DNA cytosine methylation at the lexA promoter of Escherichia coli is stationary phase specific

The bacterial DNA damage response pathway (SOS response) is composed of a network of genes regulated by a single transcriptional repressor, LexA. The lexA promoter, itself, contains two LexA operators, enabling negative feedback. In Escherichia coli, the downstream operator contains a conserved DNA cytosine methyltransferase (Dcm) site that is predicted to be methylated to 5-methylcytosine (5mC) specifically during stationary phase growth, suggesting a regulatory role for DNA methylation in the SOS response. To test this, we quantified 5mC at the lexA locus, and then examined the effect of LexA on Dcm activity, as well as the impact of this 5mC mark on LexA binding, lexA transcription, and SOS response induction. We found that 5mC at the lexA promoter is specific to stationary phase growth, but that it does not affect lexA expression. Our data support a model where LexA binding at the promoter inhibits Dcm activity without an effect on the SOS regulon.

Prevalence of Quinolone Resistance in Enterobacteriaceae from Sierra Leone and the Detection ofqnrBPseudogenes and Modified LexA Binding Sites

ABSTRACTA collection of 74Enterobacteriaceaeisolates found in Bo, Sierra Leone, were tested for quinolone antibiotic susceptibility and resistance mechanisms. The majority of isolates (62%) were resistant to quinolones, and 61% harbored chromosomalgyrAand/orparCmutations. Plasmid-mediated quinolone resistance genes were ubiquitous, withqnrBandaac(6′)-Ib-crbeing the most prevalent. Mutated LexA binding sites were found in allqnrB1genes, and truncatedqnrBpseudogenes were found in the majority ofCitrobacterisolates.

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.

Quinolone Induction ofqnrVS1in Vibrio splendidus and Plasmid-CarriedqnrS1in Escherichia coli, a Mechanism Independent of the SOS System

ABSTRACTPlasmid-carriedqnrS1is derived fromVibrio splendiduschromosomalqnrVS1. qnrVS1transcripts increased 21- to 34-fold with subinhibitory concentrations of ciprofloxacin but much less with mitomycin. No LexA binding sites were upstream ofqnrS1orqnrVS1, and similar induction levels were observed inlexA-positive andlexA-negativeEscherichia colistrains with nativeqnrS1plasmid pMG306 but not with pUC18-clonedqnrS1orqnrVS1. Thus,qnrS1induction by quinolones is independent of the SOS system and requires sequence besides that of the structural gene.

Overexpression of the recA Gene Decreases Oral but Not Intraperitoneal Fitness of Salmonella enterica

ABSTRACT Transcription of the Salmonella enterica recA gene is negatively controlled by the LexA protein, the repressor of the SOS response. The introduction of a mutation (recAo6869) in the LexA binding site, in the promoter region of the S. enterica ATCC 14028 recA gene, allowed the analysis of the effect that RecA protein overproduction has on the fitness of this virulent strain. The fitness of orally but not intraperitoneally inoculated recAo6869 cells decreased dramatically. However, the SOS response of this mutant was induced normally, and there was no increase in the sensitivity of the strain toward DNA-damaging agents, bile salts, or alterations in pH. Nevertheless, S. enterica recAo6869 cells were unable to swarm and their capacity to cross the intestinal epithelium was significantly reduced. The swarming deficiency in recAo6869 cells is independent of the flagellar phase. Moreover, swimming activity of the recAo6869 strain was not diminished with respect to the wild type, indicating that the flagellar synthesis is not affected by RecA protein overproduction. In contrast, swarming was recovered in a recAo6869 derivative that overproduced CheW, a protein known to be essential for this function. These data demonstrate that an equilibrium between the intracellular concentrations of RecA and CheW is necessary for swarming in S. enterica. Our results are the first to point out that the SOS response plays a critical role in the prevention of DNA damage by abolishing bacterial swarming in the presence of a genotoxic compound.

Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays

The lexA gene of Corynebacterium glutamicum ATCC 13032 was deleted to create the mutant strain C. glutamicum NJ2114, which has an elongated cell morphology and an increased doubling time. To characterize the SOS regulon in C. glutamicum, the transcriptomes of NJ2114 and a DNA-damage-induced wild-type strain were compared with that of a wild-type control using DNA microarray hybridization. The expression data were combined with bioinformatic pattern searches for LexA binding sites, leading to the detection of 46 potential SOS boxes located upstream of differentially expressed transcription units. Binding of a hexahistidyl-tagged LexA protein to 40 double-stranded oligonucleotides containing the potential SOS boxes was demonstrated in vitro by DNA band shift assays. It turned out that LexA binds not only to SOS boxes in the promoter–operator region of upregulated genes, but also to SOS boxes detected upstream of downregulated genes. These results demonstrated that LexA controls directly the expression of at least 48 SOS genes organized in 36 transcription units. The deduced genes encode a variety of physiological functions, many of them involved in DNA repair and survival after DNA damage, but nearly half of them have hitherto unknown functions. Alignment of the LexA binding sites allowed the corynebacterial SOS box consensus sequence TcGAA(a/c)AnnTGTtCGA to be deduced. Furthermore, the common intergenic region of lexA and the differentially expressed divS-nrdR operon, encoding a cell division suppressor and a regulator of deoxyribonucleotide biosynthesis, was characterized in detail. Promoter mapping revealed differences in divS-nrdR expression during SOS response and normal growth conditions. One of the four LexA binding sites detected in the intergenic region is involved in regulating divS-nrdR transcription, whereas the other sites are apparently used for negative autoregulation of lexA expression.

SOS Regulation of qnrB Expression

ABSTRACT In the sequence upstream from qnrB (but not qnrA or qnrS) is a LexA binding site. qnrB was shown to be under SOS control by demonstrating that quinolone susceptibility decreased with increasing temperature in a strain with a recA441(Ts) allele, whereas qnrB expression increased in response to ciprofloxacin or mitomycin C in strains with an intact lexA gene.

Conservation of the LexA repressor binding site in Deinococcus radiodurans

Summary The LexA protein is a transcriptional repressor of the bacterial SOS DNA repair system, which comprises a set of DNA repair and cellular survival genes that are induced in response to DNA damage. Its varied DNA binding motifs have been characterized and reported in the Escherichia coli, Bacillus subtilis, rhizobia family members, marine magnetotactic bacterium, Salmonella typhimurium and recently in Mycobacterium tuberculosis and this motifs information has been used in our theoretical analysis to detect its novel regulated genes in radio-resistant Deinococcus radiodurans genome. This bacterium showed presence of SOS-box like consensus sequence in the upstream sequences of 3166 genes with >60% motif score similarity percentage (MSSP) on both strands. Attempts to identify LexA-binding sites and the composition of the putative SOS regulon in D. radiodurans have been unsuccessful so far. To resolve the problem we performed theoretical analysis with modifications on reported data set of genes related to DNA repair (61 genes), stress response (145 genes) and some unusual predicted operons (21 clusters). Expression of some of the predicted SOS-box regulated operon members then was examined through the previously reported microarray data which confirm the expression of only single predicted operon i.e. DRB0143 (AAA superfamily NTPase related to 5-methylcytosine specific restriction enzyme subunit McrB) and DRB0144 (homolog of the McrC subunit of the McrBC restriction modification system). The methodology involved weight matrix construction through CONSENSUS algorithm using information of conserved upstream sequences of eight known genes including dinB, tagC, lexA, recA, uvrB, yneA of B. subtilis while lexA and recA of D. radiodurans through phylogenetic footprinting method and later detection of similar conserved SOS-box like LexA binding motifs through both RSAT & PoSSuMsearch programs. The resultant DNA consensus sequence had highly conserved 14 bp SOS-box like binding site.