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At least 19 recordsLinked to original sources
PubMed1987

Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide.

Killing of Escherichia coli by hydrogen peroxide proceeds by two modes. Mode one killing appears to be due to DNA damage, has a maximum near 1 to 3 mM H2O2, and requires active metabolism during exposure. Mode two killing is due to uncharacterized damage, occurs in the absence of metabolism, and exhibits a classical multiple-order dose-response curve up to at least 50 mM H2O2 (J. A. Imlay and S. Linn, J. Bacteriol. 166:519-527, 1986). H2O2 induces the SOS response in proportion to the degree of killing by the mode one pathway, i.e., induction is maximal after exposure to 1 to 3 mM H2O2. Mutant strains that cannot induce the SOS regulon are hypersensitive to peroxide. Analysis of the sensitivities of mutants that are deficient in individual SOS-regulated functions suggested that the SOS-mediated protection is due to the enhanced synthesis of recA protein, which is rate limiting for recombinational DNA repair. Specifically, strains wholly blocked in both SOS induction and DNA recombination were no more sensitive than mutants that are blocked in only one of these two functions, and strains carrying mutations in uvrA, -B, -C, or -D, sfiA, umuC or -D, ssb, or dinA, -B, -D, -F, -G, -H, -I, or -J were not abnormally sensitive to killing by H2O2. After exposure to H2O2, mutagenesis and filamentation also occurred with the dose response characteristic of SOS induction and mode one killing, but these responses were not dependent on the lexA-regulated umuC mutagenesis or sfiA filamentation functions, respectively. Exposure of E. coli to H2O2 also resulted in the induction of functions under control of the oxyR regulon that enhance the scavenging of active oxygen species, thereby reducing the sensitivity to H2O2. Catalase levels increased 10-fold during this induction, and katE katG mutants, which totally lack catalase, while not abnormally sensitive to killing by H2O2 in the naive state, did not exhibit the induced protective response. Protection equal to that observed during oxyR induction could be achieved by the addition of catalase to cultures of naive cells in an amount equivalent to that induced by the oxyR response. Thus, the induction of catalase is necessary and sufficient for the observed oxyR-directed resistance to killing by H2O2. Although superoxide dismutase appeared to be uninvolved in this enhanced protective response, sodA sodB mutants, which totally lack superoxide dismutase, were especially sensitive to mode one killing by H2O2 in the naive state. gshB mutants, which lack glutathione, were not abnormally sensitive to killing by H2O2.

Catalase↗
PubMed1990

Genetic analyses of cellular functions required for UV mutagenesis in Escherichia coli.

In Escherichia coli, most UV and chemical mutagenesis is not a passive process and requires the participation of the umuD and umuC gene products. However, the molecular mechanism of UV mutagenesis is not yet understood and the roles of the UmuD and UmuC proteins have not been elucidated. The umuDC operon is induced by UV irradiation and regulated as part of the SOS response. Genetic evidence now indicates that RecA-mediated cleavage activates UmuD for its role in mutagenesis. The COOH-terminal fragment of UmuD is both necessary and sufficient for this role. The RecA protein appears to have a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD at the time of SOS induction. In addition, we have obtained evidence which indicates that the GroEL and GroES proteins also play a role in UV mutagenesis. Similarities of the amino acid sequence of UmuD to the sequence of gene 45 protein of bacteriophage T4 and of the sequence of UmuC to those of the gene 44 and gene 62 proteins suggest possible roles for UmuD and UmuC in mutagenesis that are supported by preliminary evidence.

Bacterial Proteins↗
PubMed1998

Molecular characterization of Gluconobacter oxydans recA gene and its inhibitory effect on the function of the host wild-type recA gene.

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+ strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-type recA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.

Amino Acid Sequence↗
PubMed1988

Inhibition of the SOS response of Escherichia coli by the Ada protein.

Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene. On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant. The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene. Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied. This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity. However, RecF- mutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene.

Alkylation↗
PubMed1996

[Bioluminescent analysis of the SOS-response of Escherichia coli cells].

We constructed a recombinant plasmid pPLS-1 to estimate the level of SOS response in Escherichia coli by the bioluminescent method. A 6.7-kb promoterless operon of bioluminescence from Photobacterium leiognathi was cloned into a pBR322 vector, in which its expression was controlled by the SOS promoter of gene cda from a plasmid ColD. The sequence between the 5'-terminal Sph1 site of the operon and start codon ATG of the luxC gene was shown to be 56 bp in length and had no effect on the level of light emission. SOS-inducing potency of six mutagenic substances was tested in E. coli strain C600(pPLS-1). The bioluminescent method proved to be very sensitive for estimating the level of SOS response. The results obtained by this method showed good correlation with results obtained by SOS Chromotest, umu-test, and Ames' test.

Amino Acid Sequence↗
PubMed1993

Anti-SOS effects induced in Bacillus subtilis by a phi 105 mutant prophage.

The presence of the mutant prophage phi 105cts23 in Bacillus subtilis strains strongly affected several biological parameters including the viability of protoplasts and the establishment of plasmid pC194. A defective inducibility of the prophage after treatments that de-repress the SOS-like response were also observed. Although these alterations suggested a Rec-deficient phenotype, homologous recombination was not impaired in these lysogenic derivatives. In fact, chromosomal DNA transformation in these competent cells was more efficient than in cells carrying the wild type prophage: cell death due to prophage induction upon competence development was lower than expected. Alterations in the response to SOS-inducing agents and to osmotic stress correlated with the presence of this particular mutant prophage or the cloned thermosensitive repressor at the permissive temperature. The induction of an anti-SOS effect is discussed.

Bacillus Phages↗
PubMed1991

DNA lesions that block DNA replication are responsible for the dnaA induction caused by DNA damage.

The initiation protein DnaA of Escherichia coli regulates its own expression autogenously by binding to a 9 bp consensus sequence, the dnaA box, between the promoters dnaAP1 and dnaAP2. In this study, we analysed dnaA regulation in relation to DNA damage and found dnaA expression to be inducible by DNA lesions that inhibit DNA replication. On the other hand, coding DNA lesions were not able to induce dnaA expression. These results suggest that an additional regulatory mechanism is involved in dnaA gene expression and that DnaA protein may play a role in cellular responses to DNA damage. Furthermore, they strongly suggest that in response to DNA replication inhibition by DNA damage, and enhanced (re)initiation capacity is induced by oriC.

Bacterial Proteins↗
PubMed1993

Participation of the SOS system in producing deletions in E. coli plasmids.

The participation of the SOS response in the deletion of palindromic and non-palindromic inserts of about 66 and 100 bp cloned within the EcoR1 site of the chloramphenicol acetyl transferase (cat) gene of plasmid pBR325 was tested after introducing the derived plasmids into strains containing different combinations of lexA, recA and umuC alleles and the auxotrophic mutation trpE65. This allowed for a comparison of deletion frequency in the plasmids, measured as the reversion of chloramphenicol sensitivity to resistance (Cms-->Cmr), to point-mutation frequency measured from the reversion of trpE65 to tryptophan independence (Trp(-)-->Trp+). We found that the spontaneous deletion frequency of palindromic inserts was increased by the overproduction of activated RecA* and UmuC+ in lexA (Def) backgrounds but the deletion of the non-palindromic inserts was unaltered. Overproduction of RecA+ had no significant effect on deletion incidence but it did increase Trp(-)-->Trp+ reversions. The SOS stimulation of palindrome deletions paralleled the SOS mutator effect of certain recA and umuC alleles on Trp(-)-->Trp+ reversions, suggesting that some form of SOS processing was responsible for the observed increases. The results further suggest that the SOS effect on deletions depends on the distinction between palindromy vs. non-palindromy, rather than on the sizes or sequences of the inserts or those of the terminal homologies bracketing them.

Base Sequence↗
PubMed1991

Genotoxic effect of alkaloids.

Because of the increased use of alkaloids in general medical practice in recent years, it is of interest to determine genotoxic, mutagenic and recombinogenic response to different groups of alkaloids in prokaryotic and eucaryotic organisms. Reserpine, boldine and chelerythrine did not show genotoxicity response in the SOS-Chromotest whereas skimmianine showed genotoxicity in the presence of a metabolic activation mixture. Voacristine isolated from the leaves of Ervatamia coronaria shows in vivo cytostatic and mutagenic effects in Saccharomyces cerevisiae hapioids cells. The Rauwolfia alkaloid (reserpine) was not able to induce reverse mutation and recombinational mitotic events (crossing-over and gene conversion) in yeast diploid strain XS2316.

Alkaloids↗
PubMed1992

Inhibition of the recBCD-dependent activation of Chi recombinational hot spots in SOS-induced cells of Escherichia coli.

Nucleotide sequences called Chi (5'-GCTGGTGG-3') enhance homologous recombination near their location by the RecBCD enzyme in Escherichia coli (Chi activation). A partial inhibition of Chi activation measured in lambda red gam mutant crosses was observed after treatment of wild-type cells with DNA-damaging agents including UV, mitomycin, and nalidixic acid. Inhibition of Chi activation was not accompanied by an overall decrease of recombination. A lexA3 mutation which blocks induction of the SOS system prevented the inhibition of Chi activation, indicating that an SOS function could be responsible for the inhibition. Overproduction of the RecD subunit of the RecBCD enzyme from a multicopy plasmid carrying the recD gene prevented the induced inhibition of Chi activation, whereas overproduction of RecB or RecC subunits did not. It is proposed that in SOS-induced cells the RecBCD enzyme is modified into a Chi-independent recombination enzyme, with the RecD subunit being the regulatory switch key.

DNA Helicases↗
PubMed1987

Site-specific methylases induce the SOS DNA repair response in Escherichia coli.

Expression of the site-specific adenine methylase HhaII (GmeANTC, where me is methyl) or PstI (CTGCmeAG) induced the SOS DNA repair response in Escherichia coli. In contrast, expression of methylases indigenous to E. coli either did not induce SOS (EcoRI [GAmeATTC] or induced SOS to a lesser extent (dam [GmeATC]). Recognition of adenine-methylated DNA required the product of a previously undescribed gene, which we named mrr (methylated adenine recognition and restriction). We suggest that mrr encodes an endonuclease that cleaves DNA containing N6-methyladenine and that DNA double-strand breaks induce the SOS response. Cytosine methylases foreign to E. coli (MspI [meCCGG], HaeIII [GGmeCC], BamHI [GGATmeCC], HhaI [GmeCGC], BsuRI [GGmeCC], and M.Spr) also induced SOS, whereas one indigenous to E. coli (EcoRII [CmeCA/TGG]) did not. SOS induction by cytosine methylation required the rglB locus, which encodes an endonuclease that cleaves DNA containing 5-hydroxymethyl- or 5-methylcytosine (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986).

5-Methylcytosine↗
PubMed1989

Modulation of the SOS response by truncated RecA proteins.

RecA protein plays several key roles in the SOS response. We have constructed truncated proteins and examined their capacity to accomplish Weigle reactivation and mutagenesis of bacteriophage lambda and recombination in Escherichia coli. Our data indicate that the 17 carboxyl terminal amino acids are not essential to RecA function. However in the presence of wild-type RecA protein, the truncated protein reduces the efficiency of recombination without affecting either mutagenesis or induction of an SOS gene or Weigle reactivation. The data presented here suggest that activation of RecA protein does not involve mixed multimers or is not affected by their presence.

Bacteriophage lambda↗
PubMed1994

DNA replication triggered by double-stranded breaks in E. coli: dependence on homologous recombination functions.

Homologous recombination-dependent DNA replication (RDR) of a lambda cos site-carrying plasmid is demonstrated in E. coli cells when the cells express lambda terminase that introduces a double-stranded break into the cos site. RDR occurs in normal wild-type cells if the plasmid also contains the recombination hotspot chi. Chi is dispensable when cells are induced for the SOS response or contain a recD mutation. recBC sbcA mutant cells are also capable of RDR induction. A recN mutation greatly reduces RDR in normal cells, but not in SOS-induced cells. RDR proceeds by the theta mode or rolling circle mode of DNA synthesis, yielding covalently closed circular plasmid monomers or linear plasmid multimers, respectively. Previously described inducible stable DNA replication is considered to be a special type of RDR that starts exclusively from specific sites (oriMs) on the chromosome.

Bacterial Proteins↗
PubMed1998

Inhibition of Escherichia coli RecA coprotease activities by DinI.

In Escherichia coli, the SOS response is induced upon DNA damage and results in the enhanced expression of a set of genes involved in DNA repair and other functions. The initial step, self-cleavage of the LexA repressor, is promoted by the RecA protein which is activated upon binding to single-stranded DNA. In this work, induction of the SOS response by the addition of mitomycin C was found to be prevented by overexpression of the dinI gene. dinI is an SOS gene which maps at 24.6 min of the E.coli chromosome and encodes a small protein of 81 amino acids. Immunoblotting analysis with anti-LexA antibodies revealed that LexA did not undergo cleavage in dinI-overexpressed cells after UV irradiation. In addition, the RecA-dependent conversion of UmuD to UmuD' (the active form for mutagenesis) was also inhibited in dinI-overexpressed cells. Conversely, a dinI-deficient mutant showed a slightly faster and more extensive processing of UmuD and hence higher mutability than the wild-type. Finally, we demonstrated, by using an in vitro reaction with purified proteins, that DinI directly inhibits the ability of RecA to mediate self-cleavage of UmuD.

Bacterial Proteins↗
PubMed1989

Competence-specific induction of the Bacillus subtilis RecA protein analog: evidence for dual regulation of a recombination protein.

The development of competence in Bacillus subtilis is accompanied by the transcriptional activation of DNA damage-inducible (din) operons and other SOS-like responses. We report here that B. subtilis Rec protein (the analog of Escherichia coli RecA), a DNA damage-inducible protein, is substantially induced when cells differentiate to a state of competence. We quantitated the induction of B. subtilis Rec protein and the B. subtilis din-22 operon (representative of all known B. subtilis din operons) during competence development in Rec+ and DNA repair-deficient strains. We present two lines of evidence that Rec protein induction in competent cells is controlled by a competence-specific mechanism that is distinct from the SOS-like regulation that controls Rec induction following DNA damage: (i) Rec protein was significantly induced in rec mutants (recA1 and recE4) that are highly deficient in Rec induction by DNA damage, and (ii) Rec protein induction during competence development was greater than maximum Rec induction by DNA damage. On the other hand, our results suggest that the din-22 operon is induced by the same (SOS-like) mechanism both during competence development and after DNA damage.

Bacillus subtilis↗
PubMed1994

Involvement of Escherichia coli DNA polymerase II in response to oxidative damage and adaptive mutation.

DNA polymerase II (Pol II) is regulated as part of the SOS response to DNA damage in Escherichia coli. We examined the participation of Pol II in the response to oxidative damage, adaptive mutation, and recombination. Cells lacking Pol II activity (polB delta 1 mutants) exhibited 5- to 10-fold-greater sensitivity to mode 1 killing by H2O2 compared with isogenic polB+ cells. Survival decreased by about 15-fold when polB mutants containing defective superoxide dismutase genes, sodA and sodB, were compared with polB+ sodA sodB mutants. Resistance to peroxide killing was restored following P1 transduction of polB cells to polB+ or by conjugation of polB cells with an F' plasmid carrying a copy of polB+. The rate at which Lac+ mutations arose in Lac- cells subjected to selection for lactose utilization, a phenomenon known as adaptive mutation, was increased threefold in polB backgrounds and returned to wild-type rates when polB cells were transduced to polB+. Following multiple passages of polB cells or prolonged starvation, a progressive loss of sensitivity to killing by peroxide was observed, suggesting that second-site suppressor mutations may be occurring with relatively high frequencies. The presence of suppressor mutations may account for the apparent lack of a mutant phenotype in earlier studies. A well-established polB strain, a dinA Mu d(Apr lac) fusion (GW1010), exhibited wild-type (Pol II+) sensitivity to killing by peroxide, consistent with the accumulation of second-site suppressor mutations. A high titer anti-Pol II polyclonal antibody was used to screen for the presence of Pol II in other bacteria and in the yeast Saccharomyces cerevisiae. Cross-reacting material was found in all gram-negative strains tested but was not detected in gram-positive strains or in S. cerevisiae. Induction of Pol II by nalidixic acid was observed in E. coli K-12, B, and C, in Shigella flexneri, and in Salmonella typhimurium.

Adaptation, Physiological↗