Characteristics of Some Multiply Recombination-Deficient Strains of Escherichia coli

Strains of Escherichia coli have been made carrying lesions in more than one gene determining recombination. The following genotypes were constructed and verified: recC22 recB21 recA(+), recC22 recB21 recA13, recC22 recB(+)recA13, and recC(+)recB21 recA13. All multiple rec(-) strains carrying recA13 were similar to AB2463, which carries recA13 alone, in their UV sensitivities, recombination deficiencies, and inabilities to induce lambda phage in a lysogen. However, whereas AB2463 shows a high rate of ultraviolet (UV)-induced deoxyribonucleic acid (DNA) breakdown, the multiple rec(-) strains showed the low level characteristic of strains carrying recC22 or recB21 alone. The strain carrying both recC22 and recB21 was similar in all properties to the single mutants, suggesting that both gene products act in the same part of the recombination and UV repair pathways. It is concluded that in a Rec(+) strain, the recA(+) product acts to inhibit DNA breakdown determined by the recC(+) and recB(+) products.     

Chromosome Transfer from F-lac+ Strains of Escherichia coli K-12 Mutant at recA, recB, or recC

The frequency of chromosome transfer from various recombination-deficient F-lac(+) donor strains was estimated by standardizing the yield of conjugants receiving a male chromosomal marker against the level of episome transfer in the mating mixture. The efficiency of chromosome transfer from newly formed F-lac(+) cells carrying recB21 or recC22 was more than 50% of the wild-type value, although it was about 10 and 20%, respectively, if the male cell lines had become established. In contrast, recA13 donors transmitted the chromosome with less than 10(-4) of the normal frequency. If chromosome transfer from F-lac(+) strains reflects the cutting and subsequent joining of homologous single strands of episomal and chromosomal deoxyribonucleic acid by recombination, these results imply that the completed unions are not made in recA cells, but can be effected with more than 50% of normal efficiency in newly formed partial diploids mutant at either recB or recC. Thus, the defective stage in recA mutants may precede strand joining, whereas the deficiency in recB or recC cells may involve a later step in recombinant formation.     

Genetic and Physical Analysis of Plasmid Recombination in Recb Recc Sbcb and Recb Recc Sbca Escherichia Coli K-12 Mutants

The effect of mutations in known recombination genes (recA, recB, recC, recE, recF, recJ, recN, recO, recQ and ruv) on intramolecular recombination of plasmids was studied in recB recC sbcB and recB recC sbcA Escherichia coli mutants. The rate of recombination of circular dimer plasmids was at least 1000-fold higher in recB recC sbcB or recB recC sbcA mutants as compared to wild-type cells. The rate was decreased by mutations in recA, recF, recJ, recO, ruv or mutS in recB recC sbcB mutants, and by mutations in recE, recN, recO, recQ, ruv or mutS in recB recC sbcA mutants. In addition to measuring the recombination rate of circular dimer plasmids, the recombination-mediated transformation of linear dimer plasmids was also studied. Linear dimer plasmids transformed recB recC sbcB and recB recC sbcA mutants 20- to 40-fold more efficiently than wild-type cells. The transformation efficiency of linear dimer plasmids in recB recC sbcB mutants was decreased by mutations in recA, recF, recJ, recO, recQ or lexA (lexA3). In recB recC sbcA mutants the transformation efficiency of linear dimers was decreased only by a recE mutation. Physical analysis of linear dimer- or circular dimer-transformed recB recC sbcB mutants revealed that all transformants contained recombinant monomer genotypes. This suggests that recombination in recB recC sbcB cells is very efficient.     

recA (Srf) suppression of recF deficiency in the postreplication repair of UV-irradiated Escherichia coli K-12.

The mechanism by which recA (Srf) mutations (recA2020 and recA801) suppress the deficiency in postreplication repair shown by recF mutants of Escherichia coli was studied in UV-irradiated uvrB and uvrA recB recC sbcB cells. The recA (Srf) mutations partially suppressed the UV radiation sensitivity of uvrB recF, uvrB recF recB, and uvrA recB recC sbcB recF cells, and they partially restored the ability of uvrB recF and uvrA recB recC sbcB recF cells to repair DNA daughter-strand gaps. In addition, the recA (Srf) mutations suppressed the recF deficiency in the repair of DNA double-strand breaks in UV-irradiated uvrA recB recC sbcB recF cells. The recA2020 and recA801 mutations do not appear to affect the synthesis of UV radiation-induced proteins, nor do they appear to produce an altered RecA protein, as detected by two-dimensional gel electrophoresis. These results are consistent with the suggestion (M. R. Volkert and M. A. Hartke, J. Bacteriol. 157:498-506, 1984) that the recA (Srf) mutations do not act by affecting the induction of SOS responses; rather, they allow the RecA protein to participate in the recF-dependent postreplication repair processes without the need of the RecF protein.     

Repair of cross-linked DNA and survival of Escherichia coli treated with psoralen and light: effects of mutations influencing genetic recombination and DNA metabolism.

Repair of cross-linked DNA was studied in Escherichia coli strains carrying mutations affecting DNA metabolism. In wild-type cells, DNA strands cut during cross-link removal were rejoined during a subsequent incubation into high-molecular-weight molecules. This rejoining was dependent on gene products involved in genetic recombination. A close correlation was found relating recombination proficiency, the rate of strand rejoining, and formation of viable progeny after DNA cross-linking by treatment with psoralen and light. Wild-type cells and other mutants which were Rec+ (sbcB, recL, recL sbcB, recB recC sbcA, recB recC sbcB, xthA1, and xthA11) rejoined cut DNA strands at a rate of 0.8 +/- 0.1 min -1 at 37 degrees C and survived 53 to 71 cross-links per chromosome. recB, recC, recB recC, recF, or polA strains showed reduced rates of strand rejoining and survived 4 to 13 cross-links per chromosome. Recombination-deficient strains (recA, recB recC sbcB recF, recB recL) and lexA failed to rejoin DNA strands after crosslink removal and were unable to form colonies after treatments producing as few as one to two cross-links per chromosome. Strand rejoining occurred normally in cells with mutations affecting DNA replication (dnaA, danB, dnaG, and dnaE) under both permissive and nonpermissive conditions for chromosome replication. In a polA polB dnaE strain strand rejoining occurred at 32 degree C but not at 42 degree C, indicating that some DNA synthesis was required for formation of intact recombinant molecules.     

R46 and pKM101 plasmid-mediated resistance to ionizing radiation in Escherichia coli

The ability of the R46 R factor and its derivative pKM101 to modify sensitivity to 60Co gamma radiation was studied. In Escherichia coli K12 both plasmids enhanced bacterial survival after 60Co gamma irradiation. This effect was dependent on recA+ genotype but not on recB+, recB+ recC+, and recF+ genotypes. 5-Fluorouracil eliminated the R46 R factor from the parent and its rec- mutant strains. These strains lost not only the antibiotic resistance coded for R46 R factor but their radioresistance as well.     

Genetic and physical studies of recombinant plasmids formed between an R plasmid of compatibility group FI and sex factor F of HfrH.

Recombinant plasmids between an R plasmid of the FI group (R162/3) and the sex factor F or HfrH were produced after the conjugal transfer of this R plasmid into HfrH. Three types of recombinant plasmids were identified after the mating of HfrH (R162/3) with recA and rec+ recipients. One specimen of each type (pIP218, pIP222, pIP226) was studied in this report. All three recombinant plasmids carry the same genetic information for resistance to antibiotics (CSSuT) retained from R162/3. pIP218 retained all the other properties from F of HfrH: derepression for pilus synthesis, mobilization of the chromosome for the proximally transferred HfrH genes (thr, leu, proA), interference with T7 propagation, and ability to be cured by acridine orange. pIP222 retained from F of HfrH the derepression for pilus synthesis and the same polarity of chromosome transfer (thr, leu, proA), while pIP226 retained the interference with T7 propagation and acridine orange curing. Physical studies revealed that replication control and/or recovery of F and pIP218 as covalent circles of deoxyribonucleic acid are similar, and are different from R162/3. The new plasmids are more likely the result of a substitutive recombination event than a fusion. We propose genetic maps of these recombinant plasmids, showing the unequal participation of the parental plasmids in their formation.     

Recombination and the Escherichia coli K-12 sex factor F.

Recombination between two Flac tra minus elements to give Flac tra plus recombinants was measured in Rec plus and Rec minus strains of Escherichia coli K-12. Polar tra mutations were used to increase the proportion of tra plus recombinants among the parental Flac tra minus elements transferred by complementation. The kinetics, measured in a rec plus strain, showed that recombination began about 1 h after the initiation of mating and was completed about 1 h later. Recombination was abolished in a recA minus strain, reduced by two-thirds in a recF minus strain, and unaffected in recB minus and recC minus strains. It is proposed that the part not due to the RecF pathway results from a RecBC- and RecF-independent system for formation of single-stranded joins. One such join could be followed either by transfer and a site-specific recombination event, or by a second single-stranded join and then transfer: in either case replication and inheritance of the recombinant molecule would be dependent upon the F transfer replication system. Chromosome mobilization by an F' element was normal in a recB plus recF minus strain, and was reduced only fourfold in a recB minus recF plus strain: in the latter strain, both the RecF pathway and the system for single-stranded joins may have contributed to mobilization. Measurement of post-conjugational chromosomal recombination in exponential-phase recipient cells carrying surface exclusion-deficient Flac mutants indicated that F does not itself determine a generalized recombination system able to replace the RecA plus product or the RecBC and RecF pathways.     

Dark-Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light III. Effect of rec Mutations on Recovery of Excision-Deficient Mutants of Escherichia coli K-12

Mutants of Escherichia coli K-12 unable to excise pyrimidine dimers from their deoxyribonucleic acid (DNA) because of a uvr mutation show a higher survival when plated on a minimal salts medium after exposure to ultraviolet radiation than when plated on a complex medium such as nutrient agar containing yeast extract. This response has been called minimal medium recovery (MMR). Recovery of uvr mutants can take place in liquid as well as on solid medium, but not in buffer or under conditions of amino acid starvation that do not permit cell growth and normal DNA replication. MMR can thus be distinguished from the recovery of recombination-deficient (rec(-)uvr(+)) derivatives of K-12 which can occur under conditions where growth is not possible. Because MMR is characteristic of excision-defective mutants, it evidently reflects a type of repair independent of excision. We have obtained genetic evidence that MMR is determined by the rec genes, which also control recombination in K-12. Cells carrying a uvr mutation together with recA13, recA56, recB21, or recC22 failed to show MMR and were more sensitive to ultraviolet radiation than either their rec(+)uvr(-) or rec(-)uvr(+) parents. The rec(+)uvr(-) derivatives obtained from recA uvr(-) strains by transduction or by reversion regained the capacity for MMR. Our results indicate that inactivation of any one of the three genes, recA, recB, or recC, prevents cells from showing MMR.     

Effects of the Escherichia coli recF suppressor mutation, recA801, on recF-dependent DNA-repair associated phenomena

In recb recC sbcB mutants genetic recombination is dependent upon the recF gene. recA801, recA802 and recA803 (formerly called srfA mutations) were originally isolated as mutations that suppress recombination deficiency caused by a recF mutation in a recB recC sbcB genetic background. Since the recA801 mutation also suppressed some of the UV sensitivity due to recF143, we sought to determine what DNA-repair pathways were actually being restored by the recA801 mutation in this genetic background. In this paper we show that the suppression of recF143 by recA801 does not extend to the recF143-mediated defects in induced repair of UV-damaged phages. In addition, we show that recA801 suppresses only slightly the recF143-associated defect in induced expression of the SOS-regulated muc genes of pKM101. These results suggest that recA801 suppresses primarily the RecF pathway of recombinational repair.     

Recipient Gene Duplication during Generalized Transduction

An Hfr13 Delta(proA-lac) deletion recipient, -Delta(proA-lac)-F-purE(+)-, has been utilized in a study of the origins of duplications formed during chromosome fragment integration. Among the Pro(-)Lac(+) transductants, some have duplications spanning the F locus. These transductants are, or segregate, strains with F' episomes carrying genes of the duplication. Some of the duplications include purE(+), a gene which is not coinherited with lac(+) during bacteriophage P1-mediated transduction. Thus recipient genes have been duplicated during recombinant formation. Crossing-over models including replication steps provide a basis for explaining the duplication process.     

Plasmid control of recombination in E. coli K 12

Summary The recombination proficiency of three recipient strains of Escherichia coli K 12 carrying different plasmids was investigated by conjugal mating with Hfr Cavalli. Some plasmids (e.g. R1drd 19, R6K) caused a marked reduction in the yield of recombinants formed in crosses with Hfr but did not reduce the ability of host strains to accept plasmid F104. The effect of plasmids on recombination was host-dependent. In Hfr crosses with AB1157 (R1-19) used as a recipient the linkage between selected and unselected proximal markers of the donor was sharply decreased. Plasmid R1-19 also decreased the yield of recombinants formed by recF, recL, and recB recC sbcA mutants, showed no effect on the recombination proficiency of recB recC sbcB mutant, and increased the recombination proficiency of recB, recB recC sbcB recF, and recB recC sbcB recL mutants. An ATP-dependent exonuclease activity was found in all tested recB recC mutants carrying plasmid R1-19, while this plasmid did not affect the activity of exonuclease I in strain AB1157 and its rec ^– derivatives. The same plasmid was also found to protect different rec ^– derivatives of the strain AB1157 against the lethal action of UV light. We suppose that a new ATP-dependent exonuclease determined by R1-19 plays a role in both repair and recombination of the host through the substitution of or competition with the exoV coded for by the genes recB and recC.     

Involvement of recA and exr Genes in the In Vivo Inhibition of the recBC Nuclease

When Escherichia coli cells are gamma irradiated they degrade their deoxyribonucleic acid (DNA). The DNA of previously gamma-irradiated T4 phage is also degraded in infected cells. The amount of degradation is not only dependent on the dose but also on the genotype of the cell. The amount of degradation is less in cells carrying a recB or a recC mutation, suggesting that most of the DNA degradation is due to the recB(+) and recC(+) gene product (exonuclease V). In some strains a previous dose of ultraviolet (UV) light followed by incubation renders the cells resistant to DNA degradation after gamma irradiation. We have shown this inhibition to take place for infecting T4 phage also. By using six strains of E. coli selected for mutations in the genes recA, exr (or lex), and uvrB, we have been able to show that the preliminary UV treatment produces no change in recA and exr cells for both endogenous DNA degradation and the degradation of infecting irradiated T4 phage DNA, i.e., inhibition was not detected in these strains. On the other hand, wild-type cells and strains carrying mutations of uvrB show inhibition in both types of experiments. Because the recA gene product and the exr(+) (lex(+)) gene product are necessary for the induction of prophage, it is possible that the phenomenon of inducible inhibition requires recA(+) and exr(+) presence. One interpretation of these results is that an inducible inhibitor may be controlled by the exr gene.     

Genetic exchange between Escherichia coli strains in the mouse intestine

Germ-free mice contaminated with selected Escherichia coli strains were used for experiments designed to demonstrate gene transfer and recombinant formation in vivo. The well-characterized conjugation system of E. coli K-12 was examined in these experiments. Contamination of germ-free mice with a polyauxotrophic F(-) strain followed by the addition of isogenic Hfr, F', or F(+) strains resulted in the appearance of all recombinant classes at frequencies that would be expected from an in vitro mating experiment. Inheritance of unselected donor markers occurred at frequencies that were dependent on linkage relationships established in experiments in vitro. The presence of Lactobacillus had no influence on gene transfer and recombinant formation in an F' x F(-) in vivo mating. The R factor ROR-1 was transferred from E. coli strain M7-18 to an E. coli F(-) strain in the mouse intestine.     

General recombination in Escherichia coli K-12: in vivo role of RecBC enzyme.

Heterozygous lacZ- merodiploids of Escherichia coli K-12 have been used to study the role of the RecBC enzyme in general recombination. The transcribable intermediate assay detects the product of early steps in recombination without requiring the formation of viable recombinant colonies. Recombination is initiated by infection with lambda precA+. We have found that transcribable intermediate formation in crosses between F42 lac and chromosomal lac is dependent on F fertility functions and an active RecBC enzyme. Thus, the products of the recB and recC genes are required in early steps of recombination between these two substrates. Introduction of the F42 lac donor DNA by conjugation immediately after infection with lambda precA+ abolishes the requirement for an active RecBC enzyme.     

Heteroduplex analysis of tra delta f'' plasmids and the mechanism of their formation.

Four tra delta FargG+ plasmids, derived from matings between Hfr AB312 and a recA recipient, have been shown to have deletions of at least 50% of the F genome, including the region in which the tra genes map. The mutant plasmids do contain the F genes required for plasmid maintenance. Correlations can be made between, on the one hand, the F genes present on the tradelta F' plasmids and the F genes transferred early by an Hfr donor, and, on the other hand, the F genes deleted from the tradelta F' plasmids and the F genes transferred late by an Hfr donor. A biased representation of proximally and distally transferred chromosomal markers among the tradelta F' elements was also demonstrated. Taken Taken together, the asymmetrical representation of Hfr genes and the cis dominance of the Tra phenotype of these mutants can best be explained by the hypothesis that the tradelta F' plasmids are formed by repliconation of the transferred exogenote in a recA recipient.     

Biochemical and genetic studies of recombination proficiency in Escherichia coli K12

Molecular Genetics and Genomics - Residual genetic recombination is carried out by recB - recC - mutants of E. coli. Recombinants (for one gene) formed by a recB - recC - parent were shown to be as...     

Genetic mapping of the ilv7434 mutation providing threonine deaminase resistance to isoleucine inhibition in Escherichia coli

Location of previously isolated ilv7434 mutation was determined by use of transductional shortening of the F'14 episome. The ilv7434 mutation causes resistance of threonine deaminase (coded for by ilvA gene) to feed-back inhibition by isoleucine. Another phenotype characteristics of the ilv7434 mutant is the ability to feed a lawn of isoleucine auxotrophs in the cross-streak test. The F'14 strain AB1206 carrying ilv7434 mutation was used as a donor for making transductionally shortened episomes in recA recipient. These shortened F'14 episomes containing variable segments of the ilv cluster were then tested for their ability to transfer ilv7434 phenotype by complementation with ilv recA recipients. The data of complementation test suggest that ilv7434 is situated between ilvD and ilvC genes. One of 20 tested shortened episomes carrying, as shown by complementation test, incomplete ilvA gene was found to transfer ilv7434 phenotype by recombination with ilvA527 recA+ recipient. These data allow to conclude that ilv7434 mutation is located within the ilvA gene.     

A recB recC sbcB recJ host prevents recA-independent deletions in recombinant cosmid DNA propagated in Escherichia coli.

Segments of DNA are deleted from recombinant cosmid DNAs with high frequency during propagation in standard recA Escherichia coli hosts. An attempt has been made to derive an appropriate strain of E. coli, suitable for cosmid cloning, in which such deletions do not occur. We examined the effects of a series of host recombinational mutations on the deletion process, using six independent recombinant cosmids that carry inserts of mouse, Chinese hamster, or human DNA. Various E. coli host cells carrying the recombinant cosmids were cultured serially in liquid medium, and the recombinant cosmid DNAs were extracted from the host cells and analyzed by agarose gel electrophoresis and by gene transfer of the DNAs into cultured mammalian cells. Of the mutations examined, only a recB recC sbcB recJ (or recN) quadruple combination of host mutations prevented the deletion of DNA segments. The recombinant cosmid DNAs propagated in E. coli hosts that carried this combination of mutations were functionally as well as structurally intact. We propose that the recJ (and/or recN) gene is involved in some aspect of the events that lead to deletions of cosmid DNA in a recB recC sbcB genetic background.