Convenient construction of strains useful for transducing recA mutations with bacteriophage P1

Abstract The generalized transducing phage P1 grew well on heterozygous Escherichia coli carrying recA srlC 300::Tn 10 on the chromosome and recA ⁺ on a pBR322-derived plasmid. Because of the close linkage of Tn 10 to recA mutations, including recA 1, recA 13, recA 56, recA deletion and recA allele of E. coli BNG30, the latter can be moved to other strains in transductional crosses for selective resistance to tetracycline.     

RecA-dependent increased precise excision of Tn10 in Salmonella typhimurium.

UV irradiation induced the precise excision of Tn10 inserted in met, trp or srl in a Salmonella typhimurium strain; mitomycin C was also found to induce the frequency of precise excision of Tn10 from srl or met. Precise excision of Tn10 was not increased by either UV or mitomycin C in a recA mutant. Similarly, a recA mutant derived from a uvrD strain showed a drastic reduction in the high spontaneous levels of precise excision of Tn10 of this strain. These results indicate that recA is involved in the increased precise excision of Tn10. In contrast to point mutations excision of Tn10 was found to be UV inducible in a top mutant.     

Deletions Generated by the Transposon Tn10 in the srl recA Region of the ESCHERICHIA COLI K-12 Chromosome

A negative regulatory gene for the srl operon (srlR) was recognized by the characteristics of an insertion mutation generated by the transposon Tn10 determining tetracycline resistance. This finding is discussed in light of previous hypotheses on the regulation of the srl genes, which mediate metabolism of glucitol (i.e., sorbitol). Mapping showed that the order of genes in this region is: srlR srlD srlC recA alaS. Using two different methods, five mutations of both srl and recA were detected. The phenotype conferred by these mutations, UV sensitivity and extreme recombination deficiency, is characteristic of standard recA point mutants. Three of the mutations were deletions that also removed the genes for tetracycline resistance of the nearby transposon. A fourth mutation ended at a distance from Tn10 sufficient to allow separation of the two by recombination following P1 transduction; our tests did not allow us to conclude whether this mutation was an inversion or a deletion. The fifth mutation was a deletion that seemed to end immediately adjacent to the boundary of Tn10, proximal to recA. Mechanisms for the generation of these srl recA mutations are discussed.     

Conjugational hyperrecombination achieved by derepressing the LexA regulon,altering the properties of RecA protein and inactivating mismatch repair in Escherichia coli K-12

The frequency of recombinational exchanges (FRE) that disrupt co-inheritance of transferred donor markers in Escherichia coli Hfr by F(-) crosses differs by up to a factor of two depending on physiological factors and culture conditions. Under standard conditions we found FRE to be 5.01 +/- 0.43 exchanges per 100-min units of DNA length for wild-type strains of the AB1157 line. Using these conditions we showed a cumulative effect of various mutations on FRE. Constitutive SOS expression by lexA gene inactivation (lexA71::Tn5) and recA gene mutation (recA730) showed, respectively, approximately 4- and 7-fold increases of FRE. The double lexA71 recA730 combination gave an approximately 17-fold increase in FRE. Addition of mutS215::Tn10, inactivating the mismatch repair system, to the double lexA recA mutant increased FRE to approximately 26-fold above wild-type FRE. Finally, we showed that another recA mutation produced as much SOS expression as recA730 but increased FRE only 3-fold. We conclude that three factors contribute to normally low FRE under standard conditions: repression of the LexA regulon, the properties of wild-type RecA protein, and a functioning MutSHL mismatch repair system. We discuss mechanisms by which the lexA, recA, and mutS mutations may elevate FRE cumulatively to obtain hyperrecombination.     

Studies on Tn10 transposition and excision in DNA-repair mutants of Salmonella typhimurium

Transposition of Tn10 in polA, recA, uvrB, mutH and uvrD mutants of Salmonella typhimurium was studied by a mating-out assay mediated by R plasmid pKM101. A decrease in transposition frequency was observed with polA, recA and uvrD mutants; uvrB and mutH mutants showed frequencies somewhat higher than control values. No effect of dimethyl sulfoxide, sodium acetate or nitrofurazone on Tn10 transposition was observed with this assay. Precise excision of Tn10 from srl202::Tn10 in these DNA-repair mutants was also studied. An increase in excision frequency of about 20 or 150 times in 2 different polA mutants, and a smaller increase, of about 2 or 15 times over control values, was detected in mutH and uvrD mutants, respectively.     

Postexcision transposition of the transposon Tn10 in Escherichia coli K12

An experimental analysis of the fate of transposon Tn10 after excision from a proA::Tn10 site localized on the plasmid F' leads to the conclusions: 1. The precise excision is a progressive process. Its probability is estimated per time unit. 2. An excised Tn10 is always integrated into a different genetic locus. 2. An excised Tn10 is always integrated into a different genetic locus. 3. The kinetics of postexcision transposition are sometimes very slow. The excised transposon is inherited in one cell line in spite of cell multiplication. 4. The processes of excision and secondary insertion have no absolute requirement for the recA+ genotype but they are strongly enhanced in recA+ cells. 5. The kinetics of postexcision transposition are strongly dependent on the genetic site from which the transposon was excised. 6. The probability of postexcision transposition is fully determined by the probability of excision and depends on the genotype of the host and many other factors.     

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.     

Participation of DNA polymerase II in the increased precise excision of Tn10

In this work the involvement of polymerase II (Pol II) in the precise excision of Tn10 stimulated by a dnaB252 thermosensitive (Ts) mutant at the permissive temperature, by a uvrD mutant, or by mitomycin C (MMC) or ultraviolet (UV) light treatment, was investigated. A deltapolB::kan mutant showed a significant decrease in the excision of Tn10 induced by the dnaB mutation, or by MMC or UV treatment, indicating the participation of Pol II in this type of deletion process. However, no effect of Pol II was evidenced in the excision of Tn10 stimulated by the uvrD mutation. The effect of the polB mutation on Tn10 precise excision induced by all these treatments was compared to that of mutations in repair-recombination genes recF and recA. The results reveal that the degree of participation of these genes varies depending on the agent that stimulates the deletion event.     

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.     

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.     

Convenient construction of recA deletion derivatives of Escherichia coli.

Two Escherichia coli K-12 Hfr strains have been constructed which transfer a recA deletion, which is highly linked to a Tn10 insertion conferring tetracycline resistance, early during conjugation. These strains transfer the recA deletion in opposite directions with different origins of transfer, allowing for preservation of desirable recipient strain markers either clockwise or counterclockwise of recA.     

Induction of transposon Tn1 translocations as affected by different mutagens

Transposition of ampicillin Tn1 transposon is repressed under normal conditions and occurs with low frequency (10(-4) per cell). Treatment of Escherichia coli cells with 22 c+mytomycin C, nitrosoguanidine and UV light induced transposition of Tn1 into different replicons. The degree of induction depended upon the strain of bacteria and the replicon which the transposon was inserted into. Mutation recA did not influence spontaneous translocation of the transposon but fully repressed induction of transposition during the period of mutagen treatment. In the present paper, the connection of inducible transposition process with the recA and lexA inducible functions of E. coli is discussed.     

recA is required in the induction of pectin lyase and carotovoricin in Erwinia carotovora subsp. carotovora.

Pectin lyase (PNL) and the bacteriocin carotovoricin (CTV) were induced in Erwinia carotovora subsp. carotovora 71 by the DNA-damaging agents mitomycin C, nalidixic acid, and UV light. To determine whether the recA product was involved in the expression of these damage-inducible phenotypes, we cloned the E. carotovora subsp. carotovora recA+ gene, inactivated it by Tn5 insertion, and constructed an E. carotovora subsp. carotovora recA::Tn5 strain by gene replacement via homologous recombination. The RecA- strain was more sensitive to methyl methanesulfonate, nitroquinoline oxide, and UV light than its RecA+ parent. The recA mutation did not affect the production of pectate lyase, polygalacturonase, cellulase, and protease or the ability to cause soft rot of potato tubers. With this mutant, unlike with the RecA+ parent strain, PNL and CTV were not induced by mitomycin C or detected in potato tuber tissue. The RecA+ phenotype, including the inducibility of PNL and CTV, could, however, be restored in the mutant in trans by the recA+ gene from either E. carotovora subsp. carotovora or Escherichia coli. We conclude that, in E. carotovora subsp. carotovora, the recA product is required in the induction of PNL and CTV.     

Mutagenic DNA repair in Escherichia coli. XIII. Proofreading exonuclease of DNA polymerase III holoenzyme is not operational during UV mutagenesis

We have introduced a mutD5 mutation (which results in defective 3'-5'-exonuclease activity of the epsilon proofreading subunit of DNA polymerase III holoenzyme) into excision-defective Escherichia coli strains with varying SOS responses to UV light. MutD5 increased the spontaneous mutation frequency in all strains tested, including recA430, umuC122::Tn5, and umuC36 derivatives. It had no effect on UV mutability or immutability in any strain or on misincorporation revealed by delayed photoreversal in UV-irradiated umuC36, umuC122::Tn5, or recA430 bacteria. It is concluded that the epsilon proofreading subunit of DNA polymerase III holoenzyme is excluded, inhibited, or inoperative during misincorporation and mutagenesis after UV.     

The Mechanism of Reca Pola Lethality: Suppression by Reca-Independent Recombination Repair Activated by the Lexa(def) Mutation in Escherichia Coli

The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5' -> 3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF(+) is essential for this suppression pathway. recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by δrecA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of δrecA polA25::spc cells to UV damage by ~10(4)-fold. lexA(Def) also restores P1 transduction proficiency to the δrecA polA25::spc mutant to a level that is 7.3% of the recA(+) wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants.     

Dissociation of unstable cointegrate plasmids formed during transposition of IS1 element: the role of IS1 encoded recombinase

The properties of IS1/Tn9'-mediated cointegrates between plasmids pDK57 (pBR322:: :: Tn9') and pRP3.1--the deletion derivative of RP1 were investigated. It was found that IS1/Tn9'-mediated integration of pDK57 into the active transcribed regions of pRP3.1 (in particular kan and tet genes) leads to formation of unstable cointegrates capable of resolving in E. coli K-12 rec+ and recA cells. The structure of dissociation products of unstable cointegrates was studied. According to the data received in rec+ cells, the unstable cointegrates mainly produced plasmids pDK57 and pBR322::IS1--Cms-derivative of pDK57 as resolution products. In recA cells the cointegrates dissociate in different ways, and this process leads to the formation of not only pDK57 and pBR322::IS1, but also to the production of the deletion derivatives of these plasmids as well as to the derivatives of pDK57 and pBR322::IS1, containing duplications of IS1 or separate parts of Tn9'. It was concluded that the IS1-specific recombinase is involved in the dissociation (resolution) of unstable IS1/Tn9'-mediated cointegrates. This recombinase recognizes the sites localized in both inverted termini of IS1 as well as in the adjacent DNA segments. Hence, it is possible, that the IS1 recombinase is involved also in the generation of IS1-adjacent delations.     

Regularities of excising transposon Tn10 in rec-mutant E. coli cells exposed to gamma radiation

The regularities of gamma-induced excision of transposon Tn10 in different rec-strains of E. coli cells after gamma-irradiation have been studied. The survival of cells and relative frequency of the Tn10 elimination as a function of the 137Cs gamma-radiation doses were investigated. RecN and recA-mutants of E. coli were used for study of the role of rec-genes in the gamma-induced transposon excision. It was shown that the induced excision in the recN mutant was reduced. The transposon excision in the recA mutant was not revealed. The obtained results let to conclude that recA, and recN genes are involved not only in DNA repair processes but also in the gamma-induced transposon excision in bacteria.