Reciprocal and non-reciprocal homologous recombination between Escherichia coli chromosomal DNA and ultraviolet light-irradiated plasmid DNA

Plasmid DNA substrates were used to study ultraviolet (UV)-induced recombination events in Escherichia coli host cells. Plasmids derived from pBR322, containing all or part of the lac operon of E. coli, were irradiated with ultraviolet light before transformation into E. coli strains of different recA and lacY genotypes. Recombinational exchanges were identified by phenotypic changes in lactose utilization and were confirmed by restriction analysis of isolated plasmids. Ultraviolet-induced reciprocal plasmid-chromosome recombination occurred at a slightly higher frequency then non-reciprocal chromosome-to-plasmid recombination, and at a much higher frequency than non-reciprocal plasmid-to-chromosome recombination. These frequencies did not depend on segregative mechanisms. The asymmetry of non-reciprocal exchange was not due to the particular arrangement of wild-type and lacY1 alleles because the same results were observed when these were interchanged. The host recA gene was required for plasmid-chromosome recombination, and slightly enhanced plasmid survival. Evidence for plasmid replication prior to recombination was found in reciprocal recombinants, but rarely in the non-reciprocal recombinants analyzed. Irradiation of competent bacterial host cells prior to transformation did not effectively induce plasmid-chromosome recombination.     

Enhancement of UV survival, UV- and MMS-mutability, precise excision of Tn10 and complementation of umuC by plasmids of different incompatibility groups

29 conjugative resistance and colicin plasmids from 19 different incompatibility (Inc) groups were examined for their ability to enhance post-ultraviolet (UV) survival and UV- and methyl methanesulfonate(MMS)-induced mutability in Salmonella typhimurium LT2 strains. 14 Muc+ plasmids enhanced each of the survival and mutation-related properties tested, while 14 Muc- plasmids showed no enhancing effects in any tests. One Muc+ plasmid, pRG1251 (IncH1), enhanced post-UV survival and each of the mutation-related properties tested, except MMS-induced mutagenesis. Two further noteworthy plasmids, R391 (IncJ) and R394 (IncT), produced apparent strain-dependent effects in S. typhimurium which differed from those reported to have been found in Escherichia coli. Plasmid R391 enhanced post-UV survival in S. typhimurium, in contrast to its UV-sensitizing effects in E. coli. In both hosts plasmid R391 enhanced UV- and MMS-induced mutagenesis. Plasmid R394 had no enhancing effects on UV survival or UV- and MMS-induced mutagenesis in S. typhimurium, in contrast to its reported enhancement of MMS-induced mutagenesis in E. coli. Conjugal transfer of R394 to E. coli strain AB1157 and assays of mutagenesis-related traits supported results observed in S. typhimurium. Muc+ plasmids were found to enhance the frequency of precise excision of the transposon Tn10 when inserted within hisG or trpA in S. typhimurium strains. Precise excision could be further enhanced in S. typhimurium by UV-irradiation. Analysis of Tn10 mutants with altered IS10 ends indicated that intact inverted repeats at the ends of Tn10 were required for efficient enhancement of precise excision.     

Characterization of the umu-complementing operon from R391.

In addition to conferring resistances to antibiotics and heavy metals, certain R factors carry genes involved in mutagenic DNA repair. These plasmid-encoded genes are structurally and functionally related to the chromosomally encoded umuDC genes of Escherichia coli and Salmonella typhimurium. Three such plasmid operons, mucAB, impCAB, and samAB, have been characterized at the molecular level. Recently, we have identified three additional umu-complementing operons from IncJ plasmid R391 and IncL/M plasmids R446b and R471a. We report here the molecular characterization of the R391 umu-complementing operon. The nucleotide sequence of the minimal R plasmid umu-complementing (rum) region revealed an operon of two genes, rumA(R391) and rumB(R391), with an upstream regulatory signal strongly resembling LexA-binding sites. Phylogenetic analysis revealed that the RumAB(R391) proteins are approximately equally diverged in sequence from the chromosomal UmuDC proteins and the other plasmid-encoded Umu-like proteins and represent a new subfamily. Genetic characterization of the rumAB(R391) operon revealed that in recA+ and recA1730 backgrounds, the rumAB(R391) operon was phenotypically indistinguishable from mucAB. In contrast, however, the rumAB(R391) operon gave levels of mutagenesis that were intermediate between those given by mucAB and umuDC in a recA430 strain. The latter phenotype was shown to correlate with the reduced posttranslational processing of the RumA(R391) protein to its mutagenically active form, RumA'(R391). Thus, the rumAB(R391) operon appears to possess characteristics that are reminiscent of both chromosome and plasmid-encoded umu-like operons.     

Mutational and recombinational events in carcinogen-modified plasmid DNA. Influence of host-cell repair genes

A plasmid containing the STR operon has been modified in vitro (i) by irradiation with UV light, (ii) by reaction with ethyl methanesulphonate (EMS), (iii) by reaction with N-acetoxy-2-acetylaminofluorene (AcO-AAF), (iv) by reaction with (+/-)trans-benzo[a]pyrene-7, 8-dihydrodiol-9,10-epoxide (BPDE), and (v) by heating at 70 degrees C to produce apurinic sites. Suitably modified plasmid DNA was then used to transform both repair-proficient and repair-deficient strains of Escherichia coli, and the mutation frequency in the plasmid-encoded rspL+ gene measured. The influence of host mutations in the uvrB+, recA+, umuC+ and lexA+, genes on the mutation frequency have been investigated. Transformation into a uvrB strain significantly decreased survival and increased the level of mutations observed for UV- and AcO-AAF-modified plasmid DNA, while only a small increase in mutation frequency was seen with EMS-modified DNA and no increase in mutation frequency with plasmid DNA containing apurinic sites. Mutagenesis in UV- and BPDE-modified DNA (and probably also DNA containing apurinic sites) was totally dependent on he recA+ gene product, while EMS and AcO-AAF induced mutagenesis was only partially independent on the recA+ gene. Transformation of UV- or BPDE-modified DNA into a umuC or lexA strain, on the other hand, showed no change in mutation frequency from that observed with wild-type strain. Pre-irradiation of the wild-type host with UV light before transformation led to a significant increase in mutation frequency for UV- and BPDE-modified plasmid DNA. These results are discussed in terms of mutational or recombinational pathways which may be available to act on modified plasmid DNA, and suggest that the majority of the mutational events measured in this system are due to recombination between homologous regions on the plasmid and chromosomal DNA.     

Homologous recombination and mutagenesis of gamma-irradiated plasmid DNA in Escherichia coli host cells

Plasmid DNA was used to study gamma-radiation-induced recombination and mutagenesis in Escherichia coli host cells. Plasmid pBRP1, a derivative of pBR322 containing the lac operon of E. coli, was irradiated with 60Co gamma rays prior to transformation into E. coli strains of different recA and lac genotypes. Plasmid-chromosome recombination was assayed in lacY1 host cells, whereas plasmid mutagenesis was assayed in delta lac host cells lacking chromosomal sequences homologous to the plasmid. Both recombinant and mutant plasmids were identified by the phenotypic changes in lactose utilization, and confirmed by restriction analysis of isolated plasmids. Plasmid-chromosome recombination was induced to high levels (about 20% of survivors at 700 Gy) and was dependent on the host recA gene. Plasmid mutagenesis occurred at lower levels (about 1.5% of survivors at 600 Gy) and was relatively independent of the recA gene. Plasmid survival was unaffected by the presence or absence of host recA mutations or the potential for plasmid-chromosome recombination.     

The CAM-OCT plasmid enhances UV responses of Pseudomonas aeruginosa recA mutants.

The effect of the CAM-OCT plasmid on responses to UV irradiation of Pseudomonas aeruginosa recA mutants was characterized. Mutant alleles examined included rec-1, rec-2, and recA7::Tn501. The plasmid substantially enhanced both survival and mutagenesis of RecA- cells after treatment with UV light. Survival of the RecA-(CAM-OCT) cells after UV irradiation was intermediate between that seen in the wild-type P. aeruginosa PAO1 and the increased survival seen in PAO1(CAM-OCT) cells. Mutability was quantitated by the reversion to carbenicillin resistance of strains carrying a bla(Am) mutation on a derivative of plasmid RP1. UV-induced mutagenesis of CAM-OCT carrying recA mutants occurred at levels comparable to that seen in PAO1(CAM-OCT). The ability of CAM-OCT plasmid to suppress the recombination deficiency in recA mutants was tested by assaying for bacteriophage F116L-generalized transduction of a Tn7 insertion in the alkane utilization genes of CAM-OCT. Transduction of the Tn7 insertion was not detected in RecA-(CAM-OCT) strains but was easily seen in PAO1(CAM-OCT), indicating that the plasmid does not encode a recA analog. The results indicate that the CAM-OCT UV response genes are expressed in RecA- cells, which differs from results seen with other UV response-enhancing plasmids. The results suggest that CAM-OCT either encodes several UV responses genes itself or induces chromosomal UV response genes by an alternate mechanism.     

Effects of newly synthesised platinum(ii) complexes on mitochondrial functions

Protease deficient recA431 mutants of Escherichia coli are defective in their capacity for induction of SOS responses and were intermediate in their sensitivities to ultraviolet light (UV) and cis-platinum(II)diamminodichloride (cis-PDD). Survival after treatment determined as colony forming ability was greater in rec⁺ strains and decreased in recA13 mutants which are defective in both recA proteolytic and recombination capabilites. In contrast, recA431 mutants were as sensitive to N-methyl-N′-nitro-N-nitrosoguanidine (NTG) as the recA13 cells. When cells carried either the pKM101 or N3 plasmid, survival after treatment with the three mutagens was increased. Presence of these plasmids in cells also resulted in hypermutagenicity as indicated by reversion of the argE3 mutation using a modified Ames test. Mutagenesis by NTG and cis-PDD was increased, as was survival of cells treated with UV light, cis-PDD and NTG in both recA⁺ and recA431 (protease deficient) strains. No plasmid mediated enhancement of mutagenesis or cell survival was observed in recA13 mutants. Thus, the ability of the plasmids to enhance cell survival and mutagenesis was dependent on recombination proficiency of the recA gene product and not its regulatory proteolytic activity. Unlike UV or NTG, presence of one of these plasmids was needed to detect reversion of the argE3 mutation by cis-PDD.     

Isolation and analysis of a circular form of the IncJ conjugative transposon-like elements, R391 and R997: implications for IncJ incompatibility

The incompatibility between the chromosomally integrating, conjugative transposon-like, IncJ elements R997 (ampicillin resistant) and R391 (kanamycin resistant) was examined by constructing strains harbouring both elements. Unusually, recA(+) strains harbouring the resistance determinants of both elements could be isolated but all strains lacked detectable extrachromosomal DNA. The phenotypic characteristics and transfer patterns observed suggested the formation of recombinant hybrids rather than strains harbouring both elements independently. Formation of strains harbouring two IncJ elements in a recA background was thus examined and resulted in the visualisation of extrachromosomal DNA. When R391 was transferred to a recA strain containing integrated R997, both elements co-existed stably and resulted in the isolation of a plasmid of 93.9 kb. When R997 was transferred to a recA strain harbouring an integrated R391, a plasmid of 85 kb was isolated. Comparison of restriction patterns for both elements revealed many common and several distinct fragments indicating a close physical relationship. These data suggest that although IncJ elements normally integrate at a unique site in the Escherichia coli chromosome, they possess the ability for autonomous replication which becomes manifest in a recA background when this site is occupied. This observation has implications for the nature of the incompatibility associated with IncJ elements and also provides a reliable method for isolating IncJ elements for molecular characterisation.     

Post-replication repair and recombination in uvrA umuC strains of Escherichia coli are enhanced by vanillin, an antimutagenic compound

Effects of vanillin on UV killing of umuC mutant strains of E. coli were investigated in order to analyze the antimutagenic role of vanillin in mutagenesis. UV-irradiated uvrA umuC cells showed higher survival when plated on medium containing vanillin rather than medium without vanillin. This increased survival associated with exposure to vanillin was observed more clearly in uvrA umuC lexA(Ind-) and uvrA umuC recF strains. However, the effect was inhibited by additional recB recC mutations and completely blocked by an additional recA mutation. As far as tested the increased survival of UV-treated cells by vanillin was dependent on a capacity for genetic recombination. The effect of vanillin on recombination frequency between 2 plasmid DNA, pATH4 (Cmr Tcs) and pBMX7 (Apr Tcs), in a uvrA umuC background was investigated. A significantly higher frequency of plasmid recombination was observed when vanillin was present in the culture medium. These findings suggest that the antimutagenic effect of vanillin is the result of enhancement of a recA-dependent, error-free, pathway of post-replication repair.     

Effect of repair deficiency and R plasmids on spontaneous and radiation-induced mutability in Pseudomonas aeruginosa.

The effect of R plasmids on spontaneous and radiation (ultraviolet and gamma)-induced mutability in Pseudomonas aeruginosa was studied in strains containing the radiation-sensitive markers polA3 or rec-2 and the revertable auxotrophic markers hisO27 and trpB1. In the absence of an R plasmid, the radiation-induced mutability was dependent on the recA+ genotype and independent of the polA+ genotype, whereas spontaneous mutability was similar in all genetic backgrounds. R plasmids pPL1, R2, and pMG15 increased the ultraviolet radiation survival and ultraviolet-induced mutability of wild-type and polA host cells but did not alter either effect in a recA mutant. These R plasmids also increased the gamma radiation survival and gamma-induced mutability of wild-type host cells bud pMG15 also enhanced the level of spontaneous mutagenesis in wild-type host cells but not in a polA or recA mutant. These data suggested that a common plasmid gene product(s) may participate in various recA-dependent, error-prone deoxyribonucleic acid repair pathways of P. aeruginosa. The properties of a mutant R plasmid, pPL2, originally selected because it lacked enhanced ultraviolet-induced mutability, supported this conclusion.     

Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function

A recA deletion mutant of Mycobacterium smegmatis has been isolated by homologous recombination using a sacB counterselection strategy. Deletion of the recA gene from the chromosome was demonstrated by Southern hybridizations and by polymerase chain reaction (PCR). Western analysis using anti-RecA antibodies confirmed that the RecA protein was not made by the mutant strain. The recA deletion strain exhibited enhanced sensitivity to UV irradiation and failed to undergo homologous recombination. The results obtained from the recombination assays suggest that in wild-type M. smegmatis the majority of colonies arise from single cross-over homologous recombination events with only a very minor contribution from random integrations. The deficiencies in UV survival and recombination were complemented by introduction of the cloned M. smegmatis recA gene. Overexpression of RecA was found to be toxic in the absence of recX , which is found downstream of and co-transcribed with recA and is thus also affected by the deletion of recA . The M. smegmatis recA deletion strain was also complemented by the M. tuberculosis recA gene with or without its intein; most importantly, the frequency of double cross-over homologous recombination events was identical regardless of whether the M. tuberculosis recA gene contained or lacked the intein. Thus, the low frequency of homologous recombination observed in M. tuberculosis is not due to the presence of an intein-coding sequence in its recA gene per se .     

Cloning of the Serratia marcescens recA gene and construction of a Serratia marcescens recA mutant

A recombinant plasmid, pSM2513, containing an 8.5 kb DNA insert was isolated from a genomic library of Serratia marcescens by using interspecific complementation. This plasmid conferred resistance to methyl methanesulphonate and UV irradiation upon recA mutants of Escherichia coli and enhanced recombination proficiency, as measured by Hfr-mediated conjugation, in recA mutants of E. coli. Furthermore, when recA mutants of E. coli harbouring pSM2513 were subjected to UV irradiation, filamentation of the cells was observed. This did not occur upon UV irradiation of the same mutants harbouring the cloning vector alone. These results imply that the S. marcescens recA gene on pSM2513 is functionally similar to the E. coli recA gene in several respects. Restriction enzyme analysis and subcloning studies revealed that the S. marcescens recA gene was located on a 2.7 kb Bg/II-KpnI fragment of pSM2513, and its gene product of approximately 39 kDa resembled the E. coli RecA protein in molecular mass. Using transformation-mediated marker rescue, a recA mutant of S. marcescens was successfully constructed; its proficiency both in homologous recombination and in DNA repair was abolished compared with its parent.     

Mobilization of R387 Inc K conjugative plasmid by the conjugative plasmid R64 Inc I

Plasmid aggregate (R387, R64) was constructed in E. coli K12 strain. Plasmid R387 Inc K was stimulated to conjugational transfer by plasmid R64 Inc I. This stimulation was caused neither by recombination between both plasmids nor by trans-complementation of R387 conjugational systems by gene(s) product(s) of R64 plasmid. The observed phenomenon resembled rather mobilization of nonconjugative plasmids by conjugative ones. As in mobilization, the observed increase in R387 transfer frequency could take place only when both interacting plasmids were present in donor cells. Moreover, the entry exclusion system functioning in recipient cells, toward stimulating R64 plasmid affected strongly the conjugational transfer of stimulated R387 plasmid. Analogous phenomenon was observed during mobilization of nonconjugative plasmids by conjugative ones.     

Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant.

A recombinant plasmid carrying the recA gene of Aeromonas caviae was isolated from an A. caviae genomic library by complementation of an Escherichia coli recA mutant. The plasmid restored resistance to both UV irradiation and to the DNA-damaging agent methyl methanesulfonate in the E. coli recA mutant strain. The cloned gene also restored recombination proficiency as measured by the formation of lac+ recombinants from duplicated mutant lacZ genes and by the ability to propagate a strain of phage lambda (red gam) that requires host recombination functions for growth. The approximate location of the recA gene on the cloned DNA fragment was determined by constructing deletions and by the insertion of Tn5, both of which abolished the ability of the recombinant plasmid to complement the E. coli recA strains. A. caviae recA::Tn5 was introduced into A. caviae by P1 transduction. The resulting A. caviae recA mutant strain was considerably more sensitive to UV light than was its parent. Southern hybridization analysis indicated that the A. caviae recA gene has diverged from the recA genes from a variety of gram-negative bacteria, including A. hydrophila and A. sobria. Maxicell labeling experiments revealed that the RecA protein of A. caviae had an Mr of about 39,400.     

Loss of RecA function affects the ability of Escherichia coli to maintain recombinant plasmids containing a Ter site.

In the Escherichia coli chromosome, DNA replication forks arrested by a Tus-Ter complex or by DNA damage are reinitiated through pathways that involve RecA and numerous other recombination functions. To examine the role of recombination in the processing of replication forks arrested by a Tus-Ter complex, the requirements for recombination-associated gene products were assessed in cells carrying Ter plasmids, i.e., plasmids that contain a Ter site oriented to block DNA replication. Of the E. coli recombination functions tested, only loss of recA conferred an observable phenotype on cells containing a Ter plasmid, which was inefficient transformation and reduced ability to maintain a Ter plasmid when Tus was expressed. Given the current understanding of replication reinitiation, the simplest explanation for the restriction of Ter plasmid maintenance was a reduced ability to restart plasmid replication in a recA tus(+) background. However, we were unable to detect a difference in the efficiency of replication arrest by Tus in recA-proficient and recA-deficient cells, which suggests that the inability to restart arrested replication forks is not the cause of the restriction on growth, but is due to an additional function provided by RecA. Other explanations for restriction of Ter plasmid maintenance were examined, including plasmid multimerization, plasmid rearrangements, and copy number differences. The most likely cause of the restriction on Ter plasmid maintenance was a reduced copy number in recA cells that was detected when the copy number was measured in relation to an external control. Possibly, loss of RecA function leads to improper processing of replication forks arrested at a Ter site, leading to the generation of degradation-prone substrates.     

recA-dependent and recA-independent N-ethyl-N-nitrosourea mutagenesis at a plasmid-encoded herpes simplex virus thymidine kinase gene in Escherichia coli

We have compared isogenic recA13/recA+ Escherichia coli K-12 strains for the induction by N-ethyl-N-nitrosourea (ENU) of forward mutations at a plasmid-encoded herpes simplex virus type 1 thymidine kinase (HSV-tk) gene. Treatment of plasmid-bearing bacteria with ENU resulted in a dose-dependent increase in the mutant frequencies of the chromosomal udk locus and of the plasmid HSV-tk locus in both recA13 and recA+ strains. Although the recA13 strain was considerably more sensitive to the cytotoxic effects of ENU treatment than was the recA+ strain, the ENU-induced mutation frequency at both loci was greater for the recA+ strain than for the recA13 strain. When plasmid DNA modified by in vitro reaction with ENU was used to transform recA13, recA+, and UV pre-irradiated recA+ strains, an increase in the HSV-tk mutant frequency was observed in all 3 cases. The induction of mutations in recA13 and recA+ strains followed a similar dose-response, while the ENU-induced HSV-tk mutant frequency was significantly greater for UV pre-irradiated recA+ bacteria. These results indicate that fixation of ENU-induced premutagenic lesions can occur by both recA-dependent and recA-independent pathways.     

Amplified UvrA protein can ameliorate the ultraviolet sensitivity of an Escherichia coli recA mutant.

When a recA strain of Escherichia coli was transformed with the multicopy plasmid pSF11 carrying the uvrA gene of E. coli, its extreme ultraviolet (UV) sensitivity was decreased. The sensitivity of the lexA1 (Ind(-)) strain to UV was also decreased by pSF11. The recA cells expressing Neurospora crassa UV damage endonuclease (UVDE), encoding UV-endonuclease, show UV resistance. On the other hand, only partial amelioration of UV sensitivity of the recA strain was observed in the presence of the plasmid pNP10 carrying the uvrB gene. Host cell reactivation of UV-irradiated lambda phage in recA cells with pSF11 was as efficient as that in wild-type cells. Using an antibody to detect cyclobutane pyrimidine dimers, we found that UV-irradiated recA cells removed dimers from their DNA more rapidly if they carried pSF11 than if they carried a vacant control plasmid. Using anti-UvrA antibody, we observed that the expression level of UvrA protein was about 20-fold higher in the recA strain with pSF11 than in the recA strain without pSF11. Our results were consistent with the idea that constitutive level of UvrA protein in the recA cells results in constitutive levels of active UvrABC nuclease which is not enough to operate full nucleotide excision repair (NER), thus leading to extreme UV sensitivity.     

Effect of recB- and recA-mutations on phage restriction in various modification-restriction plasmid systems of E. coli

The effect of recB and recA mutations on lambda vir and P1 vir restriction by different restriction-modification plasmid systems of E. coli was studied. It was shown that effect of R1 plasmid coded restriction-modification in E. coli K12 and E. coli B strains and pJA4620 plasmid coded restriction in E. coli K12 is observed only in RecB+ strain. Phenomenon of restriction-modification determined by R124, R245 plasmids does not depend of recB mutation. Effect of recA mutation has not been found in cultures harbouring R1, R245, R124 pJA4620 plasmids.     

Plasmid-dosage effects on ultraviolet, visible light and methyl methanesulfonate mutagenesis in Salmonella typhimurium

Salmonella typhimurium LT2 strains bearing plasmids pKM101, R64 or pColIb-P9 demonstrated enhanced UV survival when compared with strains not bearing plasmids. A strain of S. typhimurium bearing both pKM101 and pColIb-P9 survived UV irradiation slightly better than either of the single-plasmid strains. Spontaneous reversion of the hisG46 and trpE8 missense alleles was enhanced in each single-plasmid strain, and for the dual-plasmid strain containing pKM101 and pColIb-P9 enhancement represented a near additivity of the response seen for the single-plasmid strains. Following exposure to UV or visible-light irradiation, reversion of hisG46 and trpE8 was also enhanced in each single-plasmid strain, but quantitatively greater in the dual-plasmid strain and was equal to or slightly greater than additive the responses of the single-plasmid strains. In contrast to visible-light irradiation, UV exposure resulted in two phenotypic Trp+-revertant classes. One Trp+ class, having normal colony size (2.0 mm) and similar in number to His+ revertants, was comprised of intragenic revertants of trpE8, while the predominant Trp+ class, having smaller colony size (0.8 mm), represented intergenic suppressor revertants, illuminating the differences in mutation and/or repair specificity for UV and visible-light exposure. Methyl methane-sulfonate (MMS)-induced reversion of hisG46 was similar in effect to that seen with UV or visible-light irradiation. Plasmids pKM101 or pColIb-P9 enhanced the frequency of hisG46 reversion, while a more than additive response was seen in a strain with both plasmids. Furthermore, MMS-induced reversion of hisG46 was also observed to be greatest in a strain bearing plasmid R64 (incompatibility group I alpha) and pKM101, when compared with single-plasmid strains bearing either R64 or pKM101.