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At least 253 records · Page 14Linked to original sources
PubMed1997

The VITOTOX test, an SOS bioluminescence Salmonella typhimurium test to measure genotoxicity kinetics.

A new test to detect genotoxicity, that we refer to as the VITOTOX test, was developed. Four gene fusions that are based on the Escherichia coli recN promoter were constructed and evaluated for their SOS response-dependent induction. The wild-type recN promoter, a derivative mutated in the second LexA binding site, a derivative with a mutated -35 region, and a derivative from which both the second LexA binding site and the -35 region were mutated, were cloned upstream of the promoterless Vibrio fischeri luxCDABE operon of pMOL877, in such a way that lux became under transcriptional control of the recN promoter derivatives. The inducibility by the SOS response of the promoter constructs was tested in both E. coli and in the Ames test Salmonella typhimurium strains TA98, TA100 and TA104. In all strains, the highest sensitivity and induction was observed with the plasmids pMOL1067 and pMOL1068, that contain the lux operon under control of the recN promoter mutated in the second LexA binding site, or a recN promoter with a mutated -35 region, respectively. Therefore, strains containing pMOL1067 or pMOL1068 were further used for genotoxicity testing. With the VITOTOX test, genotoxicity was detected within 1-4 h. The VITOTOX test is very sensitive: for most products tested, the minimal detectable concentration (MDC) values were considerably lower (5 to > 100 times) than those described for the Ames test and the SOS chromotest. A good correlation was observed with the results from the Ames tests, but certain PAHs that are not mutagenic in the Ames test were genotoxic in the VITOTOX test. With the VITOTOX strains, the kinetics of SOS induction can be determined. This feature made it possible to distinguish between compounds in mixtures of genotoxic products so long as they had different induction kinetics.

Bacterial Proteins↗
PubMed2002

Characterization of Xanthomonas axonopodis pv. citri LexA: recognition of the LexA binding site.

Levels of l exA transcripts are markedly increased upon exposure of Xanthomonas axonopodis pathovar citri ( X. a. pv. citri) to the DNA-damaging agent mitomycin C. Preliminary electrophoretic mobility-shift data led us to propose that binding of LexA protein to the sequence upstream of the lexA coding region is responsible for low promoter activity in the uniduced state. We determined that the LexA protein binds to the region located between the transcription start site and the translation initiation codon of the lexA gene of X. a. pv. citri. Using a DNase I footprinting technique, we identified a 19-bp palindromic sequence, TTAGTAGTAATACTACTAA (TTAGN(11)CTAA), located in this region as the binding sequence for the LexA protein of X. a. pv. citri, and showed that the two halves of the palindrome have to be in the inverted repeat orientation to permit binding of LexA. We also showed that almost any mutation in this sequence, including changes in the length of the spacer region of the palindrome, destroyed its ability to bind LexA both in vitro and in vivo.

Bacterial Proteins↗
PubMed1997

Production of radiation-resistant E. coli strains by daily X-irradiation.

Exposure of E. coli AB1157 (a K-12 wild-type strain) to very large, daily X-ray doses has produced mutant strains resistant to both X-rays and UV photons. Results reported here are with KS0(160), the most resistant strain isolated thus far. Relative to its parent, KS0(160) is about 2.3 x more resistant to X-rays and about 2.2 x more resistant to ultraviolet photons (ratios of sensitivities in air). Two other characteristics of KS0(160) make its responses to X-rays different from those of AB1157: KS0(160) has an oxygen enhancement ratio of only 1.8 compared with 2.7 for its parent; glycerol reduces the sensitivity of AB1157 by about 75% (in air), but the maximum effect in KS0(160) is only a 49% reduction in response. P1 transduction experiments showed that all the acquired resistance in KS0(160) is lost when SOS repair activity is genetically blocked by an inserted lexAl, indicating that the mutation(s) associated with the acquired resistance in KS0(160) are in wild-type genes involved with induced DNA repair (i.e. SOS repair activity).

Dose-Response Relationship, Radiation↗
PubMed1994

Interspecies regulation of the recA gene of gram-negative bacteria lacking an E. coli-like SOS operator.

The recA genes of Agrobacterium tumefaciens, Rhizobium meliloti, Rhizobium phaseoli and Rhodobacter sphaeroides, species belonging to the alpha-group bacteria of the Proteobacteria class, have been fused in vitro to the lacZ gene of Escherichia coli. By using a mini-Tn5 transposon derivative, each of these recA-lacZ fusions was introduced into the chromosome of each of the four species, and into that of E. coli. The recA genes of three of the alpha bacteria are induced by DNA damage when inserted in A. tumefaciens, R. phaseoli or R. meliloti chromosomes. The expression of the recA gene of R. sphaeroides is DNA damage-mediated only when present in its own chromosome; none of the genes is induced in E. coli. Likewise, the recA gene of E. coli is not induced in any of the four alpha species. These data indicate that A. tumefaciens, R. meliloti and R. phaseoli possess a LexA-like repressor, which is able to block the expression of their recA genes, as well as that of R. sphaeroides, but not the recA gene of E. coli. The LexA repressor of R. sphaeroides does not repress the recA gene of A. tumefaciens, R. meliloti, R. phaseoli or E. coli.

Agrobacterium tumefaciens↗
PubMed1994

SeqA: a negative modulator of replication initiation in E. coli.

In E. coli, replication initiates at a genetically unique origin, oriC. Rapidly growing cells contain multiple oriC copies. Initiation occurs synchronously, once and only once per cell cycle at all origins present. Secondary initiations are prevented by a sequestration process that acts uniquely on newly replicated origins, which are marked because they are hemimethylated at GATC sites. We report the identification of a gene required for sequestration and demonstrate that this gene, seqA, also serves as a negative modulator of the primary initiation process. All previously identified in vivo initiation factors play positive roles. Thus, precise control of replication initiation may involve a balance between positive and negative elements. We suggest that SeqA might be a cooperativity factor, acting to make the replication initiation process dependent upon cooperative interactions among components.

Amino Acid Sequence↗
PubMed2004

Regulation of transposition in bacteria.

Mobile genetic elements (MGEs) play a central role in the evolution of bacterial genomes. Transposable elements (TE: transposons and insertion sequences) represent an important group of these elements. Comprehension of the dynamics of genome evolution requires an understanding of how the activity of TEs is regulated and how their activity responds to the physiology of the host cell. This article presents an overview of the large range of, often astute, regulatory mechanisms, which have been adopted by TEs. These include mechanisms intrinsic to the element at the level of gene expression, the presence of key checkpoints in the recombination pathway and the intervention of host proteins which provide a TE/host interface. The multiplicity and interaction of these mechanisms clearly illustrates the importance of limiting transposition activity and underlines the compromise that has been reached between TE activity and the host genome. Finally, we consider how TE activity can shape the host genome.

Bacteria↗
PubMed1991

Escherichia coli mfd mutant deficient in "mutation frequency decline" lacks strand-specific repair: in vitro complementation with purified coupling factor.

Mutation frequency decline (MFD) is the rapid decrease in the frequency of certain induced nonsense suppressor mutations occurring when protein synthesis is transiently inhibited immediately after irradiation. MFD is abolished by mutations in the uvrA, -B, or -C genes, which prevent excision repair, or by a mfd mutation, which reduces the rate of excision but does not affect survival. Using an in vitro repair synthesis assay we found that although wild-type cells repair the transcribed (template) strand preferentially, mfd- cells are incapable of strand-specific repair. The deficiency in strand-selective repair of mfd- cell extract was corrected by adding highly purified "transcription-repair coupling factor" to the reaction mixture. We conclude that mfd is, most likely, the gene encoding the transcription-repair coupling factor.

Alleles↗
PubMed1992

Regulatory mutants and transcriptional control of the Serratia marcescens extracellular nuclease gene.

The extracellular nuclease of Serratia marcescens is regulated in a complex fashion. Unlike most catabolic enzymes, it appears not to be substrate regulated. However we have shown it to be regulated by an SOS-like system in S. marcescens. Additionally nuclease expression is regulated in a growth-phase-dependent manner. In this work we demonstrate that growth-phase-dependent regulation is at the transcriptional level. The putative LexA-binding site which mediates SOS regulation is shown to act as an operator site in vivo. The boundaries of a minimal promoter, still regulated by growth phase and SOS regulation, are defined along with the transcriptional start site. However, a region upstream of the nuclease promoter is shown to enhance significantly the expression of nuclease.

Amino Acid Sequence↗
PubMed1999

A consensus sequence for the Rhodospirillaceae SOS operators.

The sequences controlling the expression of the Rhodobacter capsulatus recA and uvrA genes belonging to the SOS DNA repair system have been identified by PCR mutagenesis. Data obtained demonstrated that the GTTCN7GTAC and GAACN7GAAC motifs present upstream of the recA gene and the GTTCN7GTTC motif found upstream of the uvrA gene are required for their respective DNA damage-mediated induction. Alignment of recA promoters of R. capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis with the uvrA promoters of R. capsulatus and R. sphaeroides has identified the consensus sequence GTTCVYVYTWTGTTC as the SOS operator site of the Rhodospirillaceae family.

Adenosine Triphosphatases↗
PubMed2001

Error-prone repair DNA polymerases in prokaryotes and eukaryotes.

DNA repair is crucial to the well-being of all organisms from unicellular life forms to humans. A rich tapestry of mechanistic studies on DNA repair has emerged thanks to the recent discovery of Y-family DNA polymerases. Many Y-family members carry out aberrant DNA synthesis-poor replication accuracy, the favored formation of non-Watson-Crick base pairs, efficient mismatch extension, and most importantly, an ability to replicate through DNA damage. This review is devoted primarily to a discussion of Y-family polymerase members that exhibit error-prone behavior. Roles for these remarkable enzymes occur in widely disparate DNA repair pathways, such as UV-induced mutagenesis, adaptive mutation, avoidance of skin cancer, and induction of somatic cell hypermutation of immunoglobulin genes. Individual polymerases engaged in multiple repair pathways pose challenging questions about their roles in targeting and trafficking. Macromolecular assemblies of replication-repair "factories" could enable a cell to handle the complex logistics governing the rapid migration and exchange of polymerases.

Animals↗
PubMed1985

Expression of the SOS genes of Escherichia coli in Salmonella typhimurium.

To lysogenize Salmonella typhimurium by Lambda phage, a region of 10.2 kb of Escherichia coli DNA carrying the nusA gene was cloned in a S. typhimurium strain containing a F'112 plasmid which codifies for the lamB region of E. coli. The strain of S. typhimurium obtained in this way, was lysogenized by lambda c IndO- bacteriophage harboring either a fusion between recA1 or sfiA genes of E. coli with lacZ gene. Likewise, pSE143 plasmid with a umu C::lacZ fusion was introduced in S. typhimurium. Afterwards, induction of these SOS genes was studied. Results obtained show that the basal transcription of both recA and sfiA genes of E. coli was higher in S. typhimurium than in E. coli. Nevertheless, induction of recA and sfiA genes by UV-irradiation and mitomycin C was higher in E. coli than in S. typhimurium. On the other hand, umuC gene of E. coli presents the same basal level of transcription in both E. coli and S. typhimurium species, although induction of this gene by UV-irradiation and mitomycin C was higher in S. typhimurium than in E. coli. Therefore, the plasmid pUA25 constructed in this work may be used to introduce, using the Lambda phage as a vector, the SOS genes of E. coli in other bacterial species which may be useful to study the relationship between their respective SOS systems.

Bacterial Proteins↗
PubMed2001

Environmental regulation of recA gene expression in Porphyromonas gingivalis.

The recA gene product in Porphyromonas gingivalis is involved in DNA repair. Further, disruption of this gene can affect the proteolytic activity and expression of other virulence factors in this organism. Since several known environmental factors can influence virulence gene expression in P. gingivalis, we investigated the influence of these signals on the expression of the recA gene in this organism. A heterodiploid strain of P. gingivalis (designated FLL118) containing a transcriptional fusion of the recA promoter region and the promoterless tetracycline-resistant gene [tetA(Q)2] and xylosidase/arabinosidase (xa) gene cassette was constructed. The recA promoter activity was assessed by measurement of xylosidase activity in FLL118. The expression remained relatively constant during different growth phases, at different pH levels and in the presence of DNA-damaging agents. In response to hemin limitation and in the presence of calcium there was a moderate increase in recA promoter activity. Temperature also affected the expression. The highest level of xylosidase activity was observed in cultures at 32 degrees C with a decline of approximately 46% as growth temperature increased to 41 degrees C. Reverse transcriptase polymerase chain reaction analysis revealed that this regulation may be occurring at the transcriptional level. These results suggest that expression of the recA gene in P. gingivalis W83 is responsive to several environmental signals but is not regulated by a DNA damage-inducible SOS-like regulatory system.

Calcium↗
PubMed2005

Genetic analysis of the requirements for SOS induction by nalidixic acid in Escherichia coli.

Nalidixic acid, the prototype antibacterial quinolone, induces the SOS response by a mechanism that requires the RecBCD nuclease/helicase. A key step inferred for this induction pathway is the conversion of a drug-induced gyrase cleavage complex into a DNA break that can be processed by RecBC. We tried to clarify the nature of this step by searching for additional gene products that are specifically necessary for SOS induction following nalidixic acid treatment. A transposon library of approximately 19,000 insertion mutants yielded 18 mutants that were substantially reduced for SOS induction following nalidixic acid but not UV treatment, and which were also hypersensitive to nalidixic acid. All 18 mutants turned out to have insertions in recB or recC. As expected, recA insertion mutants were uncovered as being uninducible by either nalidixic acid or UV treatment. Insertions in 11 other genes were found to cause partial defects in SOS induction by one or both pathways, providing possible leads in understanding the detailed mechanisms of SOS induction. Overall, these results suggest that nalidixic acid-induced DNA breaks are generated either by RecBC itself, by redundant activities, and/or by an essential protein that could not be uncovered with transposon mutagenesis.

Anti-Infective Agents↗
PubMed1995

Interspecies gene exchange in bacteria: the role of SOS and mismatch repair systems in evolution of species.

Analysis of interspecies matings between S. typhimurium and E. coli indicates that the genetic barrier that separates these (and perhaps many other) related species is primarily recombinational. The structural component of this barrier is genomic sequence divergence. The mismatch repair enzymes act as potent inhibitors of interspecies recombination, whereas the SOS system acts as an inducible positive regulator. Interspecies mating triggers a RecBC-dependent SOS response in female bacteria that increases recombination mainly through overproduction of the RecA protein. Mismatch repair acts to reduce the mutation rate and recombination between similar sequences, whereas SOS acts to increase both. These opposing activities allow mismatch repair and SOS systems to determine both the rate of accumulation of sequence divergence and the extent of genetic isolation, which are the key components of the speciation process.

Biological Evolution↗
PubMed2005

Regulation of finP transcription by DNA adenine methylation in the virulence plasmid of Salmonella enterica.

DNA adenine methylase (Dam(-)) mutants of Salmonella enterica serovar Typhimurium contain reduced levels of FinP RNA encoded on the virulence plasmid. Dam methylation appears to regulate finP transcription, rather than FinP RNA stability or turnover. The finP promoter includes canonical -10 and -35 modules and depends on the sigma(70) factor. Regulation of finP transcription by Dam methylation does not require DNA sequences upstream from the -35 module, indicating that Dam acts at the promoter itself or downstream. Unexpectedly, a GATC site overlapping with the -10 module is likewise dispensable for Dam-mediated regulation. These observations indicate that Dam methylation regulates finP transcription indirectly and suggest the involvement of a host factor(s) responsive to the Dam methylation state of the cell. We provide evidence that one such factor is the nucleoid protein H-NS, which acts as a repressor of finP transcription in a Dam(-) background. H-NS also restrains transcription of the overlapping traJ gene, albeit in a Dam-independent fashion. Hence, the decreased FinP RNA content found in Dam(-) hosts of S. enterica appears to result from H-NS-mediated repression of finP transcription.

Adenine↗
PubMed2004

The small RNA IstR inhibits synthesis of an SOS-induced toxic peptide.

More than 60 small RNAs (sRNA) have been identified in E. coli. The functions of the majority of these sRNAs are still unclear. For the few sRNAs characterized, expression and functional studies indicate that they act under stress conditions. Here, we describe a novel E. coli chromosome locus that is part of the SOS response to DNA damage. This locus encodes two sRNAs, IstR-1 and IstR-2, and a toxic peptide, TisB, encoded by tisAB mRNA. Transcription of tisAB and istR-2 is SOS regulated, whereas IstR-1 is present throughout growth. IstR-1 inhibits toxicity by base-pairing to a short region in the tisAB mRNA. This antisense interaction entails RNase III-dependent cleavage, thereby inactivating the mRNA for translation. In the absence of the SOS response, IstR-1 is present in high excess over its target. However, SOS induction leads to depletion of the IstR-1 pool, concomitant with accumulation of tisAB mRNA. Under such conditions, TisB exerts its toxic effect, slowing down growth. We propose that the inhibitory sRNA prevents inadvertent TisB synthesis during normal growth and, possibly, also limits SOS-induced toxicity. Our study adds the SOS regulon to the growing list of global regulatory circuits controlled by sRNA genes.

Amino Acid Sequence↗
PubMed1992

Mutagenesis by apurinic sites in normal and ataxia telangiectasia human lymphoblastoid cells.

We used a shuttle vector based on the Epstein-Barr virus origin of plasmid replication (oriP) to determine the types of mutations induced by depurination in human cells. Plasmid DNA was incubated at pH 2 at 40 degrees C for various times to induce up to 20 apurinic (AP) sites per 9.7-kb plasmid and electroporated into lymphoblastoid cells derived from either a normal individual or an ataxia telangiectasia patient. After replication of the vector in the human cells, plasmid DNA was isolated and analyzed for mutations induced in the plasmid-encoded herpes simplex virus type 1-thymidine kinase gene. Both the frequencies and types of mutations induced by depurination were essentially identical for normal and ataxia telangiectasia cells. The mutant frequency at 20 AP sites/plasmid was 10-fold to 13-fold greater than that observed for untreated DNA. Deletion and frameshift events accounted for 46-55% of the mutants induced by depurination. The induced deletions were relatively small (median size, 100-150 bp) and characterized by short (1-5 bp) regions of sequence homology at the endpoints. These mutations and the frameshifts, a majority of which occurred in runs of identical nucleotides, are consistent with a model involving AP-site-induced template dislocation during DNA synthesis. A broad spectrum of base-substitution mutations, which accounted for 19-36% of the induced mutants, was observed. The apparent preference for insertion opposite AP sites in human cells was G (43-55%) greater than A approximately C (18-21%) greater than T (9-14%). Our results in human cells contrast markedly with those published previously for the mutational specificity of AP sites in Escherichia coli, in which a large majority of the mutants resulted from insertion of an A opposite the abasic site.

Apurinic Acid↗
PubMed2001

The Escherichia coli SOS gene sbmC is regulated by H-NS and RpoS during the SOS induction and stationary growth phase.

sbmC, an Escherichia coli gene, belongs to the SOS regulon, whose product is involved in cell susceptibility to microcin B17 and its expression is induced at the onset of the stationary growth phase. In the present work, we have investigated the regulation of sbmC expression during SOS induction and the stationary growth phase using a single-copy sbmC'-'lacZ fusion. The SOS induction of sbmC is profoundly diminished in the hns mutant and less diminished in the rpoS mutant. The strain with hns, rpoS double mutation, showed a similar level of sbmC induction to that of a strain with hns single mutation. Mutation in rpoS or hns causes the repression of the sbmC gene during the stationary growth phase. The sbmC expression in the rpoS mutant strain was approximately twofold lower than that in the hns mutant and the rpoS hns double mutant showed a similar level of sbmC expression to mutants deficient in rpoS alone. Interestingly, the sbmC'-'lacZ expression in the exponential growth phase was not derepressed in the hns mutant background. Transformation of hns and rpoS mutants with plasmids carrying histone-like nucleoid protein (H-NS) and RpoS effectively restored the sbmC expression to the wild-type level, respectively. The gel mobility shift assay showed that purified H-NS protein directly bound with a high affinity to a DNA fragment carrying the sbmC promoter region. These findings demonstrate that H-NS regulates the sbmC expression via H-NS's direct binding to the promoter region. In conclusion, our data suggest that H-NS and RpoS regulate a stationary phase-inducible sbmC gene in E. coli.

Bacterial Proteins↗