Survival and SOS induction in cisplatin-treated Escherichia coli deficient in Pol II,RecBCD and RecFOR functions

Cisplatin is a potent anticancer agent forming intrastrand-crosslinks in DNA. The efficacy of cisplatin in chemotherapy can be limited by the development of tumor resistances such as elevated DNA repair or damage tolerance. In Escherichia coli, cisplatin treatment causes induction of the SOS regulon resulting in elevated levels of DNA Pol II, DNA Pol IV, DNA Pol V, the cell division inhibitor SfiA (SulA), homologous recombination (HR) and DNA repair. In this work, the roles of Pol II and HR in facilitating resistance of E. coli to cisplatin are studied. SOS induction levels were measured by beta-galactosidase assays in cisplatin-treated and untreated E. coli PQ30 that has the lacZ gene fused to the sfiA promoter. Comparative studies were carried out with derivatives of PQ30 constructed by P1 transduction that have transposon insertions in the polB gene, the recB gene blocking the RecBCD pathway of HR and genes of the RecFOR pathway of HR. Resistance of E. coli strains to cisplatin as determined by plating experiments decreased in the following order: parent PQ30 strain, polB > recO, recR, recF > recB. Both the RecBCD and RecFOR pathways of HR are important for survival when E. coli is exposed to cisplatin, because treatment of double mutants deficient in both pathways reduced colony forming ability to 37% in 6-9min in comparison to 39-120min for single mutants. Pol II and RecF appear to function in two distinct pathways to initiate replication blocked due to damage caused by cisplatin because function of Pol II was required for survival in mutants deficient in the RecFOR pathway after 2h of cisplatin treatment. In contrast, Pol II was not required for survival in recB mutants. SOS induction was delayed in RecFOR deficient mutants but occurred at high levels in the recB mutant soon after cisplatin treatment in a RecFOR-dependent way. An SfiA independent, DNA damage dependent pathway is apparently responsible for the filamentous cells observed after cisplatin or MMC treatments of these SfiA defective strains.     

On the nature of the RecBC and RecF pathways of conjugal recombination in Escherichia coli

The molecular mechanisms of the RecBC and RecF pathways for genetic recombination in E. coli were investigated by studying the kinetics of RecA protein function during conjugation. RecF recombination in recBC sbcB mutants is shown to be a much slower process than RecBC recombination in recBC+ sbcB+ strains, and is blocked by a mutation in lexA that prevents induction of RecA protein. Progress of the RecF pathway is greatly accelerated by a recAoc mutation which increases synthesis of RecA protein, but this does not restore recombination proficiency to a recBC sbcB lexA mutant. These results are interpreted to suggest that the RecF pathway directs integration of single-stranded Hfr DNA into the recipient chromosome whereas the RecBC pathway catalyses the exchange of largely double stranded DNA. This is consistent with the known stoichiometry of RecA protein catalysed heteroduplex DNA formation in vitro and with the delayed replication of RecF pathway recombinants which approximates to the time required for one round of DNA replication to generate homoduplex DNA. The regulation of the RecF pathway by lexA repressor is discussed in relation to the factors that govern the relative utilization of the two recombination pathways in wild-type cells.     

A loss of uvrA function decreases the induction of the SOS functions recA and umuC by mitomycin C in Escherichia coli

We have studied the levels of recA and umuC protein synthesis in Escherichia coli as a probe for regulatory and mechanistic events involved in mitomycin C mutagenesis. Both RecA and UmuC protein induction were greatly stimulated by mitomycin C in the wild-type strain, reached a peak at about 60 min for the recA gene, and at 90 min for the umuC gene, respectively, and maintained a plateau. The induction was blocked by recA and lexA(Ind-) mutations that conferred no mutagenesis on the cell. Mutation affecting uvrA protein markedly decreased induction of the recA gene as well as the umuC gene by mitomycin C. The results established that UvrA protein is involved in the induction of recA and umuC, and account, at least in part, for the mitomycin C nonmutability of uvrA mutants.     

RecBC and RecF recombination pathways and the induced precise excision of Tn10 in Escherichia coli

Mitomycin C (MMC) treatment or mutations in uvrD enhance the frequency of Tn10 precise excision. We have shown previously that several repair-recombination genes, such as recA, ruv and recF are involved in the induced excision process. In this study, we find that other genes belonging to the RecBC and RecF sexual recombination pathways also participate in this process since mutations in recB, sbcB or recO diminish, though to different degrees, the frequency of Tn10 precise excision induced by MMC treatment or by uvrD mutants. Pairwise combinations of some of these mutations were also tested for Tn10 induced precise excision; most of these double mutants showed additive effects in reducing the frequency of the excision process. The results of these studies suggest that recombinational-repair genes, particularly recF, sbcB and recO have different roles in the induced excision of Tn10 than in recombinational mating.     

Survival and induction of recA protein in mitomycin C-treated Escherichia coli rec, lex, or uvr strains

The capability to synthesize recA protein has been tested for Escherichia coli treated with mitomycin C. recA protein was assayed using an immunoradiometric assay (Paoletti, C., Salles, B., and Giacomoni, P. U. (1982) Biochimie 64, 239-246). Mitomycin C-treated wild type E. coli can express recA gene in a similar quantitative fashion, independently of the growth media used in this work; glucose did not inhibit induction of recA protein in cells growing in synthetic media. Wild type E. coli recovering from energy starvation displays a similar qualitative capability to induce the synthesis of recA protein independently of the stage of growth at which the cells are treated with the drug. At midexponential phase, the cells appear to have an enhanced capability to synthesize recA protein. The relationship between survival and capability to synthesize recA protein was explored for E. coli lex, rec, and/or uvr mutants, after treatment with mitomycin C. A good correlation was found, except for a recB mutant and for an ethidium-sensitive strain, both able to produce as much recA protein as the wild type but 100-fold more sensitive to the drug. A similarly satisfactory correlation was found when plotting the survival after UV irradiation versus the capability of synthetizing recA protein with the exception of an uvrA strain and of a lexA strain.     

Mechanisms of recombination by the RecBC and the RecF pathways following conjugation in Escherichia coli K12.

Summary The recombinational processes directed by the RecBC and the RecF pathways following conjugation in E. coli have been compared. The viable recombinant products of the RecF pathway show a higher incidence of mismatch correction, higher percentage of heterogeneous clones produced by single ex-conjugants and a much slowere rate of integration and segregation compared to the RecBC pathway. There are reasons to suspect that the product of recB and recC genes may be necessary for conversion of the single stranded donor DNA in the zygote to double stranded DNA. Theoretical considerations suggest that an exchange involving only one strand of DNA may be a much slower process, with more stringent homology requirement for the entire exchanged segment, than a double strand exchange of a comparable length; the latter should be much faster, with stringent homology requirements for only the terminal regions of the exchanged segments. It is suggested that the RecF pathway mainly mediates replacement of relatively long stretches of single strands of recipient DNA by the corresponding strands of donor DNA while the RecBC pathway mediates exchange of mostly double stranded DNA between the donor and the recipient; in addition, the RecBC pathway may also catalyze the integration of very small segments of single strands of the donor DNA. A model based on the above basic hypothesis is described. It is further suggested that the enzymes exonucleaseV and exonucleaseI control the relative yields of the recombinants produced by the two pathways by regulating the supply of the donor substrates required by these pathways; the former diverts the potential substrate of the RecF pathway (single stranded DNA) to the duplex substrates of the RecBC pathway while the latter destroys the substrates of the RecF pathway, especially in absence of exonucleaseV.     

Expression of the SOS response following simultaneous treatment with methyl-nitrosoguanidine and mitomycin C in Escherichia coli

Simultaneous treatment of Escherichia coli cultures with methyl-nitrosoguanidine and mitomycin C induces recA-dependent inhibition of respiration but not inhibition of cell division. This pattern of SOS functions expression is the same as that is found following treatment with methyl-nitrosoguanidine alone and contrary to the pattern induced after mitomycin C addition. The same result is obtained when a culture of E. coli RecA441 (formerly tif) is shifted to 42 degrees C and treated simultaneously with methyl-nitrosoguanidine. The suppressor effect of this compound over the pattern of SOS functions expression induced by mitomycin C or high temperature in recA441 mutants is directly related to the increase in its dose. Moreover, the division temperature-sensitive mutant ftsA treated with methyl-nitrosoguanidine and high temperature does not show any decrease in its normal filamentous growth when cultured at 42 degrees C. This indicates that the effect of methyl-nitrosoguanidine on the recA-independent inhibition of cell division is not due to any indiscriminate effect of this compound over the division process. These results suggest that the specific kind of lesion caused in DNA is very important in determining which SOS function is induced.     

Decomposition of ribosomal particles in Escherichia coli treated with mitomycin C

Exposure of cells of Escherichia coli to mitomycin C (5 mug/ml) resulted in a marked change in the sedimentation profiles of the cell-free extracts, indicating a specific decomposition of ribosomal particles. When the extracts were prepared in the presence of 0.01 m Mg(++) and analyzed by sucrose density gradient centrifugations, the 100S fraction disappeared rapidly from the treated cells. The 70S ribosomes were also degraded, but more slowly, with a concomitant accumulation of a fraction having a sedimentation coefficient of about 50S. However, decomposition of the 70S ribosomes was preceded by an almost complete loss of the 50S ribosomal subunits, as revealed by sedimentation analyses in the presence of 10(-4)m Mg(++). Synthesis of the ribosomes in the treated cells was also suppressed, being demonstrated by a lower incorporation of uracil-2-(14)C into the ribosomal fractions. However, the change in the ribosomal profile in the treated cells apparently resulted from the decomposition of pre-existing ribosomes, rather than from the inhibition of the net synthesis of ribosomes. Sedimentation analyses and chromatography of the nucleic acids extracted from the treated cells indicated extensive but delayed degradation of the ribosomal ribonucleic acid (RNA), but not of the soluble RNA or deoxyribonucleic acid fractions. Altered structure of the ribosomes in the treated cells was also indicated by their lower melting temperature, broadened thermal profile, higher electrophoretic mobility, and extreme sensitivity to ribonuclease treatment, compared with normal ribosomes. The synthesis of messenger RNA was inhibited progressively with time in the treated cells.     

Stimulation of recombination between homologous sequences on carcinogen-treated plasmid DNA and chromosomal DNA by induction of the SOS response in Escherichia coli K12

Summary Previous studies have shown that transformation of Escherichia coli by plasmid DNA modified in vitro by carcinogens leads to RecA-dependant recombination between homologous plasmid and chromosomal DNA sequences. The mechanism of this recombination has now been studied using recombination-deficient mutants, and the influence of induction of the SOS response on the level of recombination investigated. Plasmid pNO1523, containing the str ⁺ operon (Sm^s), has been modified in vitro by either irradiation with UV light, or by reaction with (±) trans-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) and used to transform streptomycin-resistant hosts. The formation of Amp^r transformants which also carry streptomycin resistance was used as a measure of the level of recombination between plasmid and chromosomal DNA. Transformation of recB and recC mutants produced no change in the level of recombination while in the recF mutant a significant decrease was observed compared to the wild type host. Thermal induction of the SOS response in tif-1 and tif-1 umuC mutants followed by transformation led to a four-fold increase in recombination in both cases. The results suggest that the streptomycin-resistant transformants arise exclusively via a recombinational pathway which is largely dependant on the recF gene product, and that this pathway is influenced by induction of the SOS response. These results are discussed in terms of the mechanism of this recombination.     

xni-deficient Escherichia coli are proficient for recombination and multiple pathways of repair

Single-strand-dependent DNA exonucleases play important roles in DNA repair and recombination in all organisms. In Escherichia coli the redundant functions provided by the RecJ, ExoI, ExoVII and ExoX exonucleases are required for mismatch repair, UV resistance and homologous recombination. We have examined whether the xni gene product, the single-strand exonuclease ExoIX, is also a member of this group. We find that deletion of xni has no effect on the above processes, or on resistance to oxidative damage, even in combination with other exonuclease mutations. We conclude that the xni gene product does not belong to this group of nucleases that play redundant roles in DNA recombination and repair.     

RecFOR function is required for DNA repair and recombination in a RecA loading-deficient recB mutant of Escherichia coli

The RecA loading activity of the RecBCD enzyme, together with its helicase and 5' --> 3' exonuclease activities, is essential for recombination in Escherichia coli. One particular mutant in the nuclease catalytic center of RecB, i.e., recB1080, produces an enzyme that does not have nuclease activity and is unable to load RecA protein onto single-stranded DNA. There are, however, previously published contradictory data on the recombination proficiency of this mutant. In a recF(-) background the recB1080 mutant is recombination deficient, whereas in a recF(+) genetic background it is recombination proficient. A possible explanation for these contrasting phenotypes may be that the RecFOR system promotes RecA-single-strand DNA filament formation and replaces the RecA loading defect of the RecB1080CD enzyme. We tested this hypothesis by using three in vivo assays. We compared the recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants. We show that RecFOR functions rescue the repair and recombination deficiency of the recB1080 mutant and that RecA loading is independent of RecFOR in the recB1080 recD double mutant where this activity is provided by the RecB1080C(D(-)) enzyme. According to our results as well as previous data, three essential activities for the initiation of recombination in the recB1080 mutant are provided by different proteins, i.e., helicase activity by RecB1080CD, 5' --> 3' exonuclease by RecJ- and RecA-single-stranded DNA filament formation by RecFOR.     

The appearance of the UmuD'C protein complex in Escherichia coli switches repair from homologous recombination to SOS mutagenesis

The process of SOS mutagenesis in Escherichia coli requires (i) the replisome enzymes, (ii) RecA protein, and (iii) the formation of the UmuD'C protein complex which appears to help the replisome to resume DNA synthesis across a lesion. We found that the UmuD'C complex is an antagonist of RecA-mediated recombination. Homologous recombination in an Hfr x F^- cross decreased as a function of the UmuD'C cell concentration; this effect was challenged by increasing RecA concentration. Recombination of a u.v.-damaged F-lac with the lac gene of an F^- recipient was reduced by increasing the UmuD'C concentration while lac mutagenesis increased, showing an inverse relationship between recombination and SOS mutagenesis. We explain our data with the following model. The kinetics of appearance of the UmuD'C complex after DNA damage is slow, reaching a maximum after an hour. Within that period, excision and recombinational repair have had time to occur. When the UmuD'C concentration relative to the number of residual RecA filaments, not resolved by recombinational repair, becomes high enough, UmuD'C proteins provide a processive factor for the replisome to help replication bypass and repel the standing RecA filament. Thus, at a high enough concentration, the UmuD'C complex will switch repair from recombination to SOS mutagenesis.     

uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway

It has previously been shown that recombination between tandem repeats is not significantly affected by a recA mutation in Escherichia coli . Here, we describe the activation of a RecA-dependent recombination pathway in a hyper-recombination mutant. In order to analyse how tandem repeat deletion may proceed, we searched for mutants that affect this process. Three hyper-recombination clones were characterized and shown to be mutated in the uvrD gene. Two of the mutations were identified as opal mutations at codons 130 and 438. A uvrD  ::Tn 5 mutation was used to investigate the mechanism of deletion formation in these mutants. The uvrD -mediated stimulation of deletion was abolished by a lexAind3 mutation or by inactivation of either the recA , recF , recQ or ruvA genes. We conclude that (i) this stimulation requires SOS induction and (ii) tandem repeat recombination in uvrD mutants occurs via the RecF pathway. In uvrD ⁺ cells, constitutive expression of SOS genes is not sufficient to stimulate deletion formation. This suggests that the RecF recombination pathway activated by SOS induction is antagonized by the UvrD protein. Paradoxically, we observed that the overproduction of UvrD from a plasmid also stimulates tandem repeat deletion. However, this stimulation is RecA independent, as is deletion in a wild-type strain. We propose that the presence of an excess of the UvrD helicase favours replication slippage. This work suggests that the UvrD helicase controls a balance between different routes of tandem repeat deletion.     

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.     

Impact of homologous and non-homologous recombination in the genomic evolution of Escherichia coli

ABSTRACT: BACKGROUND: Escherichia coli is an important species of bacteria that can live as a harmless inhabitantof the guts of many animals, as a pathogen causing life-threatening conditions or freely inthe non-host environment. This diversity of lifestyles has made it a particular focus ofinterest for studies of genetic variation, mainly with the aim to understand how acommensal can become a deadly pathogen. Many whole genomes of E. coli have beenfully sequenced in the past few years, which offer helpful data to help understand how thisimportant species evolved. RESULTS: We compared 27 whole genomes encompassing four phylogroups of Escherichia coli (A,B1, B2 and E). From the core-genome we established the clonal relationships between theisolates as well as the role played by homologous recombination during their evolutionfrom a common ancestor. We found strong evidence for sexual isolation between three lineages (A+B1, B2, E), which could be explained by the ecological structuring of E. coliand may represent on-going speciation. We identified three hotspots of homologousrecombination, one of which had not been previously described and contains the aroCgene, involved in the essential shikimate metabolic pathway. We also described the roleplayed by non-homologous recombination in the pan-genome, and showed that thisprocess was highly heterogeneous. Our analyses revealed in particular that the genomes ofthree enterohaemorrhagic (EHEC) strains within phylogroup B1 have converged fromoriginally separate backgrounds as a result of both homologous and non-homologousrecombination. CONCLUSIONS: Recombination is an important force shaping the genomic evolution and diversification ofE. coli, both by replacing fragments of genes with an homologous sequence and also byintroducing new genes. In this study, several non-random patterns of these events wereidentified which correlated with important changes in the lifestyle of the bacteria, andtherefore provide additional evidence to explain the relationship between genomicvariation and ecological adaptation.     

Induction of protein synthesis in Escherichia coli following UV- or gamma-irradiation, mitomycin C treatment or tif Expression.

Summary The rate of synthesis of total cellular proteins has been studied by pulse labelling cells at various periods after irradiation with UV or -rays, after treatment with mitomycin C (MMC) or after expression of the temperature sensitive mutation tif. Subsequent gel electrophoresis and autoradiography reveals changes in the rate of synthesis of several proteins. The most striking change is in a protein of molecular weight 40,000, protein X, which has been previously most extensively studied in cells treated with nalidixic acid (Gudas, 1976). Synthesis of large quantities of protein X is induced by UV, -rays, MMC treatment or tif expression in rec ⁺ but not recA cells. A feature of recA cells is that they break down their DNA excessively after irradiation or MMC treatment. However, if protein synthesis following irradiation is prohibited by chloramphenicol, post-irradiation degradation becomes excessive in recA ⁺ cells. This inverse relationship between DNA degradation and new protein synthesis is consistent with the hypothesis that an induced protein such as X is responsible for controlling DNA degradation following irradiation. Protein X is not induced in a lexB mutant following MMC treatment. In this respect the lexB mutant behaves like lexA and recA mutants in that the ability to induce protein X can be correlated with excessive DNA degradation.Studies on the induction of proteins in inf, tif and tif sfi mutants fail to reveal any correlation between induction of protein X and either the induction of prophage or septation.