Widespread distribution of a lexA-regulated DNA damage-inducible multiple gene cassette in the Proteobacteria phylum

The SOS response comprises a set of cellular functions aimed at preserving bacterial cell viability in front of DNA injuries. The SOS network, negatively regulated by the LexA protein, is found in many bacterial species that have not suffered major reductions in their gene contents, but presents distinctly divergent LexA-binding sites across the Bacteria domain. In this article, we report the identification and characterization of an imported multiple gene cassette in the Gamma Proteobacterium Pseudomonas putida that encodes a LexA protein, an inhibitor of cell division (SulA), an error-prone polymerase (DinP) and the alpha subunit of DNA polymerase III (DnaE). We also demonstrate that these genes constitute a DNA damage-inducible operon that is regulated by its own encoded LexA protein, and we establish that the latter is a direct derivative of the Gram-positive LexA protein. In addition, in silico analyses reveal that this multiple gene cassette is also present in many Proteobacteria families, and that both its gene content and LexA-binding sequence have evolved over time, ultimately giving rise to the lexA lineage of extant Gamma Proteobacteria.     

Lex marks the spot: the virulent side of SOS and a closer look at the LexA regulon

The SOS response that responds to DNA damage induces many genes that are under LexA repression. A detailed examination of LexA regulons using genome-wide techniques has recently been undertaken in both Escherichia coli and Bacillus subtilis. These extensive and elegant studies have now charted the extent of the LexA regulons, uncovered many new genes, and exposed a limited overlap in the LexA regulon between the two bacteria. As more bacterial genomes are analysed, more curiosities in LexA regulons arise. Several notable examples include the discovery of a LexA-like protein, HdiR, in Lactococcus lactis, organisms with two lexA genes, and small DNA damage-inducible cassettes under LexA control. In the cyanobacterium Synechocystis, genetic and microarray studies demonstrated that a LexA paralogue exerts control over an entirely different set of carbon-controlled genes and is crucial to cells facing carbon starvation. An examination of SOS induction evoked by common therapeutic drugs has shed new light on unsuspected consequences of drug exposure. Certain antibiotics, most notably fluoroquinolones such as ciprofloxacin, can induce an SOS response and can modulate the spread of virulence factors and drug resistance. SOS induction by beta-lactams in E. coli triggers a novel form of antibiotic defence that involves cell wall stress and signal transduction by the DpiAB two-component system. In this review, we provide an overview of these new directions in SOS and LexA research with emphasis on a few themes: identification of genes under LexA control, the identification of new endogenous triggers, and antibiotic-induced SOS response and its consequences.     

Inducible SOS response system of DNA repair and mutagenesis in Escherichia coli

Chromosomal DNA is exposed to continuous damage and repair. Cells contain a number of proteins and specific DNA repair systems that help maintain its correct structure. The SOS response was the first DNA repair system described in Escherichia coli induced upon treatment of bacteria with DNA damaging agents arrest DNA replication and cell division. Induction of the SOS response involves more than forty independent SOS genes, most of which encode proteins engaged in protection, repair, replication, mutagenesis and metabolism of DNA. Under normal growth conditions the SOS genes are expressed at a basal level, which increases distinctly upon induction of the SOS response. The SOS-response has been found in many bacterial species (e.g., Salmonella typhimurium, Caulobacter crescentus, Mycobacterium tuberculosis), but not in eukaryotic cells. However, species from all kingdoms contain some SOS-like proteins taking part in DNA repair that exhibit amino acid homology and enzymatic activities related to those found in E. coli. but are not organized in an SOS system. This paper presents a brief up-to-date review describing the discovery of the SOS system, the physiology of SOS induction, methods for its determination, and the role of some SOS-induced genes.     

Expression of Serratia marcescens extracellular proteins requires recA.

A previously described regulatory mutation which abolishes expression of the extracellular nuclease of Serratia marcescens is shown to be a mutation of the Serratia recA gene. The defect in nuclease expression could be restored by introducing a plasmid carrying the recA gene of Escherichia coli. The DNA sequence of the Serratia gene is very similar to that of the E. coli gene. The putative LexA-binding site of the Serratia recA gene is almost identical to that of E. coli, along with the promoter. A similar LexA-binding site can also be found upstream of the nuclease gene. As expected from this finding, we show that nuclease expression can be induced by SOS-inducing agents such as mitomycin C. Although inducible in S. marcescens, the nuclease was expressed only at the uninduced levels in E. coli and could not be induced by mitomycin C. The extracellular chitinase and lipase were similarly affected by the mutations altering nuclease expression and were also induced by mitomycin C.     

An SOS response induced by high pressure in Escherichia coli

Although pressure is an important environmental parameter in microbial niches such as the deep sea and is furthermore used in food preservation to inactivate microorganisms, the fundamental understanding of its effects on bacteria remains fragmentary. Our group recently initiated differential fluorescence induction screening to search for pressure-induced Escherichia coli promoters and has already reported induction of the heat shock regulon. Here the screening was continued, and we report for the first time that pressure induces a bona fide SOS response in E. coli, characterized by the RecA and LexA-dependent expression of uvrA, recA, and sulA. Moreover, it was shown that pressure is capable of triggering lambda prophage induction in E. coli lysogens. The remnant lambdoid e14 element, however, could not be induced by pressure, as opposed to UV irradiation, indicating subtle differences between the pressure-induced and the classical SOS response. Furthermore, the pressure-induced SOS response seems not to be initiated by DNA damage, since DeltarecA and lexA1 (Ind-) mutants, which are intrinsically hypersensitive to DNA damage, were not sensitized or were only very slightly sensitized for pressure-mediated killing and since pressure treatment was not found to be mutagenic. In light of these findings, the current knowledge of pressure-mediated effects on bacteria is discussed.     

Expression of canonical SOS genes is not under LexA repression in Bdellovibrio bacteriovorus

The here-reported identification of the LexA-binding sequence of Bdellovibrio bacteriovorus, a bacterial predator belonging to the delta-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 delta-Proteobacteria LexA proteins but binds to the cyanobacterial LexA repressor. This places B. bacteriovorus LexA at the base of the delta-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.     

SOS repair and DNA supercoiling influence the genetic stability of DNA triplet repeats in Escherichia coli

Molecular mechanisms responsible for the genetic instability of DNA trinucleotide sequences (TRS) account for at least 20 human hereditary disorders. Many aspects of DNA metabolism influence the frequency of length changes in such repeats. Herein, we demonstrate that expression of Escherichia coli SOS repair proteins dramatically decreases the genetic stability of long (CTG/CAG)n tracts contained in plasmids. Furthermore, the growth characteristics of the bacteria are affected by the (CTG/CAG)n tract, with the effect dependent on the length of the TRS. In an E. coli host strain with constitutive expression of the SOS regulon, the frequency of deletions to the repeat is substantially higher than that in a strain with no SOS response. Analyses of the topology of reporter plasmids isolated from the SOS+ and SOS- strains revealed higher levels of negative supercoiling in strains with the constitutively expressed SOS network. Hence, we used strains with mutations in topoisomerases to examine the effect of DNA topology upon the TRS instability. Higher levels of negative DNA supercoiling correlated with increased deletions in long (CTG/CAG)n, (CGG/CCG)n and (GAA/TTC)n. These observations suggest a link between the induction of bacterial SOS repair, changes in DNA topology and the mechanisms leading to genetic instability of repetitive DNA sequences.     

Regulation of the SOS response in Bacillus subtilis: evidence for a LexA repressor homolog.

The inducible SOS response for DNA repair and mutagenesis in the bacterium Bacillus subtilis resembles the extensively characterized SOS system of Escherichia coli. In this report, we demonstrate that the cellular repressor of the E. coli SOS system, the LexA protein, is specifically cleaved in B. subtilis following exposure of the cells to DNA-damaging treatments that induce the SOS response. The in vivo cleavage of LexA is dependent upon the functions of the E. coli RecA protein homolog in B. subtilis (B. subtilis RecA) and results in the same two cleavage fragments as produced in E. coli cells following the induction of the SOS response. We also show that a mutant form of the E. coli RecA protein (RecA430) can partially substitute for the nonfunctional cellular RecA protein in the B. subtilis recA4 mutant, in a manner consistent with its known activities and deficiencies in E. coli. RecA430 protein, which has impaired repressor cleaving (LexA, UmuD, and bacteriophage lambda cI) functions in E.coli, partially restores genetic exchange to B. subtilis recA4 strains but, unlike wild-type E. coli RecA protein, is not capable of inducing SOS functions (expression of DNA damage-inducible [din::Tn917-lacZ] operons or RecA synthesis) in B. subtilis in response to DNA-damaging agents or those functions that normally accompany the development of physiological competence. Our results provide support for the existence of a cellular repressor in B. subtilis that is functionally homologous to the E. coli LexA repressor and suggest that the mechanism by which B. subtilis RecA protein (like RecA of E. coli) becomes activated to promote the induction of the SOS response is also conserved.     

抑制E.coli的SOS应答对恩诺沙星抗药性的影响

通过敲除SOS应答启动蛋白基因rec A,探讨SOS应答对E.coli恩诺沙星抗药性的影响,并体外评价Rec A抑制剂和恩诺沙星联用对细菌协同抑制作用的影响.利用Red重组系统,构建E.coli ATCC 25922的rec A缺失菌株E.coli ATCC 25922/?rec A;在恩诺沙星压力下,利用荧光定量PCR测定SOS应答系统相关基因rec A和umu C表达量的变化.用微量肉汤稀释法测定恩诺沙星等常用抗生素对两个菌株的MIC变化;利用梯度平板法测定恩诺沙星对两个菌株抗药性变异的影响;合成Rec A抑制剂,并评估其与恩诺沙星联合抑制E.coli生长及其抗药性的作用.结果表明,E.coli ATCC 25922/?rec A菌株对恩诺沙星的最低抑菌浓度值降低至原始菌株的1/8;经药物处理后,在梯度平板上,rec A缺失菌株较野生型不易产生抗药性;荧光定量PCR表明,rec A缺失菌株或在Rec A抑制剂作用下,SOS应答系统受到一定的抑制.敲除rec A,使菌株对恩诺沙星的抗药性和抗药率均明显降低;Rec A抑制剂在一定程度上能抑制SOS应答,起到协同抑菌作用.     

大肠杆菌（Escherichia coli）LexA蛋白的结构与功能

LexA蛋白首先在大肠杆菌（Escherichia coli）中作为SOS反应的重要调节因子之一被发现. LexA蛋白含有202个氨基酸,由N端DNA结合结构域和C端催化核心结构域构成. 细胞中LexA蛋白大都以二聚体形式存在,并且有可切割和不可切割两种构象. 在正常生理条件下,LexA特异性结合16 bp的保守序列5′-CTGTN8ACAG-3′,即SOS盒,抑制约50个基因的表达. 当发生DNA损伤时,活化的RecA蛋白通过稳定LexA蛋白可切割构象,促进LexA蛋白Ala84-Gly85间肽键的切割,产生的C端LexA85 202和N端LexA1 84被蛋白酶ClpXP和Lon快速降解. LexA蛋白切割后,SOS基因以一定的顺序开始表达,并且完成DNA损伤修复. 本文回顾和总结了LexA蛋白分子结构,自我切割分子机制和影响因素,以及在SOS反应中的作用等方面的研究进展. 同时,也讨论了LexA蛋白在原核细胞中的进化保守性.     

The single-stranded DNA-binding protein of Escherichia coli.

The single-stranded DNA-binding protein (SSB) of Escherichia coli is involved in all aspects of DNA metabolism: replication, repair, and recombination. In solution, the protein exists as a homotetramer of 18,843-kilodalton subunits. As it binds tightly and cooperatively to single-stranded DNA, it has become a prototypic model protein for studying protein-nucleic acid interactions. The sequences of the gene and protein are known, and the functional domains of subunit interaction, DNA binding, and protein-protein interactions have been probed by structure-function analyses of various mutations. The ssb gene has three promoters, one of which is inducible because it lies only two nucleotides from the LexA-binding site of the adjacent uvrA gene. Induction of the SOS response, however, does not lead to significant increases in SSB levels. The binding protein has several functions in DNA replication, including enhancement of helix destabilization by DNA helicases, prevention of reannealing of the single strands and protection from nuclease digestion, organization and stabilization of replication origins, primosome assembly, priming specificity, enhancement of replication fidelity, enhancement of polymerase processivity, and promotion of polymerase binding to the template. E. coli SSB is required for methyl-directed mismatch repair, induction of the SOS response, and recombinational repair. During recombination, SSB interacts with the RecBCD enzyme to find Chi sites, promotes binding of RecA protein, and promotes strand uptake.     

Inference of gene regulatory networks and compound mode of action from time course gene expression profiles

MOTIVATION: Time series expression experiments are an increasingly popular method for studying a wide range of biological systems. Here we developed an algorithm that can infer the local network of gene-gene interactions surrounding a gene of interest. This is achieved by a perturbation of the gene of interest and subsequently measuring the gene expression profiles at multiple time points. We applied this algorithm to computer simulated data and to experimental data on a nine gene network in Escherichia coli. RESULTS: In this paper we show that it is possible to recover the gene regulatory network from a time series data of gene expression following a perturbation to the cell. We show this both on simulated data and on a nine gene subnetwork part of the DNA-damage response pathway (SOS pathway) in the bacteria E. coli. CONTACT: dibernardo@tigem.it SUPLEMENTARY INFORMATION: Supplementary data are available at http://dibernado.tigem.it     

UV induction of LexA independent proteins which could be involved in SOS repair

The SOS response is induced in E coli following treatments that interfere with DNA replication. The response is under the control of the recA and the lexA genes. Strains defective in LexA repressor constitutively express SOS proteins. However, SOS repair does not reach its maximum level in these strains. Instead, an activation of RecA protein and de novo protein synthesis are required for full repair. We have analyzed by 2-dimensional gel electrophoresis the induction of proteins after UV irradiation of lexA(Def) bacteria. Proteins which might participate in SOS repair are induced under these conditions.     

Auto-regulation of DNA degrading bacteriocins: molecular and ecological aspects

Colicins, proteinaceous antibiotics produced by Escherichia coli, specifically target competing strains killing them through one of a variety of mechanisms, including pore formation and nucleic acid degradation. The genes encoding colicins display a unique form of expression, which is tightly regulated, involving the DNA damage response regulatory system (the SOS response system), confined to stressful conditions and released by degradation of the producing cell. Given their lethal nature, colicin production has evolved a sophisticated system for repression and expression. While exploring the expression of 13 colicins we identified a novel means of induction unique to strains that kill by DNA degradation: these colicinogenic strains mildly poison themselves inflicting DNA damage that induces their DNA repair system (the SOS system), and their own expression. We established that among the four known DNase colicins (E2, E7, E8 and E9), three act to induce their own production. Using different stresses we show that this form of self-regulation entails high cost when growth conditions are not optimal, and is not carried out by individual cells but is a population-mediated trait. We discuss this novel form of colicins’ regulation and expression, and its possible molecular mechanism and evolutionary implications.