An SOS Regulon under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria

ABSTRACTThe SOS response is a transcriptional regulatory network governed by the LexA repressor that activates in response to DNA damage. In theBetaproteobacteria, 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 betaproteobacteriumSideroxydans lithotrophicus.In silicoandin vitroanalyses show that LexA targets six genes by recognizing a binding motif with the consensus sequence GAACGaaCGTTC, which is strongly reminiscent of theBacillus subtilisLexA-binding motif. We confirm that the closely relatedGallionella capsiferriformansshares the same LexA-binding motif, andin silicoanalyses indicate that this motif is also conserved in theNitrosomonadalesand theMethylophilales. 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 theBetaproteobacteria. However, theirlexAgene is unrelated to the standardBetaproteobacterialexA, and there is evidence of its spread through lateral gene transfer. In contrast to other reported cases of noncanonical LexA-binding motifs, the regulon ofS. lithotrophicusis comparable in size and function to that of many otherBetaproteobacteria, suggesting that a convergent SOS regulon has reevolved under the control of a new LexA protein. Analysis of the DNA-binding domain ofS. lithotrophicusLexA 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 theB. subtilis-type motif an optimal interface for multiple LexA sequences.IMPORTANCEUnderstanding 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.

Regulating colicin synthesis to cope with stress and lethality of colicin production

Colicins are plasmid-encoded bacteriocins active against Escherichia coli and closely related species of Enterobacteriaceae. They promote microbial diversity and genetic diversity in E. coli populations. Colicin synthesis is characteristically repressed by the LexA protein, the key regulator of the SOS response. As colicins are released by cell lysis, generally two LexA dimers binding to two overlapping SOS boxes control untimely expression. Nevertheless, genetic organization of the colicin clusters, additional transcription regulators as well as post-transcriptional mechanisms involving translational efficiency of the lysis and activity genes fine-tune colicin expression and protect against lethality of colicin production.

Cohabitation of Two Different lexA Regulons in Pseudomonas putida

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.

SOS Regulation of the Type III Secretion System of Enteropathogenic Escherichia coli

ABSTRACT Genomes of bacterial pathogens contain and coordinately regulate virulence-associated genes in order to cause disease. Enteropathogenic Escherichia coli (EPEC), a major cause of watery diarrhea in infants and a model gram-negative pathogen, expresses a type III secretion system (TTSS) that is encoded by the locus of enterocyte effacement (LEE) and is necessary for causing attaching and effacing intestinal lesions. Effector proteins encoded by the LEE and in cryptic prophage are injected into the host cell cytoplasm by the TTTS apparatus, ultimately leading to diarrhea. The LEE is comprised of multiple polycistronic operons, most of which are controlled by the global, positive regulator Ler. Here we demonstrated that the LEE2 and LEE3 operons also responded to SOS signaling and that this regulation was LexA dependent. As determined by a DNase I protection assay, purified LexA protein bound in vitro to a predicted SOS box located in the divergent, overlapping LEE2/LEE3 promoters. Expression of the lexA1 allele, encoding an uncleavable LexA protein in EPEC, resulted in reduced secretion, particularly in the absence of the Ler regulator. Finally, we obtained evidence that the cryptic phage-located nleA gene encoding an effector molecule is SOS regulated. Thus, we demonstrated, for the first time to our knowledge, that genes encoding components of a TTSS are regulated by the SOS response, and our data might explain how a subset of EPEC effector proteins, encoded in cryptic prophages, are coordinately regulated with the LEE-encoded TTSS necessary for their translocation into host cells.

Identification of the Acidobacterium capsulatum LexA box reveals a lateral acquisition of the Alphaproteobacteria lexA gene

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 GTTCN7GTTC 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

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.

The pKO2 Linear Plasmid Prophage of Klebsiella oxytoca

ABSTRACT Temperate bacteriophages with plasmid prophages are uncommon in nature, and of these only phages N15 and PY54 are known to have a linear plasmid prophage with closed hairpin telomeres. We report here the complete nucleotide sequence of the 51,601-bp Klebsiella oxytoca linear plasmid pKO2, and we demonstrate experimentally that it is also a prophage. We call this bacteriophage φKO2. An analysis of the 64 predicted φKO2 genes indicate that it is a fairly close relative of phage N15; they share a mosaic relationship that is typical of different members of double-stranded DNA tailed-phage groups. Although the head, tail shaft, and lysis genes are not recognizably homologous between these phages, other genes such as the plasmid partitioning, replicase, prophage repressor, and protelomerase genes (and their putative targets) are so similar that we predict that they must have nearly identical DNA binding specificities. The φKO2 virion is unusual in that its phage λ-like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The φKO2 genome also carries putative homologues of bacterial dinI and umuD genes, both of which are involved in the host SOS response. We show that these divergently transcribed genes are regulated by LexA protein binding to a single target site that overlaps both promoters.