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.     

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.     

Identification of the recR locus of Escherichia coli K-12 and analysis of its role in recombination and DNA repair

Summary A new recombination gene called recR has been identified and located near dnaZ at minute 11 on the current linkage map of Escherichia coli. The gene was detected after transposon mutagenesis of a recB sbcB sbcC strain and screening for insertion mutants that had a reduced efficiency of recombination in Hfr crosses. The recR insertions obtained conferred a recombination deficient and extremely UV sensitive phenotype in both recB recC sbcA and recB recC sbcB sbcC genetic backgrounds. recR derivatives of recBC ⁺ sbc ⁺ strains were proficient in conjugational and transductional recombination but deficient in plasmid recombination and sensitive to UV light. Strains carrying recR insertions combined with mutations uvrA and other rec genes revealed that the gene is involved in a recombinational process of DNA repair that relies also on recF and recO, and possibly recJ, but which is independent of recB, recC and recD. The properties of two other insertions, one located near pyrE and the other near guaA, are discussed in relation to their proximity to recG and xse (the gene for exonuclease VII), respectively.     

Genetic analysis and molecular cloning of the Escherichia coli ruv gene

Summary The genetic organisation of the ruv gene, a component of the SOS system for DNA repair and recombination in Escherichia coli, was investigated. New point mutations as well as insertions and deletions were generated using transposon Tn10 inserted in eda as a linked marker for site specific mutagenesis, or directly as a mutagen. The mutations were ordered with respect to one another and previously isolated ruv alleles by means of transductional crosses. The direction of chromosome mobilization from ruv:: Mud(Ap^R lac)strains carrying F42lac ⁺ established that ruv is transcribed in a counterclockwise direction. Recombinant phages able to restore UV resistance to ruv mutants were identified, and the ruv ⁺ region was subcloned into a low copy number plasmid. The ruv ⁺ plasmid was able to correct the extreme radiation sensitivity and recombination deficiency of ruv recBC sbcB strains.     

Rec-dependent and Rec-independent recombination of plasmid-borne duplication in Escherichia coli K12

Summary Plasmidic recombination in E. coli K12 has been previously demonstrated to be dependent on the host rec genotype. The construction of plasmids that carry a duplication within an antibiotic-resistance gene is described. Recombination between the direct repeats recreates an active antibiotic-resistance gene, allowing quantitative analysis of recombination frequencies in a closely related set of E. coli K12 strains carrying various rec mutations. Using this system, intraplasmidic recombination of a duplication within the pBR322 tetracycline-resistance gene is shown to be rec-dependent while recombination of a similar duplication within the kanamycin-resistance gene of Tn903 is shown to be independent of recA, recB, recC, recE, recF and sbcB.     

Isolation and characterization of dam+ revertants and suppressor mutations that modify secondary phenotypes of dam-3 strains of Escherichia coli K-12

Summary Bacteria mutant in the dam (DNA adenine methylation) gene and in either recA or recB or recC genes are inviable (Virm^- phenotype). From crosses between dam-3 bacteria and recA1 or recB21 recC22 strains, Vrm⁺ recombinants were recovered. Among these recombinants, Dam⁺ revertants were present which did not show the phenotypes normally associated with dam-3 bacteria. Three classes of indirectly suppressed strains (dam-3 genotype) were also recovered which showed alterations in the secondary phenotypes normally associated with dam-3 bacteria. These strains contained a second unlinked mutation in either mutL or mutS or sin. In addition, mutation in either sbcA or sbcB suppresses the Vrm^- phenotype of dam-3 recB21 recC22 strains.     

Intramolecular homologous recombination of linearized plasmids in Escherichia coli K12

Summary The efficacy of linear DNA as a substrate for general homologous recombination was demonstrated using BamHI-linearized pKLC8.5, a plasmid that carries internal direct repeats flanking the unique BamHI site. An analogous plasmid, pKLC2.31, was used in a parallel and comparative study of intramolecular homologous recombination in circular DNA substrates. When the rec ⁺ wild-type strain, AB1157, and its isogenic rec ^– derivatives were transformed with linear pKLC8.5 DNA, intramolecular homologous recombination was independent of recA, recB, recN, recO and exonuclease III (xth-1) functions. Although the recBCsbcA and recBCsbcBC cells were both very recombination proficient, only linear but not circular DNA was used as substrate for intramolecular homologous recombination in the recBCsbcA cells. In both the recBCsbcA and recBCsbcBC genetic backgrounds, the recombination frequencies for linearized pKLC8.5 DNA were 100%. A notable difference between the two strains was that none of the recBCsbcA transformants obtained with circular pKLC8.5 DNA were Tc^s recombinants, whereas 11% of the corresponding recBCsbcBC transformants were Tc^s recombinants. The sbcB mutation was responsible for the recombination proficiency of the recBCsbcBC cells. Unlike the case in recBCsbcA cells, intramolecular homologous recombination of linear DNA in the recBCsbcBC cells was dependent on recA and recF as well as recN and recO gene functions, but was independent of recJ and reeL gene functions.     

Properties of strains of Escherichia coli K12 carrying mutant lex and rec alleles

Summary Strains of Escherichia coli K12 have been constructed which carry the lex-3 mutation in combination with recA56, recB21, or recC22. The lex ^– recA ^–strain is equally as sensitive to ultraviolet light (UV) and ionizing radiation and as recombination-deficient as the corresponding lex ⁺ recA ^–strain, whereas the lex ^– recB ^–and lex ^– recC ^–strains are somewhat more sensitive to UV and ionizing radiation than the corresponding lex ^– rec ⁺strain and have approximately the same recombination deficiencies as the respective lex ⁺ recB ^–and Lex ⁺ recC ^–strains. When cultures of the lex ^– recB ^–and lex ^–recC^–strains are UV-irradiated, they degrade their DNA at the low rate characteristic of lex ⁺ recB ^–and lex ⁺ recC ^–single mutants, in contrast to the high rate of degradation seen with lex ^– rec ⁺single mutants.These results imply that the lex ⁺and recA ⁺products act in the same pathway of DNA repair and that both are needed to limit the DNA breakdown due to the recB ⁺/C⁺nuclease.     

A protein, connected with the integrity of the recF gene in Escherichia coli K-12

Summary A protein of molecular weight 74,000, called protein Z, has been identified in cells of the genotype recB21 recC22 sbcB15 by SDS-polyacrylamide gel electrophoresis. This protein has not been detected in cells of the genotype recB21 recC22 sbcB15 recF144. The transductional transfer of recF144 into the rec ⁺ cells leads to the disappearance of the protein Z band. These results demonstrate that the recF gene is essential for protein Z synthesis. Of two recF ^– mutants studied, recF144 completely lacks protein Z, while recF143 preserves a functionally inactive protein Z, probably resulting from a missense mutation.The recF144 cells are characterizied by a very low frequency of genetic exchange between the donor and recipient chromosomes after conjugation. The scale of the genetic map for these cells is 3-fold higher than for wild-type cells.     

Identification of the genes coding for the second-largest subunits of RNA polymerases I and III of Drosophila melanogaster

Summary Colony forming ability of Escherichia coli strains carrying the rnh-339::cat mutant allele is strongly dependent on the recBCD and sbcB genes. A mutation inactivating either the RecBCD nuclease or exonuclease I (sbcB) is sufficient to restrict severely the efficiency of plating of strains carrying the rnh-339::cat mutation. Combining a non-lethal temperature-sensitive mutation in the RecBCD nuclease, recB270 (Ts) or recC271 (Ts), with rnh-339::cat renders strains temperature sensitive for growth, even though rnh ⁺ strains with the recB270 (Ts) or recC271 (Ts) alleles are viable at 42 C. The recombinational functions of the RecBCD nuclease can be excluded as the source of lethality on the basis of the following observations. Introduction of a recombination proficient, exonuclease defective recD1009 allele or production of the phage GamS protein (an inhibitor of the RecBCD exonuclease activity) in an rnh-339::cat strain dramatically delays or impairs the ability of such strains to form colonies. Restoration of recombination proficiency by inclusion of an sbcB15 mutation with recB21 recC22 mutations does not restore the ability of the rnh-339::cat mutant strains to plate normally. A recBCD ⁺ strain bearing the rnh-339::cat and sbcB15 mutations forms very few visible colonies after 24 h but forms colonies at normal frequencies after 48 h of incubation. Finally, plating efficiencies of strains are unaffected when the RecBCD recombination pathway is inactivated by introduction of recA56 into an rnh-339::cat strain. These results imply that the defective growth of rnh-339::cat recBCD strains is due to a defect in repair and not recombination mediated by either the RecBCD or the RecF pathway.     

Interplasmidic and intraplasmidic recombination in Escherichia coli K-12

Summary The construction of plasmids which facilitate the study of interplasmidic and intraplasmidic recombination is described. In this system, a single recombination event between two mutated Te^r genes on separate plasmids or on one plasmid leads to a change in the host phenotype from sensitivity to resistance to tetracycline.Recombination proficiencies have been determined for different E. coli K-12 strains: both interplasmidic and intraplasmidic recombination are independent of the recBC gene product. RecA mutations decrease the proficiency of plasmidic recombination 40–100 fold. Intraplasmidic and interplasmidic recombination via the recE pathway are more efficient than via the recBC pathway. Intraplasmidic recombination, but not interplasmidic recombination via the recE pathway is independent of a functional recA product.     

The Genetic Dependence of Recombination in Recd Mutants of Escherichia Coli

RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations.     

Hyper-recombination in dam mutants of Escherichia coli K-12

Summary F-prime heterogenotes of dam-3 bacteria segregate F-prime homogenotes at a frequency 30–200 times higher than the isogenic dam ⁺ strain. A hyperrecombination mutant which shows increased recombination between chromosomal duplications was characterized as a dam mutant. The dam-3 allele causes a reduction in linkage of proximal unselected markers in transconjugants and increases the recombination frequency between a pair of closely linked markers. It is concluded that dam mutations confer a hyperrecombination phenotype to the cell.     

Involvement in DNA repair of the ruvA gene of Escherichia coli

Summary The ruv operon of Escherichia coli consists of two genes, orfl1 and ruv, which encode 22 and 37 kilodalton proteins, respectively, and are regulated by the SOS system. Although the distal gene, ruv, is known to be involved in DNA repair, the function of orf1 has not been studied. To examine whether orf1 is also involved in DNA repair, we constructed a strain with a deletion of the entire ruv operon. The strain was sensitive to UV even after introduction of low copy number plasmids carrying either orf1 or ruv, but UV resistance was restored by introduction of a plasmid carrying both orfl and ruv. These results suggest that orf1 as well as ruv is involved in DNA repair. Therefore, orf1 and ruv should be renamed ruvA and ruvB, respectively.     

Apparent gene conversion in an Escherichia coli rec+ strain is explained by multiple rounds of reciprocal crossing-over

Summary Gene conversion, the non-reciprocal transfer of sequence information between homologous DNA sequences, has been reported in lower eukaryotes, mammals and in Escherichia coli. In an E. coli rec ⁺ strain, we established a plasmid carrying two different deleted neo genes (neoDL and neoDR) in an inverted orientation and then selected for homologous recombination events that had reconstructed an intact neo ⁺ gene. We found some plasmids that had apparently experienced intramolecular gene conversion. Further evidence, however, suggests that they are products of multiple rounds of reciprocal crossing-over,apparently involving two plasmid molecules. First, most of the Neo⁺ clones contained multiple types of Neo⁺ plasmids, although the frequency of producing the neo ⁺ clones was low. Second, all the neo ⁺ clones also contained, as a minority, one particular form of dimer, which can be formed by reciprocal crossing-over between neoDL of one plasmid molecule and neoDR of another plasmid molecule. Third, in reconstruction experiments, we cloned and purified this dimer and transferred it back into the rec ⁺ cells. The dimer gave rise to clones containing multiple types of neo ⁺ recombinant monomers, including those apparent gene conversion types, and containing only few molecules of this dimer plasmid.     

A molecular model for conjugational recombination in Escherichia coli K12

Summary Conjugational recombination in Escherichia coli was investigated by measuring lacZ ⁺ product, -galactosidase, in crosses between lacZ mutants. Enzyme production in both Hfr and F-prime crosses was detected very soon after transfer of the donor lacZ allele. The level of enzyme activity was reduced by no more than two-fold when the recipient carried a recB mutation. With an F-prime donor, recombination appeared to be restricted largely to a short period immediately after transfer, with little evidence of recombination during subsequent exponential growth of the transconjugant cells. These observations are interpreted to suggest that recA dependent recombination is able to initiate with high efficiency at gaps present in the donor DNA before synthesis of a complementary strand is completed, and independently of recB function. A molecular model for conjugational recombination based on this idea is presented in terms of the known activities of recA and recBC products. Some of the predictions of the model are tested by analysing the recombinant genotypes produced in Hfr crosses with multiply marked strains.     

Hyper-recombination in uvrD mutants of Escherichia coli K-12

Summary A mutant strain of E. coli which was isolated initially because of its strong hyper-recombination phenotype was shown to carry a lesion in uvrD. The presence of this mutation, designated uvrD210, increased the frequency of recombination between chromosomal duplications in F-prime repliconant cells and reduced linkage between closely linked markers in crosses with Hfr donors. A comparable hyper-rec phenotype was demonstrated in strains carrying other alleles of uvrD previously referred to as mutU4, uvr502 and recL152. The recombination activity of a uvrD210 strain was abolished by mutation of recA but the mutator activity associated with this allele proved to be independent of recA. It is suggested that uvrD mutations reduce the fidelity of DNA replication and that the accumulation of lesions in the newly synthesized strand provides additional sites for initiating recombination.     

DNA degradation in minicells of Escherichia coli K-12

Summary The properties of minicell producing mutants of Escherichia coli deficient in gentic recombination were examined. Experiments were designed to test recombinant formation in conjugal crosses, survival following UV-irradiation in cells, and the state of DNA metabolism in minicells. The REC^- phenotypes are unaffected by min ⁺/^- genotypes in whole cells. In contrast to minicells produced by rec ⁺ parental cells, minicells from a recB21 strain have limited capacity to degrade linear, Hfr transferred DNA. The lack of a functional recA gene product, presumably involved in inhibiting the recBC nuclease action(s), permits unrestricted Hfr DNA breakdown in minicells produced by a recA1 strain. This results in an increase in TCA soluble products and in the formation of small DNA molecules that sediment near the top of an alkaline sucrose gradient. Unlike the linear DNA, circular duplex DNA from plasmids R64-11 or dv, segregated into the minicells, is resistant to breakdown. By using in vitro criteria, and [³²P]-labelled linear DNA from bacteriophage T₇ for substrate, we found that the ATP-dependent exonuclease of the recBC complex (exo V) is present in rec ⁺ and recA^- minicells, and is lacking in the recB21 mutant. In fact, the absence of a functional exo V in recBC^- minicells results in isolation of larger than average Hfr DNA from minicells. We suggest that recombination (REC) enzymes segregate into the polar minicells at the time of minicell biogenesis. This system should be useful for studies on DNA metabolism and functions of the recBC and recA gene products.Paper 1 in series, see Khachatourians et al., 1974.     

recB recJ mutants ofSalmonella typhimurium are deficient in transductional recombination, DNA repair and plasmid maintenance

recB recJ mutants ofSalmonella typhimurium are deficient in transduction of chromosomal markers and ColE1-derived plasmids, and also in the maintenance of ColE1 and F plasmids. Plasmid instability is less severe inrecD recJ strains; ColE1 plasmid DNA preparations from these strains show an increased yield of high molecular weight (HMW) linear multimers and a concomitant reduction in plasmid monomers compared to the wild type. Plasmids remain unstable inrecA recD recJ mutants; since these do not produce HMW linear concatemers, we propose that a decrease in monomer production leads to plasmid instability.recB recJ strains also display decreased viability, a component of which may be related to their deficiency in DNA repair. In contrast to their severe defects in recombination, DNA repair and plasmid maintenance,recB recJ mutants ofS. typhimurium behave similarly to the wild type in the segregation of chromosome duplications. The latter observation suggests that neither RecBCD nor RecJ functions are required for chromosomal recombination events that do not involve the use of free ends as recombination substrates.     

Effect of ruv mutations on recombination and DNA repair in Escherichia coli K12

Summary Mutation of the ruv gene of E. coli is associated with sensitivity to radiation, and filamentous growth after transient inhibition of DNA synthesis. The filamentation of ruv strains is abolished by mutations in sfiA or sfiB that prevent SOS induced inhibition of cell division, but this does not restore resistance to UV radiation. Double mutants carrying both ruv and uvr mutations are considerably more sensitive to UV radiation than the single mutants, but there is no additive effect of ruv with recA, recF, recB, or recC mutations. ruv mutations have little effect on conjugal recombination in wild-type strains but confer recombination deficiency and extreme sensitivity to ionizing radiation in recBC sbcB strain. These results, together with the fact that ruv strains are excision proficient and mutable by UV light, are interpreted to suggest that the ruv ⁺ product is involved in recombinational repair of damaged DNA rather than in cell division as suggested by Otsuji et al. (1974).