Constitutive expression of the SOS response in recA718 mutants of Escherichia coli requires amplification of RecA718 protein.

In recA718 lexA+ strains of Escherichia coli, induction of the SOS response requires DNA damage. This implies that RecA718 protein, like RecA+ protein, must be converted, by a process initiated by the damage, to an activated form (RecA) to promote cleavage of LexA, the cellular repressor of SOS genes. However, when LexA repressor activity was abolished by a lexA-defective mutation [lexA(Def)], strains carrying the recA718 gene (but not recA+) showed strong SOS mutator activity and were able to undergo stable DNA replication in the absence of DNA damage (two SOS functions known to require RecA activity even when cleavage of LexA is not necessary). lambda lysogens of recA718 lexA(Def) strains exhibited mass induction of prophage, indicative of constitutive ability to cleave lambda repressor. When the cloned recA718 allele was present in a lexA+ strain on a plasmid, SOS mutator activity and beta-galactosidase synthesis under LexA control were expressed in proportion to the plasmid copy number. We conclude that RecA718 is capable of becoming activated without DNA damage for cleavage of LexA and lambda repressor, but only if it is amplified above its base-line level in lexA+ strains. At amplified levels, RecA718 was also constitutively activated for its roles in SOS mutagenesis and stable DNA replication. The nucleotide sequence of recA718 reveals two base substitutions relative to the recA+ sequence. We propose that the first allows the protein to become activated constitutively, whereas the second partially suppresses this capability.     

Adhesins of Leptospira interrogans Mediate the Interaction to Fibrinogen and Inhibit Fibrin Clot Formation In Vitro

We report in this work that Leptospira strains, virulent L. interrogans serovar Copenhageni, attenuated L. interrogans serovar Copenhageni and saprophytic L. biflexa serovar Patoc are capable of binding fibrinogen (Fg). The interaction of leptospires with Fg inhibits thrombin- induced fibrin clot formation that may affect the haemostatic equilibrium. Additionally, we show that plasminogen (PLG)/plasmin (PLA) generation on the surface of Leptospira causes degradation of human Fg. The data suggest that PLA-coated leptospires were capable to employ their proteolytic activity to decrease one substrate of the coagulation cascade. We also present six leptospiral adhesins and PLG- interacting proteins, rLIC12238, Lsa33, Lsa30, OmpL1, rLIC11360 and rLIC11975, as novel Fg-binding proteins. The recombinant proteins interact with Fg in a dose-dependent and saturable fashion when increasing protein concentration was set to react to a fix human Fg concentration. The calculated dissociation equilibrium constants (K_D) of these reactions ranged from 733.3±276.8 to 128±89.9 nM for rLIC12238 and Lsa33, respectively. The interaction of recombinant proteins with human Fg resulted in inhibition of fibrin clot by thrombin-catalyzed reaction, suggesting that these versatile proteins could mediate Fg interaction in Leptospira. Our data reveal for the first time the inhibition of fibrin clot by Leptospira spp. and presents adhesins that could mediate these interactions. Decreasing fibrin clot would cause an imbalance of the coagulation cascade that may facilitate bleeding and help bacteria dissemination     

In silico identification of common putative drug targets in <Emphasis Type="Italic">Leptospira interrogans</Emphasis>

Infectious diseases are the leading causes of death worldwide. Hence, there is a need to develop new antimicrobial agents. Traditional method of drug discovery is time consuming and yields a few drug targets with little intracellular information for guiding target selection. Thus, focus in drug development has been shifted to computational comparative genomics for identifying novel drug targets. Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. Availability of L. interrogans serovars and human genome sequences facilitated to search for novel drug targets using bioinformatics tools. The genome sequence of L. interrogans serovar Copenhageni has 5,124 genes while that of serovar Lai has 4,727 genes. Through subtractive genomic approach 218 genes in serovar Copenhageni and 158 genes in serovar Lai have been identified as putative drug targets. Comparative genomic approach had revealed that 88 drug targets were common to both the serovars. Pathway analysis using the Kyoto Encyclopaedia of Genes and Genomes revealed that 66 targets are enzymes and 22 are non-enzymes. Sixty two common drug targets were predicted to be localized in cytoplasm and 16 were surface proteins. The identified potential drug targets form a platform for further investigation in discovery of novel therapeutic compounds against Leptospira.     

recO and recR mutations delay induction of the SOS response in Escherichia coli

RecF, RecO and RecR, three of the important proteins of the RecF pathway of recombination, are also needed for repair of DNA damage due to UV irradiation. recF mutants are not proficient in cleaving LexA repressor in vivo following DNA damage; therefore they show a delay of induction of the SOS response. In this communication, by measuring the in vivo levels of LexA repressor using anti-LexA antibodies, we show that recO and recR mutant strains are also not proficient in LexA cleavage reactions. In addition, we show that recO and recR mutations delay induction of β-galactosidase activity expressed from a lexA-regulated promoter following exposure of cells to UV, thus further supporting the idea that recF, recO and recR gene products are needed for induction of the SOS response.     

SOS induction in mycobacteria: analysis of the DNA-binding activity of a LexA-like repressor and its role in DNA damage induction of the recA gene from Mycobacterium smegmatis

The protein encoded by the lexA gene from Mycobacterium leprae was overproduced in Escherichia coli . The recombinant protein bound to the promoter regions of the M. leprae lexA , M. leprae recA and M. smegmatis recA genes at sites with the sequences 5'-GAACACATGTTT and 5'-GAACAGGTGTTC, which belong to the 'Cheo box' family of binding sites recognized by the SOS repressor from Bacillus subtilis . Gel mobility shift assays were used to confirm that proteins with the same site specificity of DNA binding are also present in Mycobacterium tuberculosis and M. smegmatis . Complex formation was impaired by mutagenic disruption of the dyad symmetry of the M. smegmatis recA Cheo box. LexA binding was also inhibited by preincubation of the M. smegmatis and M. tuberculosis extracts with anti- M. leprae LexA antibodies, suggesting that the mycobacterial LexA proteins are functionally conserved at the level of DNA binding. Finally, exposure of M. smegmatis to DNA-damaging agents resulted in induction of the M. smegmatis recA promoter with concomitant loss of DNA binding of LexA to its Cheo box, confirming that this organism possesses the key regulatory elements of a functional SOS induction system.     

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.     

Suppression of the E. coli SOS response by dNTP pool changes

The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30⁺-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction.     

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.     

Analyses of binding sequences of the two LexA proteins of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar citri

Xanthomonas axonopodis pv. citri (X. axonopodis pv. citri) possesses two lexA genes, designated lexA1 and lexA2. Electrophoretic mobility shift data show that LexA1 binds to both lexA1 and lexA2 promoters, but LexA2 does not bind to the lexA1 promoter, suggesting that LexA1 and LexA2 play different roles in regulating the expression of SOS genes. In this study, we have determined that LexA2 binds to a 14-bp dyad-spacer-dyad palindromic sequence, 5'-TGTACAAATGTACA-3', located at nucleotides -41 to -28 relative to the translation start site of lexA2 of X. axonopodis pv. citri. The two spacer nucleotides in this sequence can be changed from AA to TT without affecting LexA2 binding; all other base deletions or substitutions abolish LexA2 binding. The LexA1 binding sequence in the promoter region of lexA2 is TTAGTACTAAAGTTATAA and is located at -133 to -116, and that in the lexA1 gene is AGTAGTAATACTACT located at nucleotides -19 to -5 relative to the translation start site of lexA1. Any base change in the latter sequence abolishes LexA1 binding.     

Induction of the SOS response in Escherichia coli inhibits Tn5 and IS50 transposition.

In response to DNA damage or the inhibition of normal DNA replication in Escherichia coli, a set of some 20 unlinked operons is induced through the RecA-mediated cleavage of the LexA repressor. We examined the effect of this SOS response on the transposition of Tn5 and determined that the frequency of transposition is reduced 5- to 10-fold in cells that constitutively express SOS functions, e.g., lexA(Def) strains. Furthermore, this inhibition is independent of recA function, is fully reversed by a wild-type copy of lexA, and is not caused by an alteration in the levels of the Tn5 transposase or inhibitor proteins. We isolated insertion mutations in a lexA(Def) background that reverse this transposition defect; all of these mapped to a new locus near 23 min on the E. coli chromosome.     

The Leptospira interrogans lexA gene is not autoregulated

Footprinting and mutagenesis experiments demonstrated that Leptospira interrogans LexA binds the palindrome TTTGN(5)CAAA found in the recA promoter but not in the lexA promoter. In silico analysis revealed that none of the other canonical SOS genes is under direct control of LexA, making the leptospiral lexA gene the first described which is not autoregulated.