ESCHERICHIA COLI Gene Induction by Alkylation Treatment

Searches for alkylation-inducible (aid) genes of Escherichia coli have been conducted by screening random fusions of the Mu-dl(ApR lac) phage for fusions showing increased beta-galactosidase activity after treatment with methylating agents, but not after treatments with UV-irradiation. In this report we describe gene fusions that are specifically induced by alkylation treatments. Nine new mutants are described, and their properties are compared with the five mutants described previously. The total of 14 fusion mutants map at five distinct genetic loci. They can be further subdivided on the basis of their induction by methyl methanesulfonate (MMS) and N-methyl-N' -nitro-N-nitrosoguanidine (MNNG). alkA, aidB and aidD are induced by both agents and appear to be regulated by ada. Neither aidC nor aidI is regulated by ada. Moreover, since aidC is induced only by MNNG and aidI is induced only by MMS, these two genes are likely to be individually regulated. Thus, there appear to be at least three different regulatory mechanisms controlling aid genes.     

SOS induction of the gene sulA is partly inhibited in Escherichia coli K12 cells overproducing the RecA protein

A study was made of the SOS induction of the gene sulA of Escherichia coli K12 in relation to the gene dosage of the gene recA. In experiments the sulA::lacZ fusion strain PQ37 and derivatives of PQ37 with the multi-copy plasmids pDR1453 or pBR322 were used. The SOS response was induced with nitrofurantoin, SOS induction of the gene sulA was determined on the basis of the amount of beta-galactosidase synthesized, i.e. by the SOS chromotest (Quillardet et al., 1982a). It was found in this work that cells with the plasmid pDR1453, which contain the gene recA of E. coli K12 (Sancar and Rupp, 1979), have a decreased SOS induction of the gene sulA. Cells with the plasmid pBR322 do not exhibit this decrease. Inactivation of the gene recA in the plasmid pDR1453 with preservation of the functional gene recA in the chromosome leads to a restoration of 'standard' SOS induction of the gene sulA. The results show that the amount of the gene product of the gene recA affects the SOS induction of the gene sulA.     

Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant

We isolated a new recF mutant of Escherichia coli K-12 by insertion of transposon Tn5 into the recF gene. This recF400::Tn5 allele displayed the same phenotypic characteristics as the classic recF143 mutation. By using Mu d(Ap lac) fusions, the induction of nine SOS genes, including recA, umuC, dinA, dinB, dinD, dinF, recN, and sulA, by UV irradiation and nalidixic acid was examined. Induction of eight genes by the two agents was impaired by recF400::Tn5 to different extents. The ninth fused SOS gene, dinF, was no longer inducible by UV when combined with recF400::Tn5. The generally impaired SOS response in recF strains did not result from weak induction of recA protein synthesis, since a recA operator-constitutive mutation did not alleviate the inhibitory effect of the recF mutation. The results suggest that recF plays a regulatory role in the SOS response. It is proposed that this role is to optimize the signal usage by recA protein to become a protease.     

Oxidative and SOS-response in Escherichia coli and their interference]

Interference between the oxidative and SOS responses in Escherichia coli was studied. The oxidative response involves both reactive oxygen scavenging system and DNA repair systems which are distinct from either the SOS or adaptive response to alkylating agents. The oxyR gene is a positive regulatory gene for the oxidative response and controls at least 9 proteins which are induced by treatment with H2O2. This gene is not a portion of the SOS regulon that involves at least 17 different genes in E. coli and controls the SOS response--another inducible and nonspecific repair activity. The SOS response was measured in E. coli PQ37 by means of a sfiA: :lacZ operon fusion according to "SOS Chromotest" in a completely automated system "Bioscreen C" (Labsystems, Finland). Our data have shown that: 1) H2O2 was a potent inducer of sfiA gene--one of the SOS genes; 2) there was strong negative effect of the oxidative response on the subsequent induction of the SOS response. In common with our previous findings it should be concluded that there is an interference between the SOS response--on the one hand, and the adaptive and oxidative responses--on the other. The nonspecific heat shock response is proposed to be a main key in these interferences.     

Gene expression in E. coli after treatment with streptozotocin

Gene induction by the methylating agents streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was evaluated in E. coli fusion mutants. These mutants have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents and were previously selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methyl methanesulfonate or MNNG. The results demonstrate that STZ differs from MNNG and MNU in failing to induce aidC expression. Further, expression of aidC after exposure to MNU and MNNG occurs only in nonaerated cultures; aeration blocks the induction. Induction of aidD, alkA, aidB, and sfiA expression occurs with all 3 agents although at markedly lower concentrations of MNNG and STZ compared to MNU. alkA and to a lesser extent aidD mutants of E. coli strains were more sensitive to these agents, while no differences were evident between wild-type and aidB or aidC fusion mutants.     

Antibiotic-induced SOS response promotes horizontal dissemination of pathogenicity island-encoded virulence factors in staphylococci

Although mobile genetic elements have a crucial role in spreading pathogenicity-determining genes among bacterial populations, environmental and genetic factors involved in the horizontal transfer of these genes are largely unknown. Here we show that SaPIbov1, a Staphylococcus aureus pathogenicity island that belongs to the growing family of these elements that are found in many strains, is induced to excise and replicate after SOS induction of at least three different temperate phages, 80alpha, phi11 and phi147, and is then packaged into phage-like particles and transferred at high frequency. SOS induction by commonly used fluoroquinolone antibiotics, such as ciprofloxacin, also results in replication and high-frequency transfer of this element, as well as of SaPI1, the prototypical island of S. aureus, suggesting that such antibiotics may have the unintended consequence of promoting the spread of bacterial virulence factors. Although the strains containing these prophages do not normally contain SaPIs, we have found that RF122-1, the original SaPIbov1-containing clinical isolate, contains a putative second pathogenicity island that is replicated after SOS induction, by antibiotic treatment, of the prophage(s) present in the strain. Although SaPIbov1 is not induced to replicate after SOS induction in this strain, it is transferred by the antibiotic-activated phages. We conclude that SOS induction by therapeutic agents can promote the spread of staphylococcal virulence genes.     

Coupling of DNA replication and cell division: sulB is an allele of ftsZ.

Treatments that damage DNA in Escherichia coli result in the inhibition of cell division. This inhibition is controlled by the lexA-recA regulatory circuit and can be specifically uncoupled by the mutations sulA (sfiA) and sulB (sfiB), which map at 21 and 2 min, respectively. Presently it is thought that sulA codes for an inducible inhibitor of cell division, the expression of which is controlled directly by the lexA repressor. In this report, it is shown that sulB is an allele of ftsZ, an essential cell division gene. A sulB mutation leads to an altered ftsZ gene product which is slightly thermosensitive and has an altered mobility on polyacrylamide gels. It is suggested that the altered ftsZ gene product is resistant to the sulA inhibitor, thus permitting cell division after induction of the SOS response. It is also shown that an increase in the gene dosage of ftsZ delays the onset of filamentation after SOS induction.     

lon incompatibility associated with mutations causing SOS induction: null uvrD alleles induce an SOS response in Escherichia coli

The uvrD gene in Escherichia coli encodes a 720-amino-acid 3'-5' DNA helicase which, although nonessential for viability, is required for methyl-directed mismatch repair and nucleotide excision repair and furthermore is believed to participate in recombination and DNA replication. We have shown in this study that null mutations in uvrD are incompatible with lon, the incompatibility being a consequence of the chronic induction of SOS in uvrD strains and the resultant accumulation of the cell septation inhibitor SulA (which is a normal target for degradation by Lon protease). uvrD-lon incompatibility was suppressed by sulA, lexA3(Ind(-)), or recA (Def) mutations. Other mutations, such as priA, dam, polA, and dnaQ (mutD) mutations, which lead to persistent SOS induction, were also lon incompatible. SOS induction was not observed in uvrC and mutH (or mutS) mutants defective, respectively, in excision repair and mismatch repair. Nor was uvrD-mediated SOS induction abolished by mutations in genes that affect mismatch repair (mutH), excision repair (uvrC), or recombination (recB and recF). These data suggest that SOS induction in uvrD mutants is not a consequence of defects in these three pathways. We propose that the UvrD helicase participates in DNA replication to unwind secondary structures on the lagging strand immediately behind the progressing replication fork, and that it is the absence of this function which contributes to SOS induction in uvrD strains.     

Novel mechanism for UV sensitivity and apparent UV nonmutability of recA432 mutants: persistent LexA cleavage following SOS induction.

The recA432 mutant allele was isolated (T. Kato and Y. Shinoura, Mol. Gen. Genet. 156:121-131, 1977) by virtue of its defect in cellular mutagenesis (Mut-) and its hypersensitivity to damage by UV irradiation (UVs), which were phenotypes expected for a recA mutant. However, we found that in a different genetic background (lexA51 sulA211 uvrB+), recA432 mutants expressed certain mutant phenotypes but not the Mut- and UVs phenotypes (D.G. Ennis, N. Ossanna, and D.W. Mount, J. Bacteriol. 171:2533-2541, 1989). We present several lines of evidence that these differences resulted from the sulA genotype of the cell and that the apparent UVs and Mut- phenotypes of the sulA+ derivatives resulted from lethal filamentation of induced cells because of persistent derepression of sulA. First, transduction of sulA(Def) mutations into the recA432 strains restored cellular mutagenesis and resistance to UV. Second, recA432 sulA+ strains underwent filamentous death following SOS-inducing treatments. Third, cleavage of LexA repressor in a recA432 strain continued at a rapid rate long after UV induction, at a time when cleavage of the repressor in the recA+ parental strain had substantially declined. Fourth, we confirmed that a single mutation (recA432) conferring both the UVs and Mut- phenotypes mapped to the recA gene. These findings indicate that the RecA432 mutant protein is defective in making the transition back to the deactivated state following SOS induction; thus, the SOS-induced state of recA432 mutants is prolonged and can account for an excess of SulA protein, leading to filamentation. These results are discussed in the context of molecular models for RecA activation for LexA and UmuD cleavage and their roles in the control of mutagenesis and cell division in the SOS response.     

Roles of Ruva, Ruvc and Recg Gene Functions in Normal and DNA Damage-Inducible Replication of the Escherichia Coli Chromosome

Induction of the SOS response in Escherichia coli activates normally repressed DNA replication which is termed inducible stable DNA replication (iSDR). We previously demonstrated that initiation of iSDR requires the products of genes, such as recA, recB and recC, that are involved in the early stages of homologous recombination. By measuring the copy number increase of the origin (oriM1) region on the chromosome, we show, in this study, that initiation of iSDR is stimulated by mutations in the ruvA, ruvC and recG genes which are involved in the late stages of homologous recombination. Continuation of iSDR, on the other hand, is inhibited by these mutations. The results suggest that Holliday recombination intermediates, left on the chromosome due to abortive recombination, arrest replication fork movement. Low levels of iSDR and sfiA (sulA) gene expression were also observed in exponentially growing ruvA, ruvC and recG mutants, suggesting that the SOS response is chronically induced in these mutants. We propose that replication forks are arrested in these mutants, albeit at a low frequency, even under the normal (uninduced) conditions.     

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.     

The pcsA gene is identical to dinD in Escherichia coli.

The pcsA68 mutant of Escherichia coli is a cold-sensitive mutant which forms long filaments with a large nucleoid in the central region at 20 degrees C. We here show that (i) the coding region for the pcsA gene is identical with orfY located upstream of pyrE and can be deleted without loss of viability; (ii) pcsA is also identical to dinD, a DNA damage-inducible gene, whose expression is regulated by the LexA-RecA system; (iii) the cold-sensitive phenotype of the pcsA68 mutation is suppressed by delta recA or lexA1 (Ind-) mutation, but not by sulA inactivation; (iv) overproduction of PcsA68 leads to inhibition of cell growth in recA+ and delta recA strains at 20 and 37 degrees C, but PcsA+ does not show such an effect at any temperature; (v) SOS response is induced in the pcsA68 mutant cells at 20 degrees C. We discuss the possible function of the pcsA gene, comparing it with the sulA or the dif-xerCD function. We also describe a new method for gene disruption with positive and negative selection.     

Physiology of the SOS response: kinetics of lexA and recA transcriptional activity following induction

The products of the lexA and recA genes play central roles in the regulation of the Escherichia coli SOS response. We have measured the rate of mRNA synthesis from each gene at intervals following various inducing treatments in order to obtain a more precise timing of the induction process. Further, we provide quantitative evidence for kinetics of decay from fully induced levels of mRNA synthesis to basal levels as the cells shut down the SOS response which are in agreement with previously published data on the expression of specific SOS functions. The induction kinetics of lexA and recA gene expression are parallel except for nalidixic acid (NAL) treatment, with the actual levels of lexA mRNA synthesis being about 10-fold lower than that of recA. Reestablishment of repression from RecA commenced over 30 min earlier than from lexA. These results are fully consistent with the model that the functions result from the increased gene expression.     

Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response.

The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N4-ethenocytosine; epsilon C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in delta recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the epsilon C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in delta recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in delta umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recA-independent alternative induction pathway for SOS gene.     

Alkylating Agents Induce Uvm, a Reca-Independent Inducible Mutagenic Phenomenon in Escherichia Coli

Noninstructive DNA damage in Escherichia coli induces SOS functions hypothesized to be required for mutagenesis and translesion DNA synthesis at noncoding DNA lesions. We have recently demonstrated that in E. coli cells incapable of SOS induction, prior UV-irradiation nevertheless strongly enhances mutagenesis at a noninstructive lesion borne on M13 DNA. Here, we address the question whether this effect, named UVM for UV modulation of mutagenesis, can be induced by other DNA damaging agents. Exponentially growing δrecA cells were pretreated with alkylating agents before transfection with M13 single-stranded DNA bearing a site-specific ethenocytosine residue. Effect of cell pretreatment on survival of the transfected DNA was determined as transfection efficiency. Mutagenesis at the ethenocytosine site in pretreated or untreated cells was analyzed by multiplex DNA sequencing, a phenotype-independent technology. Our data show that 1-methyl-3-nitro-1-nitrosoguanidine, N-nitroso-N-methylurea and dimethylsulfate, but not methyl iodide, are potent inducers of UVM. Because alkylating agents induce the adaptive response to defend against DNA alkylation, we asked if the genes constituting the adaptive response are required for UVM. Our data show that MNNG induction of UVM is independent of ada, alkA and alkB genes and define UVM as an inducible mutagenic phenomenon distinct from the E. coli adaptive and SOS responses.     

Induction of the SOS response in Escherichia coli cells under osmotic stress and in the presence of N-methyl-N'-nitro-N-nitrosoguanidine

We investigated the dynamics of the SOS response induction and the frequency of reversions induced by the monofunctional alkylating compound N-methyl-N'-nitro-N-nitrosoguanidine in Escherichia coli cells exposed to osmotic stress for 1 h. During the stress treatment of the wild-type cultures adapted and not adapted to the alkylating agent, the maximum SOS response values and induced reversion frequencies were recorded twice. The SOS response values and induced reversion frequencies remained unchanged during the whole period after attaining the maximum values in adapted and nonadapted cells carrying a mutation in the excision repair gene. Presumably, the SOS mutagenesis mechanisms are turned on in the cells with an inactivated excision repair system earlier than in wild-type cells.