392 research outputs found
Identification of molecules that inhibit activity of Escherichia coli or Staphylococcus aureus LexA protein
No full textOdziv SOS pri bakterijah se sproži ob poškodbah DNA in bakteriji omogoči, da ohrani integriteto lastnega genoma. Poglavitni regulator odziva je transkripcijski faktor LexA, ki z vezavo na promotorska področja prepreči prepis genov, katerih produkti so vključeni v popravilo DNA. Genom bakteriofaga GIL01 vključuje gen za protein gp7, ki se veže s proteinom LexA bakterije Bacillus thuringiensis. Protein gp7 zveča afiniteto LexA do specifičnih nukleotidnih zaporedij in inhibira s proteinom RecA sproženo inaktivacijo LexA. Posledično protein gp7 vpliva na aktivacijo odziva SOS, kar potencialno zavre prilagoditev bakterij na stresne razmere v okolju, tudi pridobitev odpornosti proti antibiotikom. Namen magistrskega dela je bil prepoznati molekule, ki vplivajo na delovanje represorja LexA, podobno kot protein gp7. V ta namen smo izolirali protein gp8 temperatnega bakteriofaga GIL01 bakterije B. thuringiensis in protein OrbA temperatnega bakteriofaga ICP1 bakterije Vibrio cholerae. Rezultati nakazujejo, da se izolirana rekombinantna proteina ne vežeta s proteinom LexA gostiteljske bakterije in ne vplivata na cepitev proteina LexA. V nadaljevanju smo analizirali vpliv nekaterih izbranih malih molekul, pridobljenih iz laboratorija prof. dr. Stanislava Gobca iz Fakultete za farmacijo, na aktivnost LexA. Z uporabo površinske plazmonske resonance smo dokazali, da se učinkovina 6-hidroksiflavon (U5) s šibko afiniteto veže s proteinom LexA bakterij Escherichia coli in Staphylococcus aureus ter inhibira vezavo LexA bakterije E. coli na DNA. S poskusi in vivo, v tekoči kulturi, smo dokazali, da dodatek U5 upočasni rast bakterijske kulture E. coli ΔacrB in deluje sinergistično z antibiotikom ciprofloksacin. Učinkovina U5 je šibko zavrla tudi rast seva E. coli RW542, ki ima okvarjen gen za LexA in okvarjen promotorski element gena sulA, zato predvidevamo, da U5 inhibitorno vpliva tudi na procese, ki niso del odziva SOS.SOS response in bacteria is triggered upon DNA damage. It enables bacteria to maintain the integrity of their genome. The key regulator of the SOS response is the transcription factor LexA. LexA binds to promoter regions and represses the induction of SOS genes involved in DNA damage repair. The bacteriophage GIL01 encodes the gp7 protein, which interacts with LexA from the bacterium Bacillus thuringiensis. The gp7 protein increases the affinity of LexA for target DNA sites and inhibits RecA-induced self-cleavage of LexA. Therefore, the gp7 protein affects the induction of the SOS response which presumably inhibits the adaptation of bacteria to harsh environmental conditions, such as antibiotic stress. The aim of this work was to identify molecules that have the potential to affect the activities of LexA, similar to the gp7 protein. Therefore, we purified the recombinant proteins: the gp8 protein of the temperate bacteriophage GIL01, which infects the bacterium B. thuringiensis and the recombinant protein OrbA of the bacteriophage ICP1 that infects bacterium Vibrio cholerae. Our results indicate that the OrbA and gp8 proteins do not interact with the protein LexA of the host bacterium and do not affect the self-cleavage activity of LexA. Next, we tested the effect of selected small compounds that we obtained from the laboratory of Prof. Dr. Stanislav Gobec of the Faculty of Pharmacy, on LexA activity. Using surface plasmon resonance spectroscopy, we showed that the molecule 6-hydroxyflavone (U5) has a low affinity for the LexA protein of Escherichia coli and Staphylococcus aureus and inhibits the binding of LexA from E. coli to the target DNA sites. Using in vivo experiments, we demonstrated that U5 inhibits the growth of the E. coli ΔacrB strain and acts synergistically with the antibiotic ciprofloxacin. Furthermore, the molecule U5 weakly inhibited the growth of strain E. coli RW542, which carries the frameshift mutation in the lexA gene and the deleted promoter element of the sulA gene. Therefore, we hypothesize that compound U5 also affects processes other than the SOS response
The Use and Abuse of LexA by Mobile Genetic Elements
No full textThe SOS response is an essential process for responding to DNA damage in bacteria. The expression of SOS genes is under the control of LexA, a global transcription factor that undergoes self-cleavage during stress to allow the expression of DNA repair functions and delay cell division until the damage is rectified. LexA also regulates genes that are not part of this cell rescue program, and the induction of bacteriophages, the movement of pathogenicity islands, and the expression of virulence factors and bacteriocins are all controlled by this important transcription factor. Recently it has emerged that when regulating the expression of genes from mobile genetic elements (MGEs), LexA often does so in concert with a corepressor. This accessory regulator can either be a host-encoded global transcription factor, which responds to various metabolic changes, or a factor that is encoded for by the MGE itself. Thus, the coupling of LexA-mediated regulation to a secondary transcription factor not only detaches LexA from its primary SOS role, but also fine-tunes gene expression from the MGE, enabling it to respond to multiple stresses. Here we discuss the mechanisms of such coordinated regulation and its implications for cells carrying such MGEs.</p
The LexA regulated genes of the Clostridium difficile.
No full textInternational audienceBACKGROUND: The SOS response including two main proteins LexA and RecA, maintains the integrity of bacterial genomes after DNA damage due to metabolic or environmental assaults. Additionally, derepression of LexA-regulated genes can result in mutations, genetic exchange and expression of virulence factors. Here we describe the first comprehensive description of the in silico LexA regulon in Clostridium difficile, an important human pathogen. RESULTS: We grouped thirty C. difficile strains from different ribotypes and toxinotypes into three clusters according to lexA gene/protein variability. We applied in silico analysis coupled to surface plasmon resonance spectroscopy (SPR) and determined 16 LexA binding sites in C. difficile. Our data indicate that strains within the cluster, as defined by LexA variability, harbour several specific LexA regulon genes. In addition to core SOS genes: lexA, recA, ruvCA and uvrBA, we identified a LexA binding site on the pathogenicity locus (PaLoc) and in the putative promoter region of several genes involved in housekeeping, sporulation and antibiotic resistance. CONCLUSIONS: Results presented here suggest that in C. difficile LexA is not merely a regulator of the DNA damage response genes but also controls the expression of dozen genes involved in various other biological functions. Our in vitro results indicate that in C. difficile inactivation of LexA repressor depends on repressor's dissociation from the operators. We report that the repressor's dissociation rates from operators differentiate, thus the determined LexA-DNA dissociation constants imply on the timing of SOS gene expression in C. difficile
Intradomain LexA rotation is a prerequisite for DNA binding specificity
No full textAbstractIn the absence of DNA damage the LexA protein represses the bacterial SOS system. We performed molecular dynamic simulations of two LexA dimers bound to operators. Our model predicted that rotation of the LexA DNA binding domain, with respect to the dimerised C-terminal domain, is required for selective DNA binding. To confirm the model, double and quadruple cysteine LexA mutants were engineered. Electrophoretic mobility-shift assay and surface plasmon resonance showed that disulfide bond formation between the introduced cysteine residues precluded LexA specific DNA binding due to blocked domain reorientation. Our model could provide the basis for novel drug design
The LexA regulated genes of the Clostridium difficile
No full textBackground: The SOS response including two main proteins LexA and RecA, maintains the integrity of bacterial genomes after DNA damage due to metabolic or environmental assaults. Additionally, derepression of LexA-regulated genes can result in mutations, genetic exchange and expression of virulence factors. Here we describe the first comprehensive description of the in silico LexA regulon in Clostridium difficile, an important human pathogen.
Results: We grouped thirty C. difficile strains from different ribotypes and toxinotypes into three clusters according to lexA gene/protein variability. We applied in silico analysis coupled to surface plasmon resonance spectroscopy (SPR) and determined 16 LexA binding sites in C. difficile. Our data indicate that strains within the cluster, as defined by LexA variability, harbour several specific LexA regulon genes. In addition to core SOS genes: lexA, recA, ruvCA and uvrBA, we identified a LexA binding site on the pathogenicity locus (PaLoc) and in the putative promoter region of several genes involved in housekeeping, sporulation and antibiotic resistance.
Conclusions: Results presented here suggest that in C. difficile LexA is not merely a regulator of the DNA damage response genes but also controls the expression of dozen genes involved in various other biological functions. Our in vitro results indicate that in C. difficile inactivation of LexA repressor depends on repressor`s dissociation from the operators. We report that the repressor`s dissociation rates from operators differentiate, thus the determined LexA-DNA dissociation constants imply on the timing of SOS gene expression in C. difficile
RAP: a new computer program for de novo identification of repeated sequences in whole genomes
No full textMOTIVATION: DNA repeats are a common feature of most genomic sequences. Their de novo identification is still difficult despite being a crucial step in genomic analysis and oligonucleotides design. Several efficient algorithms based on word counting are available, but too short words decrease specificity while long words decrease sensitivity, particularly in degenerated repeats.
RESULTS: The Repeat Analysis Program (RAP) is based on a new word-counting algorithm optimized for high resolution repeat identification using gapped words. Many different overlapping gapped words can be counted at the same genomic position, thus producing a better signal than the single ungapped word. This results in better specificity both in terms of low-frequency detection, being able to identify sequences repeated only once, and highly divergent detection, producing a generally high score in most intron sequences.
AVAILABILITY: The program is freely available for non-profit organizations, upon request to the authors.
CONTACT: [email protected]
SUPPLEMENTARY INFORMATION: The program has been tested on the Caenorhabditis elegans genome using word lengths of 12, 14 and 16 bases. The full analysis has been implemented in the UCSC Genome Browser and is accessible at http://genome.cribi.unipd.it
The SOS Response Master Regulator LexA Is Associated with Sporulation, Motility and Biofilm Formation in Clostridium difficile.
No full textThe LexA regulated SOS network is a bacterial response to DNA damage of metabolic or environmental origin. In Clostridium difficile, a nosocomial pathogen causing a range of intestinal diseases, the in-silico deduced LexA network included the core SOS genes involved in the DNA repair and genes involved in various other biological functions that vary among different ribotypes. Here we describe the construction and characterization of a lexA ClosTron mutant in C. difficile R20291 strain. The mutation of lexA caused inhibition of cell division resulting in a filamentous phenotype. The lexA mutant also showed decreased sporulation, a reduction in swimming motility, greater sensitivity to metronidazole, and increased biofilm formation. Changes in the regulation of toxin A, but not toxin B, were observed in the lexA mutant in the presence of sub-inhibitory concentrations of levofloxacin. C. difficile LexA is, therefore, not only a regulator of DNA damage but also controls many biological functions associated with virulence
Lytic gene expression in the temperate bacteriophage GIL01 is activated by a phage-encoded LexA homologue
No full textThe GIL01 bacteriophage is a temperate phage that infects the insect pathogen Bacillus thuringiensis. During the lytic cycle, phage gene transcription is initiated from three promoters: P1 and P2, which control the expression of the early phage genes involved in genome replication and P3, which controls the expression of the late genes responsible for virion maturation and host lysis. Unlike most temperate phages, GIL01 lysogeny is not maintained by a dedicated phage repressor but rather by the host's regulator of the SOS response, LexA. Previously we showed that the lytic cycle was induced by DNA damage and that LexA, in conjunction with phage-encoded protein gp7, repressed P1. Here we examine the lytic/lysogenic switch in more detail and show that P3 is also repressed by a LexA-gp7 complex, binding to tandem LexA boxes within the promoter. We also demonstrate that expression from P3 is considerably delayed after DNA damage, requiring the phage-encoded DNA binding protein, gp6. Surprisingly, gp6 is homologous to LexA itself and, thus, is a rare example of a LexA homologue directly activating transcription. We propose that the interplay between these two LexA family members, with opposing functions, ensures the timely expression of GIL01 phage late genes.</p
Bacteriophage GIL01 gp7 interacts with host LexA repressor to enhance DNA binding and inhibit RecA-mediated auto-cleavage
No full textThe SOS response in Eubacteria is a global response to DNA damage and its activation is increasingly associated with the movement of mobile genetic elements. The temperate phage GIL01 is induced into lytic growth using the host's SOS response to genomic stress. LexA, the SOS transcription factor, represses bacteriophage transcription by binding to a set of SOS boxes in the lysogenic promoter P1. However, LexA is unable to efficiently repress GIL01 transcription unless the small phage-encoded protein gp7 is also present. We found that gp7 forms a stable complex with LexA that enhances LexA binding to phage and cellular SOS sites and interferes with RecA-mediated auto-cleavage of LexA, the key step in the initiation of the SOS response. Gp7 did not bind DNA, alone or when complexed with LexA. Our findings suggest that gp7 induces a LexA conformation that favors DNA binding but disfavors LexA auto-cleavage, thereby altering the dynamics of the cellular SOS response. This is the first account of an accessory factor interacting with LexA to regulate transcription.Finnish Centre of Excellence in Biological Interactions [252411]; (cofunded under the Marie Curie Actions of the European Commission) [FP7-COFUND];EMBO[ASTF 286-2013 to N.F.]; Academy of Finland [251106 to J.K.B.]; Slovenian Research Agency [P1-0207 to M.B. and I0-0021 to V.H. and G.A.]; Spanish Ministry of Economy and Competitiveness [BFU2011-23645 to M.S.]. Funding for open access charge: Academy of Finland.Peer Reviewe
LexA and AsnC can bind to <i>cea8</i> promoter region simultaneously.
No full text<p>A) The panel shows an EMSA analysing the binding of purified LexA and AsnC protein to a P<sup>32</sup> end-labelled <i>cea8</i> fragment in the presence of L-asparagine (+ L-Asn). The concentration of AsnC in lanes 2–7 and 9–14 was 0.5, 1.05, 2.1, 4.2, 8.4 and 12.6 μM, respectively. LexA protein was present in reactions at concentration of 400 nM. The location of free DNA, the position of the wells and the various protein/DNA complexes is marked. B) The binding of purified LexA and AsnC proteins to the <i>cea8</i> promoter fragment was studied by DNase I footprinting in the presence and absence of L-asparagine (± L-Asn). AsnC was included at concentrations of 1.0, 2.1 and 4.2 μM and LexA at a concentration of 400 nM. The prominent hypersensitive bands, corresponding to positions +1 and -16, that are produced by the concurrent binding of AsnC and LexA, are starred. Red boxes indicate the position of AsnC interactions, which are affected by L-Asn. The location of the two LexA binding sites, LexA<sub>1</sub> and LexA<sub>2</sub>, is indicated by orange boxes and the -10 and -35 promoter elements by blue boxes. C) The panel shows an extended run of the footprint analysis from panel B, focusing on positions -80 to -60 upstream of the transcription start site. Labels are identical to those described above. Panels D-F) show models of the different nucleoprotein complexes formed at the <i>cea8</i> promoter by the binding of LexA and AsnC.</p
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