Genetic analysis of the recF pathway to genetic recombination in Escherichia coli k12: Isolation and characterization of mutants

A recombination proficient strain ofEscherichia coli which is recB^? recC^? sbcB^? has been subjected to mutagenesis by nitrosoguanidine. Among the recombination deficient mutants isolated one was sbcB⁺, three were recA^～ and 11 were mutants in at least four newrec genes: recF, recJ, recK and recL. recF143 and recL152 are cotransducible with ilv but they lie on opposite sides of the ilv operons as determined by F$\text{\$}\text{?}$studies. recF, recL and recK are not involved in the RecBC pathway of recombination since a recB⁺recC⁺sbcB^? strain carrying a mutation in one of these genes is recombination proficient. Hence the hypothesis that a RecF pathway of recombination can operate as a partially independent substitute for the RecBC pathway of recombination is supported. recF^?recB⁺ and recF⁺recB^? single mutants are sensitive to u.v. irradiation while the recF^?recB^? double mutant is more sensitive than either single mutant. The sensitivity of the recB^?recC^?sbcB^?recF^? strain approaches the sensitivity of a recA^? single mutant. This is interpreted to mean that there are partially independent RecF and RecBC pathways for the repair of u.v. damage. recJ and mutations were not mapped precisely; hence the mutant properties they confer can not be stated conclusively.     

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.     

Interaction of the exrA mutation with rec mutants of Escherichia coli K12

Mutants of Escherichia coli K₁₂ were constructed which carry the exrA mutation addition to the various recombination deficient mutations recA recB and recC. The double mutant containing the exrA recA genotype is found to be slightly more sensitive to UV irradiation at very low doses of UV but essentially is very similar to the exrA + recA at the high UV doses. The recombination deficiency, λ induction and DNA degradation of the exrA recA shows a slight increase in the defectiveness of each of these functions. The double mutants of exrA recB and exrA recC show an increase in UV sensitivity and recombination deficiency and λ induction. The DNA degradation following UV-irradiation of these mutants is more characteristic of the recB and recC mutant alone.These results give further support to the theory that exrA and lex are probably mutants within the same cistron and also suggest that exrA, lex and recA are involved in a common DNA repair pathway and that the gene products of all three functions are required to regulate recB+ and recC+ endonuclease induced DNA degradation.     

Involvement of Recombination Genes in Growth and Viability of Escherichia coli K-12

We have studied the growth properties of 17 isogenic strains of Escherichia coli K-12 differing only in the recA, recB, recC, and sbcA alleles. We have observed the following. (i) All recombination deficient strains have decreased growth rates and decreased viabilities compared with recombination proficient strains. The large populations of nonviable cells in Rec⁻ cultures may arise by spontaneous lethal sectoring (9). (ii) A recA mutant strain which is entirely recombination deficient and which shows high ultraviolet sensitivity and “reckless” deoxyribonucleic acid (DNA) breakdown has approximately the same growth rate and twice the viability as recB and recC mutant strains which have residual recombination proficiency, moderate ultraviolet sensitivity, and “cautious” DNA breakdown. (iii) Indirectly suppressed (sbcA⁻) recombination proficient (Rec⁺) revertants of recB and recC mutant strains have approximately normal growth rates and are three times as viable as their Rec⁻ ancestors (but not as viable as rec⁺ cells). We suggest the following hypothesis to account for the low viability of Rec⁻E. coli. Single-strand breaks in the DNA duplex, necessary for normal bacterial growth, may be repaired in a Rec⁺ cell. Failure of Rec⁻ cells to repair this normal DNA damage may lead to the observed loss of viability.     

Induction of E. coli recA protein via recBC and alternate pathways: quantitation by enzyme-linked immunosorbent assay (ELISA)

Summary An enzyme-linked immunosorbent assay (ELISA) has been adapted to measure E. coli recA protein in the 1 to 10 ng range in whole-cell sonicates, membrane extracts, and osmotic shock fluid from 2x10⁸ cells. The specific activity of recA protein is maintained at a relatively constant basal level (800 to 1,200 molecules per cell for wild-type E. coli in L-broth, salt-depleted broth and minimal media) during early-log and mid-log phase growth, but it increases by two- to ten-fold as the culture approaches saturation density. Nalidixate-induced levels are 20- to 50-fold higher, and 100-fold higher in a constitutive tif ^- spr ^-mutant.Induction of recA protein synthesis by nalidixic acid, which normally requires functional recBC enzyme, also occurs in recB ^-and recC ^-cells by pathways activated by mutation in the sbcA and sbcB indirect suppressors. In recB ^- sbcA ^-mutants, exonuclease VIII, the recE gene product, is required for induction of recA protein. Abolition of exonuclease I activity by mutation in sbcB allows induction of recA protein by nalidixate in recB ^-and recC ^-cells. Mutation in recF does not affect induction by nalidixate in RecBC⁺ cells, but it enables induction to occur in RecBC^- cells, suggesting that recF gene product is involved in regulation of recA protein.     

Analysis of the Role of Recombination and Repair in Mutagenesis of ESCHERICHIA COLI by Uv Irradiation

Multiple mutant strains have been tested for their mimicry of the UV-mutagenesis deficiency of a recA single mutant. Revertants to histidine prototrophy and clear plaque mutants of lambda were scored to determine capacity for UV-mutagenesis. Nearly normal capacity was shown by a uvr⁺ recB^- recF ^- strain, which shows almost no recA-dependent recombination, by uvr^- recB⁺ recF ^- strains, which show almost no recA-dependent repair and by a uvrA^- recB^- recF^- strain, which shows neither recA-dependent recombination nor repair. Since the uvr mutants can be assumed to show additionally no excision repair, these results may mean that UV-mutagenesis occurs during processes other than recombination and repair. Alternative hypotheses are discussed. The slight difference in mutagenic capacity was traced to the recF single mutation, which blocks the production of unmixed bursts of clear-plaque lambda mutants. Since this accounts for only about 10% of the mutations leading to clear-plaque mutants, it is suggested that there is more than one UV-mutagenic process.     

Sister Chromatid Exchange Frequencies in Escherichia coli Analyzed by Recombination at the dif Resolvase Site

Sister chromatid exchange (SCE) in Escherichia coli results in the formation of circular dimer chromosomes, which are converted back to monomers by a compensating exchange at the dif resolvase site. Recombination at dif is site specific and can be monitored by utilizing a density label assay that we recently described. To characterize factors affecting SCE frequency, we analyzed dimer resolution at the dif site in a variety of genetic backgrounds and conditions. Recombination at dif was increased by known hyperrecombinogenic mutations such as polA, dut, and uvrD. It was also increased by a fur mutation, which increased oxidative DNA damage. Recombination at dif was eliminated by a recA mutation, reflecting the role of RecA in SCE and virtually all homologous recombination in E. coli. Interestingly, recombination at dif was reduced to approximately half of the wild-type levels by single mutations in either recB or recF, and it was virtually eliminated when both mutations were present. This result demonstrates the importance of both RecBCD and RecF to chromosomal recombination events in wild-type cells.     

Evidence of abortive recombination in ruv mutants of Escherichia coli K12

Summary Genetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids. Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold. Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants. Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec ⁺ sbc ⁺ strains, depending on the plasmid used. Recombinant plasmids carrying ruv ⁺ were transferred efficiently. With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA. It is proposed that the failure to recover transconjugants in ruv recA ⁺strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.     

Recombination and Annealing Pathways Compete for Substrates in Making rrn Duplications in Salmonella enterica

Tandem genetic duplications arise frequently between the seven directly repeated 5.5-kb rrn loci that encode ribosomal RNAs in Salmonella enterica. The closest rrn genes, rrnB and rrnE, flank a 40-kb region that includes the purHD operon. Duplications of purHD arise by exchanges between rrn loci and form at a high rate (10⁻³/cell/division) that remains high in strains blocked for early steps in recombination (recA, recB, and/or recF), but drops 30-fold in mutants blocked for later Holliday junction resolution (ruvC recG). The duplication defect of a ruvC recG mutant was fully corrected by an added mutation in any one of the recA, recB, or recF genes. To explain these results, we propose that early recombination defects activate an alternative single-strand annealing pathway for duplication formation. In wild-type cells, rrn duplications form primarily by the action of RecFORA on single-strand gaps. Double-strand breaks cannot initiate rrn duplications because rrn loci lack Chi sites, which are essential for recombination between two separated rrn sequences. A recA or recF mutation allows unrepaired gaps to accumulate such that different rrn loci can provide single-strand rrn sequences that lack the RecA coating that normally inhibits annealing. A recB mutation activates annealing by allowing double-strand ends within rrn to avoid digestion by RecBCD and provide a new source of rrn ends for use in annealing. The equivalent high rates of rrn duplication by recombination and annealing pathways may reflect a limiting economy of gaps and breaks arising in heavily transcribed, palindrome-rich rrn sequences.     

Analysis of IS21-mediated mobilization of plasmid pACYC184 by R68.45 in Escherichia coli

Escherichia coli plasmids like pACYC184 or pBR325 can be mobilized by the P-type plasmid R68.45, which carries a tandem duplication of insertion element IS21, at a frequency of 10^?3–10^?5 per donor cell. Analysis of exconjugant cells revealed that plasmid mobilization occurs via cointegrate formation involving transposition of IS21. No resolution of cointegrates of pACYC184 and the P-type plasmid could be found in recA recipient cells. In the cointegrate, the E. coli plasmid is flanked by single copies of IS21 in direct orientation. After resolution of the cointegrate in recA⁺ recipients, the mobilizing plasmid R68.45 lost one copy of IS21 becoming indistinguishable from plasmid R68. It was shown that during mobilization, insertion element IS21 transposes to the mobilized plasmid. Insertion sites and orientations of IS21 in 33 pACYC184::IS21 insertion mutants have been determined: IS21 was found to be integrated in plasmid pACYC184 in different regions but only in one orientation. The IS21 tandem structure of plasmid R68.45 and its role in the mobilization process is discussed.     

Biochemical and genetic studies of recombination proficiency in Escherichia coli K12. IV. Analysis of recombinants formed by a recombination deficient (recB21 recC22) strain

Summary 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 recombination deficient as their parent, when recombination of a second gene is measured. Therefore the resididual recombination cannot be attributed to a genetically recombination proficient fraction of the parent recB ^- recC ^- culture. I conclude that each recB ^- recC ^- parent cell is capable of carrying out genetic recombination. This conclusion is consistent with the existence of an alternate (and minor) recombination mechanism in E. coli K12, independent of the recB ⁺ recC ⁺ mediated steps.The previous paper in this series was Capaldo-Kimball and Barbour, Involvement of Recombination Genes in Growth and Viability, J. Bact. April (1971).     

The role of recombination in the formation of circular oligomers of the lambda plasmid

The λdv1 plasmid forms an extensive oligomeric series of circular DNA molecules in recombination-proficient (recsu⁺) Escherichia coli. These rec⁺ [λdv1]⁺ strains can be typed into the following four classes according to which member of the oligomeric series is most frequent: monomer, dimer, trimer, and tetramer strains. Each of these strains forms a set of circular λdv1 DNA molecules in which most members belong to the series l, 2l, 3l, 4l, where l is the length of the most frequent circular DNA that characterizes the strain—i.e. l equals the length of the most frequent oligomer in the respective strain. In a given strain, the frequency of a molecular species decreases as its length becomes a larger multiple of l. For example, the dimer strains produce dimers, tetramers, hexamers, octomers, etc., in decreasing frequencies, which reach the limits of detection at about the hexadecamer.When recA^? mutations that are absolutely defective for host recombination are introduced into each of these four strains, l retains the same values as in the parent rec⁺ strain, but oligomers larger than 2l are not formed, and the frequency of the 2l oligomer is much reduced. The introduction of recB^? or recC^? mutations, which are only partially defective for host recombination, produces a much smaller perturbation of the rec⁺ distributions, and $\text{rec}{}^{\mathrm{+}}\text{recA}{}^{\mathrm{?}}$ merodiploids exhibit the rec⁺ phenotype with respect to both oligomerization and host recombination.The effects of rec^? mutations on the distribution of λdv1 oligomers and the nature of the oligomeric series produced in rec⁺ cells all indicate that an intermolecular reciprocal recombination between two circular λdv1 DNAs is the principal reaction responsible for oligomerization. It is suggested that the small residual oligomerization that yields 2l oligomers in recA^?cells results from aberrant segregation of the DNA strands at the termination of the replication of l-sized molecules.The inactivation of recA, but not of recB or C, also results in a marked reduction in the frequency of spontaneous curing which in recA⁺ [λdv1⁺]hosts leads to the segregation of [λdv^?]cells. However, spontaneous curing does not appear to be dependent upon the recombination reactions that yield the [λdv 1⁺]oligomers, since the frequency of oligomerization in recA⁺ hosts decreases with increasing l, whereas the frequency of curing increases with increasing l.     

Genetic recombination in Escherichia coli. IV. Isolation and characterization of recombination-deficiency mutants of Escherichia coli K12

19 independent recombination-deficient mutants were isolated. 7 carried mutations that mapped near or in the recB and recC genes between thyA and argA. 10 mutants carried mutations cotransducible with pheA and exhibited no complementation with recA in temporary zygotic diploids.     

Mutants of Escherichia coli with altered deoxyribonucleases. II. Isolation and characterization of mutants for exonuclease I

Mutants of Escherichia coli K12 deficient in exonuclease I (xon^?)³ were identified by enzymic assay of randomly selected, heavily mutagenized clones. From one of the six mutants of independent origin a thermolabile variant of exonuclease I was partially purified and identified, indicating that the mutation is probably in a structural gene for the enzyme. Transduction of this mutation into a recB^? recC^? strain did not result in the suppression of any of the phenotypic traits of the recipient. Although the five other mutants also appear to have temperature-sensitive exonuclease I activities in crude extracts, these enzymes were not sufficiently stable to permit purification. These latter mutations were of the xonA^? type; they produced a temperature-dependent suppression of the sensitivity to ultraviolet light and to mitomycin C manifested by a recB^? recC^? strain. None of the six mutations were of the sbcB^? type; that is, they did not suppress the recombination deficiency of a recB^? recC^? strain.In experiments with bacteriophage Plke, the six mutations were 41 to 62% cotransducible with the his region of E. coli. Heterozygous F′-merodiploids were constructed and studied for possible complementation of exonuclease I activity. All six mutations and an sbcB^? mutation were recessive to the wild-type alleles, and all were found to belong to a single complementation group. The results suggest that alterations of a structural gene for exonuclease I may result in the indirect suppression of the ultraviolet and mitomycin sensitivity manifested by recB^? recC^? strains.     

The recF-dependent endonuclease from Escherichia coli K12. Formation and resolution of pBR322 DNA multimers

Summary The instability of supercoiled pBR322 DNA obtained from different cells has been investigated. Partially purified plasmid DNA species from rec ⁺, recA and recBC sbcB cells are converted in vitro first to relaxed and then to linear molecules. The recA and recBC sbcB cells produce the best conditions for the monomerization of the pBR322 DNA and the stable maintenance of plasmids. The supercoiled pBR322 DNA from the recBC sbcB recF144 cells has been isolated preferentially in multimeric from (circular oligomers). These DNA forms are not converted to plasmid monomers and are converted to linear molecules three-fold slower than the monomer linearization in the case of the recBC sbcB cells.On the other hand, incubation of the pure pBR322 DNA with the recF-dependent protein Z (Krivonogov and Novitskaja 1982) results in the ATP-independent conversion of supercoiled plasmid DNA to relaxed and linear molecules. These results demonstrate an endonuclease activity of the recF-controlled protein Z, which may be involved in general recA-dependent recombination and formation of the pBR322 monomers in the cell.The results also show that the recF144 mutation in recBC sbcB recF and recF cells leads to the absence of detectable amounts of a 49,000 molecular weight protein.     

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

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

Improving Salmonella vector with rec mutation to stabilize the DNA cargoes

Background  

Salmonella has been employed to deliver therapeutic molecules against cancer and infectious diseases. As the carrier for target gene(s), the cargo plasmid should be stable in the bacterial vector. Plasmid recombination has been reduced in E. coli by mutating several genes including the recA, recE, recF and recJ. However, to our knowledge, there have been no published studies of the effect of these or any other genes that play a role in plasmid recombination in Salmonella enterica.     

Construction of a recF deletion mutant of Azotobacter vinelandii and its characterization

A deletion was engineered in the cloned recF gene by digestion with suitable restriction endonucleases and a tetracycline resistance gene cartridge was inserted. The mutation was subsequently transferred to the Azotobacter vinelandii chromosome by double cross-over under pressure of tetracycline selection. A recF recA mutant was also constructed in a similar manner. The mutations were found to be stable and mutation of the wild-type recF gene was confirmed by Southern blot hybridization. Both the mutants were UV sensitive and recombination deficient. Mutations in genes involved in nitrogen fixation in A. vinelandii are rather frequent and obtained comparatively easily despite of the presence of multiple identical chromosomes in A. vinelandii. It has been speculated that some kind of `homogenotization' process operates which is responsible for the `transmission' of mutation from one chromosome to all the chromosomes. This process is not affected by a mutation in recF or recA or in both recF and recA.