pv. campestris (X. c. pv. campestris) and Xanthomonas oryzae pathovar oryzae (X. o. pv. oryzae) chromosome. Sequence analysis of these gene clusters showed significant homology to the previously reported LexA, recA and recX genes. The genetic linkage and the deduced amino acid sequences of these genes displayed very high identity in different pathovars of X. campestris as well as in X. oryzae. Immunoblot analysis revealed that the over-expressed LexA Protein of X. c. pv. citri functioned as a repressor of recA expression in X. c. pv. campestris, indicating that the recombinant X. c. pv. citri LexA Protein was functional in a different X. campestris pathovar. The abundance of RecA Protein was markedly increased upon exposure of X. c. pv. campestris to mitomycin C, and an upstream region of this gene was shown to confer sensitivity to positive regulation by mitomycin C on a luciferase reporter gene construct. A symmetrical sequence of TTAGTAGTAATACTACTAA present within all three Xanthomonas LexA promoters and a highly conserved sequence of TTAGCCCCATACCGAA present in the three regulatory regions of recA indicate that the SOS box of Xanthomonas strains might differ from that of Escherichia coli.

  • Structural and functional characterization of the LexA gene of Xanthomonas campestris pathovar citri.
    Molecular genetics and genomics : MGG, 2001
    Co-Authors: Mei-kwei Yang, Tu J
    Abstract:

    The role of the LexA Protein and, specifically, its effect on recA expression were analyzed in Xanthomonas campestris pathovar citri (X.c. pv. citri). Overexpression of LexA from X.c. pv. citri, in the plant pathogen, as well as in Escherichia coli, results in increased sensitivity to the DNA-damaging agents mitomycin C and ultraviolet radiation, indicating that the recombinant X.c. pv. citri LexA Protein is functional in a different bacterial species. Immunoblot analysis revealed that the overexpressed LexA Protein functioned as a repressor of recA expression in X.c. pv. citri, and that the mitomycin C-induced increase in the abundance of RecA was accompanied by specific proteolysis of LexA that required RecA. Although the LexA Protein from X.c. pv. citri also blocked the expression of recA in E. coli, the E. coli RecA Protein was not able to support the autocatalytic cleavage of LexA from the plant pathogen. The transcription start site of the X.c. pv. citri LexA gene was identified, and the region upstream of this gene was shown to confer responsiveness to mitomycin C on a luciferase reporter gene construct. Electrophoretic mobility-shift assays demonstrated that X.c. pv. citri LexA interacts with the promoter region of X.c. pv. citri LexA, as well as with those of the recA genes of X.c. pv. citri and E. coli. These results indicate that LexA functions as a repressor of gene expression in X.c. pv. citri just as it does in E. coli.

  • Identification of a LexA gene in, and construction of a LexA mutant of, Xanthomonas campestris pv. citri.
    Current microbiology, 2000
    Co-Authors: Mei-kwei Yang, Yen-chun Yang
    Abstract:

    The LexA gene of Xanthomonas campestris pathovar citri (X.c. pv. citri) was cloned and sequenced. The 639-bp open reading frame encodes a Protein of 213 amino acids that shares substantial sequence homology with the products of previously characterized LexA genes, sharing 46% identity with the LexA Protein of Escherichia coli. Amino acids required for autocatalytic cleavage of LexA are conserved in the X.c. pv. citri Protein, whereas domains thought to mediate DNA binding differ markedly from those of LexA Proteins from E. coli and other bacteria. The X.c. pv. citri LexA Protein was overexpressed in E. coli, and SDS-polyacrylamide gel electrophoresis revealed a molecular size of 23 kDa for the purified Protein. A LexA mutant of X.c. pv. citri was constructed by gene replacement, and the basal level of recA expression in this mutant was shown to be similar to that for wild-type cells exposed to a DNA-damaging agent. These results indicate that LexA functions as a repressor of recA expression in X.c. pv. citri.

Nancy Guillén - One of the best experts on this subject based on the ideXlab platform.

  • Expression of the Bacillus subtilis dinR and recA genes after DNA damage and during competence.
    Journal of Bacteriology, 1992
    Co-Authors: Agnès Raymond-denise, Nancy Guillén
    Abstract:

    The Bacillus subtilis dinR gene product is homologous to the LexA Protein of Escherichia coli and regulates the expression of dinR and dinC. Using transcriptional fusions in the dinR and the recA genes, we have investigated the epistatic relationship between these two genes during the SOS response induced either by DNA damage or by competence. The results show that after DNA damage, induction of the expression of both recA and dinR is dependent on the activity of the DinR and RecA Proteins. A RecA-dependent activity on DinR is proposed as the initial event in the induction of the SOS network. In contrast, the competence-related induction of dinR and recA appears to involve two distinct mechanisms. While one mechanism corresponds to the classical regulation of the SOS response, the other appears to involve an activating factor. Moreover, this factor is active in cells in which competence is prevented by a mutation in the regulatory gene comA.

  • Identification of dinR, a DNA damage-inducible regulator gene of Bacillus subtilis.
    Journal of Bacteriology, 1991
    Co-Authors: Agnès Raymond-denise, Nancy Guillén
    Abstract:

    A Bacillus subtilis strain deficient in homologous recombination was isolated from a library of Tn917lac insertion mutants. The interrupted locus consists of an open reading frame encoding a 22,823-dalton polypeptide. Analysis of the deduced amino acid sequence revealed 34% identity and 47.3% similarity with the LexA Protein from Escherichia coli. The gene was designated dinR. It is located between the recA and thyA genetic markers, at 162 degrees on the B. subtilis chromosome. The dinR gene was shown to be expressed during the entire B. subtilis cellular cycle with at least a threefold increase when cells develop competence. In addition, the use of a merodiploid strain, in which a copy of the wild-type dinR gene coexists with a dinR-lacZ transcriptional fusion, demonstrated that dinR is an SOS gene and that the SOS-induced expression of dinR occurred only when a wild-type copy of dinR was present. In addition, DinR seems to regulate the expression of dinC, another SOS gene.

Kazuo Yamamoto - One of the best experts on this subject based on the ideXlab platform.

Susana Campoy - One of the best experts on this subject based on the ideXlab platform.

  • Additional file 9: of Expansion of the SOS regulon of Vibrio cholerae through extensive transcriptome analysis and experimental validation
    2018
    Co-Authors: Evelyne Krin, Susana Campoy, Sebastian Pierlé, Odile Sismeiro, Bernd Jagla, Marie-agnès Dillies, Hugo Varet, Oihane Irazoki, Zoé Rouy, Stéphane Cruveiller
    Abstract:

    EMSA performed between LexA Protein and various promoter region in presence (+) and absence (−) of LexA Protein and with a LexA box DNA control (SOS-Box). (PPTX 40364 kb

  • An SOS Regulon under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria
    Journal of bacteriology, 2015
    Co-Authors: Neus Sanchez-alberola, Jordi Barbé, Susana Campoy, David Emerson, Ivan Erill
    Abstract:

    The SOS response is a transcriptional regulatory network governed by the LexA repressor that activates in response to DNA damage. In the Betaproteobacteria, LexA is known to target a palindromic sequence with the consensus sequence CTGT-N8-ACAG. We report the characterization of a LexA regulon in the iron-oxidizing betaproteobacterium Sideroxydans lithotrophicus. In silico and in vitro analyses show that LexA targets six genes by recognizing a binding motif with the consensus sequence GAACGaaCGTTC, which is strongly reminiscent of the Bacillus subtilis LexA-binding motif. We confirm that the closely related Gallionella capsiferriformans shares the same LexA-binding motif, and in silico analyses indicate that this motif is also conserved in the Nitrosomonadales and the Methylophilales. Phylogenetic analysis of LexA and the alpha subunit of DNA polymerase III (DnaE) reveal that the organisms harboring this noncanonical LexA form a compact taxonomic cluster within the Betaproteobacteria. However, their LexA gene is unrelated to the standard Betaproteobacteria LexA, and there is evidence of its spread through lateral gene transfer. In contrast to other reported cases of noncanonical LexA-binding motifs, the regulon of S. lithotrophicus is comparable in size and function to that of many other Betaproteobacteria, suggesting that a convergent SOS regulon has reevolved under the control of a new LexA Protein. Analysis of the DNA-binding domain of S. lithotrophicus LexA reveals little sequence similarity with that of other LexA Proteins targeting similar binding motifs, suggesting that network structure may limit site evolution or that structural constrains make the B. subtilis-type motif an optimal interface for multiple LexA sequences. IMPORTANCE Understanding the evolution of transcriptional systems enables us to address important questions in microbiology, such as the emergence and transfer potential of different regulatory systems to regulate virulence or mediate responses to stress. The results reported here constitute the first characterization of a noncanonical LexA Protein regulating a standard SOS regulon. This is significant because it illustrates how a complex transcriptional program can be put under the control of a novel transcriptional regulator. Our results also reveal a substantial degree of plasticity in the LexA recognition domain, raising intriguing questions about the space of Protein-DNA interfaces and the specific evolutionary constrains faced by these elements.

  • Cohabitation of Two Different LexA Regulons in Pseudomonas putida
    Journal of Bacteriology, 2007
    Co-Authors: Marc Abella, Ivan Erill, Susana Campoy, Fernando Rojo, Jordi Barbé
    Abstract:

    In contrast to the vast majority of the members of the domain Bacteria, several Pseudomonas and Xanthomonas species have two LexA genes, whose products have been shown to recognize different LexA binding motifs, making them an interesting target for studying the interplay between cohabiting LexA regulons in a single species. Here we report an analysis of the genetic composition of the two LexA regulons of Pseudomonas putida KT2440 performed with a genomic microarray. The data obtained indicate that one of the two LexA Proteins (LexA1) seems to be in control of the conventional Escherichia coli-like SOS response, while the other LexA Protein (LexA2) regulates only its own transcriptional unit, which includes the imuA, imuB, and dnaE2 genes, and a gene (PP_3901) from a resident P. putida prophage. Furthermore, PP_3901 is also regulated by LexA1 and is required for DNA damage-mediated induction of several P. putida resident prophage genes. In silico searches suggested that this marked asymmetry in regulon contents also occurs in other Pseudomonas species with two LexA genes, and the implications of this asymmetry in the evolution of the SOS network are discussed.

  • Insights into the LexA regulon of Thermotogales
    Antonie van Leeuwenhoek, 2006
    Co-Authors: Gerard Mazón, Susana Campoy, Antonio R. Fernández De Henestrosa, Jordi Barbé
    Abstract:

    The LexA genes of Thermotoga maritima and Petrotoga miotherma , both members of the Order Thermotogales , have been cloned and their transcriptional organization, as well as the functional characteristics of their encoded products, analyzed. In both bacterial species, the LexA gene was found to be co-transcribed together with another four ( T. maritima ) or three ( P. miotherma ) upstream open-reading frames. The P. miotherma LexA was able to bind promoters of both the cognate LexA encoding operon and the uvrA gene but not to that of the recA . Conversely, LexA Protein and crude cell extracts from T. maritima were unable to bind promoters governing the expression of either its LexA or recA genes. In agreement with these observations, no functional copy of the P. miotherma LexA box, corresponding to the GANTN_6GANNAC motif, seems to be present in the T. maritima genome. Giving support to the proposal that the evolutionary branching order of the Order Thermotogales is very close to that of Gram-positive bacteria, the P. miotherma LexA Protein was still able to recognize the previously described LexA-binding sequence for Gram-positive bacteria.

  • Identification of the Acidobacterium capsulatum LexA box reveals a lateral acquisition of the Alphaproteobacteria LexA gene.
    Microbiology (Reading England), 2006
    Co-Authors: Gerard Mazón, Ivan Erill, Susana Campoy, Jordi Barbé
    Abstract:

    Acidobacterium capsulatum is the most thoroughly studied species of a new bacterial phylogenetic group designated the phylum Acidobacteria. Through a tblastn search, the A. capsulatum LexA gene has been identified, and its product purified. Electrophoretic mobility shift assays have shown that A. capsulatum LexA Protein binds specifically to the direct repeat GTTCN(7)GTTC motif. Strikingly, this is also the LexA box of the Alphaproteobacteria, but had not previously been described outside this subclass of the Proteobacteria. In addition, a phylogenetic analysis of the LexA Protein clusters together Acidobacterium and the Alphaproteobacteria, moving the latter away from their established phylogenetic position as a subclass of the Proteobacteria, and pointing to a lateral gene transfer of the LexA gene from the phylum Acidobacteria, or an immediate ancestor, to the Alphaproteobacteria. Lastly, in vivo experiments demonstrate that the A. capsulatum recA gene is DNA-damage inducible, despite the fact that a LexA-binding sequence is not present in its promoter region.