Genetic recombination in Bacillus subtilis 168: effect of recN, recF, recH and addAB mutations on DNA repair and recombination

A recN ^– (recN1) strain of Bacillus subtilis was constructed. The effects of this and recF, recH and addAB mutations on recombination proficiency were tested. Mutations in the recN, recF recH and addAB genes, when present in an otherwise Rec⁺ B. subtilis strain, did not affect genetic exchange. Strains carrying different combinations of mutations in these genes were constructed and examined for their sensitivity to 4-nitroquinoline1-oxide (4NQO) and recombination proficiency. The recH mutation did not affect the 4NQO sensitivity of recN and recF cells and it only marginally affected that of addA addB cells. However, it reduced genetic recombination in these cells 10²- to 10⁴-fold. The addA addB mutations increased the 4NQO sensitivity of recF and recN cells, but completely blocked genetic recombination of recF cells and marginally affected recombination in recN cells. The recN mutation did not affect the recombinational capacity of recF cells. These data indicate that the recN gene product is required for, DNA repair and recombination and that the recF, recH and addAB genes provide overlapping activities that compensate for the effects of single mutants proficiency. We proposed that the recF, recH, recB and addA gene products define four different epistatic groups.     

The Bacillus subtilis chromatin-associated protein Hbsu is involved in DNA repair and recombination

The Bacillus subtilis hbs gene encodes an essential chromatin-associated protein termed Hbsu. Hbsu, the counterpart of the Escherichia coli HU protein, binds DNA in a non-specific way but has a clear preference for bent, kinked or altered DNA sequences. To investigate the role of Hbsu in DNA repair and DNA recombination we have constructed a series of site-directed mutants in the hbs gene and used these mutant genes to substitute the wild-type chromosomal hbs gene. The hbs47 mutation, which codes for a mutant protein in which residue Phe-47 has been replaced by Trp, does not cause any discernible phenotype. Additional substitution of residue Arg-55 by Ala (hbs4755 mutation) rendered cells deficient in DNA repair, homologous recombination and (i protein-mediated site-specific recombination. We have also tested the effect on DNA repair of the hbs4755 mutation in combination with mutations in different functions of homologous DNA recombination (recA, recF, recG, recti and addAB). The hbs4755 mutation did not modify the sensitivity of recH and addAB cells to the DNA-damaging agents methylmethane sulphonate (MMS) or 4-nitroquinoline-1-oxide (4NQO), and it only marginally affected recF and recG cells. The hbs4755 mutation blocked intermolecular recombination in recH cells and markedly reduced it (20- to 50-fold) in recF and recG cells, but had no effect on addAB cells. Taken together, these data indicate that the Hbsu protein is required for DNA repair and for homologous DNA recombination.     

Analysis of the Bacillus subtilis recO gene: RecO forms part of the RecFLOR function

The deduced protein product of the Bacillus subtilis gene yqfI, which is 255 residues long, shares homology (25% identity) with the Escherichia coli RecO protein. A null allele of yqfI, when present in an otherwise Rec⁺ B. subtilis strain, causes cells to become highly sensitive to DNA-damaging agents, and plasmid transformation (intramolecular recombination) is reduced by 25-fold while chromosomal transformation (intermolecular recombination) is only moderately affected (2.5-fold reduction). Therefore, the yqfI gene was renamed recO and its null allele is referred to as recO1. The recO1 mutation was introduced into recombination-deficient strains representative of the epistatic groups α (recF, recR and recL strains), β (addA5 addB72), γ (recH342) and ? (recU40). The recO mutation did not affect the sensitivity of recF, recR or recL cells to DNA-damaging agents, increased the sensitivity of recU and addAB cells and abolished the DNA repair capacity of recH cells. The recO mutation did not affect intermolecular recombination in recF, recL, recH or recU cells, but reduced (by about 9-fold) the incidence of intermolecular recombination in addAB cells. The recO mutation did not affect intramolecular recombination in the addAB, recU, recF or recL cells, but reduced it by about 75-fold in recH cells. The defects caused by the recO1 mutation can be partially suppressed by a common suppressor of the recF, recL and recR phenotypes. We therefore assigned recO to epistatic group α and predict that the RecO protein acts at the same stage of recombination as the RecF, RecL and RecR proteins, in a RecFLOR complex.     

Genetic Recombination in Bacillus subtilis 168: Effects of recU and recS Mutations on DNA Repair and Homologous Recombination

Bacillus subtilis recombination-deficient mutants were constructed by inserting a selectable marker (cat gene) into the yppB and ypbC coding regions. The yppB:cat and ypbC:cat null alleles rendered cells sensitive to DNA-damaging agents, impaired plasmid transformation (25- and 100-fold), and moderately affected chromosomal transformation when present in an otherwise Rec⁺ B. subtilis strain. The yppB gene complemented the defect of the recG40 strain. yppB and ypbC and their respective null alleles were termed “recU” and “recU1” (recU:cat) and “recS” and “recS1” (recS:cat), respectively. The recU and recS mutations were introduced into rec-deficient strains representative of the α (recF), β (addA5 addB72), γ (recH342), and (recG40) epistatic groups. The recU mutation did not modify the sensitivity of recH cells to DNA-damaging agents, but it did affect inter- and intramolecular recombination in recH cells. The recS mutation did not modify the sensitivity of addAB cells to DNA-damaging agents, and it marginally affected recF, recH, and recU cells. The recS mutation markedly reduced (about 250-fold) intermolecular recombination in recH cells, and there were reductions of 10- to 20-fold in recF, addAB, and recU cells. Intramolecular recombination was blocked in recS recF, recS addAB, and recS recU cells. RecU and RecS have no functional counterparts in Escherichia coli. Altogether, these data indicate that the recU and recS proteins are required for DNA repair and intramolecular recombination and that the recF (α epistatic group), addAB (β), recH (γ), recU (), and recS genes provide overlapping activities that compensate for the effects of single mutation. We tentatively placed recS within a new group, termed “ζ.”     

Analysis of the Bacillus subtilis recO gene: RecO forms part of the RecFLOR function

The deduced protein product of the Bacillus subtilis gene yqfI, which is 255 residues long, shares homology (25% identity) with the Escherichia coli RecO protein. A null allele of yqfI, when present in an otherwise Rec⁺ B. subtilis strain, causes cells to become highly sensitive to DNA-damaging agents, and plasmid transformation (intramolecular recombination) is reduced by 25-fold while chromosomal transformation (intermolecular recombination) is only moderately affected (2.5-fold reduction). Therefore, the yqfI gene was renamed recO and its null allele is referred to as recO1. The recO1 mutation was introduced into recombination-deficient strains representative of the epistatic groups α (recF, recR and recL strains), β (addA5 addB72), γ (recH342) and ɛ (recU40). The recO mutation did not affect the sensitivity of recF, recR or recL cells to DNA-damaging agents, increased the sensitivity of recU and addAB cells and abolished the DNA repair capacity of recH cells. The recO mutation did not affect intermolecular recombination in recF, recL, recH or recU cells, but reduced (by about 9-fold) the incidence of intermolecular recombination in addAB cells. The recO mutation did not affect intramolecular recombination in the addAB, recU, recF or recL cells, but reduced it by about 75-fold in recH cells. The defects caused by the recO1 mutation can be partially suppressed by a common suppressor of the recF, recL and recR phenotypes. We therefore assigned recO to epistatic group α and predict that the RecO protein acts at the same stage of recombination as the RecF, RecL and RecR proteins, in a RecFLOR complex. Received: 5 October 1998 / Accepted: 28 January 1999     

RecBCD- RecFOR-independent pathway of homologous recombination in Escherichia coli

The RecA protein is a key bacterial recombination enzyme that catalyzes pairing and strand exchange between homologous DNA duplexes. In Escherichia coli, RecA protein assembly on DNA is mediated either by the RecBCD or RecFOR protein complexes. Correspondingly, two recombination pathways, RecBCD and RecF (or RecFOR), are distinguished in E. coli. Inactivation of both pathways in recB(CD) recF(OR) mutants results in severe recombination deficiency. Here we describe a novel, RecBCD- RecFOR-independent (RecBFI) recombination pathway that is active in ΔrecBCD sbcB15 sbcC(D) ΔrecF(OR) mutants of E. coli. In transductional crosses, these mutants show only four-fold decrease of recombination frequency relative to the wild-type strain. At the same time they recombine 40- to 90-fold better than their sbcB⁺ sbcC⁺ and ΔsbcB sbcC counterparts. The RecBFI pathway strongly depends on recA, recJ and recQ gene functions, and moderately depends on recG and lexA functions. Inactivation of dinI, helD, recX, recN, radA, ruvABC and uvrD genes has a slight effect on RecBFI recombination. After exposure to UV and gamma irradiation, the ΔrecBCD sbcB15 sbcC ΔrecF mutants show moderately increased DNA repair proficiency relative to their sbcB⁺ sbcC⁺ and ΔsbcB sbcC counterparts. However, introduction of recA730 allele (encoding RecA protein with enhanced DNA binding properties) completely restores repair proficiency to ΔrecBCD sbcB15 sbcC ΔrecF mutants, but not to their sbcB⁺ sbcC⁺ and ΔsbcB sbcC derivatives. Fluorescence microscopy with UV-irradiated recA-gfp fusion mutants suggests that the kinetics of RecA filament formation might be slowed down in the RecBFI pathway. Inactivation of 3′-5′ exonucleases ExoVII, ExoIX and ExoX cannot activate the RecBFI pathway in ΔrecBCD ΔsbcB sbcC ΔrecF mutants. Taken together, our results show that the product of the sbcB15 allele is crucial for RecBFI pathway. Besides protecting 3′ overhangs, SbcB15 protein might play an additional, more active role in formation of the RecA filament.     

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.     

Genetic recombination in Bacillus subtilis 168: effect of DeltahelD on DNA repair and homologous recombination.

The B. subtilis DeltahelD allele rendered cells proficient in transformational recombination and moderately sensitive to methyl methanesulfonate when present in an otherwise Rec(+) strain. The DeltahelD allele was introduced into rec-deficient strains representative of the alpha (recF strain), beta (addA addB), gamma (recH), epsilon (DeltarecU), and zeta (DeltarecS) epistatic groups. The DeltahelD mutation increased the sensitivity to DNA-damaging agents of addAB, DeltarecU, and DeltarecS cells, did not affect the survival of recH cells, and decreased the sensitivity of recF cells. DeltahelD also partially suppressed the DNA repair phenotype of other mutations classified within the alpha epistatic group, namely the recL, DeltarecO, and recR mutations. The DeltahelD allele marginally reduced plasmid transformation (three- to sevenfold) of mutations classified within the alpha, beta, and gamma epistatic groups. Altogether, these data indicate that the loss of helicase IV might stabilize recombination repair intermediates formed in the absence of recFLOR and render recFLOR, addAB, and recH cells impaired in plasmid transformation.     

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.     

Genetic control of recombination exchange frequency in Escherichia coli K-12.

The frequency of recombination exchanges per unit length of DNA (Freuld) can be estimated by measuring the scale of the genetic map that is the mean statistical distance between two neighboring crossovers. The scales appear to be equal for the alternative pathways of recombination, RecBCD (wild-type cells) or RecF (recBC- sbcB- sbcC- genotypes). The absolute value of the scale depends on specific experimental conditions. recR, recQ, ruv, recJ and recN genes of the RecF pathway of recombination (recBC- sbcBC- cell genotypes) do not appear to be silent in wild-type cells where the RecBCD pathway predominates. On the contrary, these genes are responsible for the Freuld. The list recF504::Kmr greater than recQ61::Tn3 greater than ruv-54 greater than recJ284::Tn10 shows decreasing efficiency in inhibiting recombination exchanges by these mutations. The recN264 mutation gives a small, but opposite effect of increasing the frequency of recombination exchanges. The effect of the recF and recQ mutations appears to be additive, but that is not the case in combinations of ruv-54 with recF504::Kmr or recQ61::Tn3.     

Genetic analysis of conjugational recombination in Escherichia coli K12 strains deficient in RecBCD enzyme

Conjugational recombination in Escherichia coli was investigated by comparing the effects of recN, recO, ruv and lexA mutations on the formation of recombinants in crosses with strains lacking RecBCD enzyme. The results presented reveal that recN and ruv mutations do not abolish residual recombination in a recB mutant, and have only a rather modest effect on recombination in recBC sbcA strains; in these respects they are quite different from recF, recJ and recO mutations. The differences between these two groups of genes are discussed in relation to the molecular exchanges needed to produce viable recombinants.     

The recombination genes addAB are not restricted to gram-positive bacteria: genetic analysis of the recombination initiation enzymes RecF and AddAB in Rhizobium etli

Single-strand gaps (SSGs) and double-strand breaks (DSBs) are the major initiation sites for recombination. In bacteria, the SSGs are repaired by RecFOR, while the DSBs are processed by RecBCD in gram-negative bacteria and AddAB in gram-positive bacteria. Unexpectedly, instead of recBCD genes, the addAB genes were found in members of the alpha-proteobacteria group (gram negative). Taking Rhizobium etli as a model, the role of recF and addAB genes in homologous recombination and repair of damaged DNA was evaluated. Inactivation of either recF or addA provoked strong sensitivity to UV radiation and mitomycin C, while an additive effect was observed in the recF-addA mutant. The DSBs generated by nalidixic acid caused low viability only in the addA mutant. The recombination frequency of large and small plasmids was reduced in the recF mutant (24- and 36-fold, respectively), whereas a slight decrease (threefold) in the addA mutant was observed. Moreover, an additive effect (47- and 90-fold, respectively) was observed in the double mutant, but it was not as dramatic as that in a recA mutant. Interestingly, the frequency of deletion and Campbell-type recombination was slightly affected in either single or double mutants. These results suggest that another pathway exists that allows plasmid and Campbell-type recombination in the absence of recF and addA genes.     

Repair of DNA double-strand breaks in Escherichia coli K12 requires a functional recN product

Summary Mutation of the recN gene of Escherichia coli in a recBC sbcB genetic background blocks conjugational recombination and confers increased sensitivity to UV light and mitomycin C. The basis for this phenotype was investigated by monitoring the properties associated with recN mutations in otherwise wild-type strains. It was established that recN single mutants are almost fully resistant to UV irradiation, and that there is no detectable defect in repair of UV lesions by excision, error-prone, or recombinational mechanisms. However, recN mutations confer sensitivity to mitomycin C and ionizing radiation both in wild-type and recB sbcB strains. The sensitivity to ionizing radiation is correlated with a deficiency in the capacity to repair DNA double-strand breaks by a UV inducible mechanism. Recombinant phages that complement the recombination and repair defects of recN recBC sbcB mutants have been identified, and the recN gene has been cloned from these phages into a low copy-number plasmid.     

Genetic Analysis of δheld and δuvrd Mutations in Combination with Other Genes in the Recf Recombination Pathway in Escherichia Coli: Suppression of a Ruvb Mutation by a Uvrd Deletion

Helicase II (uvrD gene product) and helicase IV (helD gene product) have been shown previously to be involved in the RecF pathway of recombination. To better understand the role of these two proteins in homologous recombination in the RecF pathway [recBCsbcB(C) background], we investigated the interactions between helD, uvrD and the following RecF pathway genes: recF, recO, recN and ruvAB. We observed synergistic interactions between uvrD and the recF, recN, recO and recG genes in both conjugational recombination and the repair of methylmethane sulfonate (MMS)-induced DNA damage. No synergistic interactions were detected between helD and the recF, recO and recN genes when conjugational recombination was analyzed. We did, however, detect synergistic interactions between helD and recF/recO in recombinational repair. Suprisingly, the uvrD deletion completely suppressed the phenotype of a ruvB mutation in a recBCsbcB(C) background. Both conjugational recombination efficiency and MMS-damaged DNA repair proficiency returned to wild-type levels in the δuvrDruvB9 double mutant. Suppression of the effects of the ruvB mutation by a uvrD deletion was dependent on the recG and recN genes and not dependent on the recF/O/R genes. These data are discussed in the context of two ``RecF' homologous recombination pathways operating in a recBCsbcB(C) strain background.     

Frequency of recombination exchanges in Escherichia coli K-12: genetic determinants controlling this frequency

RecF, recQ, ruv, recJ and recN genes of so called RecF pathway of recombination appear to be not silent on the RecBCD pathway also. These genes are responsible for the frequency of recombination exchanges per unit length of DNA. The list: recF::Kmr greater than recQ::Tn3 greater than ruv54 greater than recJ::Tn9 demonstrated the efficiency of inhibition of recombination exchanges by these mutations. The recN262 mutation gives a feeble contrary effect. It slightly increases the frequency of recombination exchanges.     

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.     

Overlapping functions of recD, recJ and recN provide evidence of three epistatic groups of genes in Escherichia coli recombination and DNA repair

The recD, recJ and recN genes of Escherichia coli K-12 have been shown to be involved in genetic recombination and DNA repair in this organism. Yet, mutation of any one of these genes does not seem to interfere much with the recovery of recombinants from conjugational crosses. Strains carrying all possible combinations of mutations inactivating these genes were constructed and examined for their recombination proficiency and sensitivity to UV light. The recD recJ and recJ recN double mutants are moderately sensitive to UV light and slightly deficient in recombination. A combination of mutations in all 3 genes produced strains that are very deficient in recombination (50- to 100-fold reduction) and strikingly sensitive to UV light. We conclude that these genes provide overlapping activities that compensate for one another in the single mutants. On the basis of these and other data, recombination genes are classified into 3 epistatic groups that define activities which function pre-synaptically or post-synaptically to promote genetic exchanges catalysed by RecA.     

Involvement of recF, recO, and recR Genes in UV-Radiation Mutagenesis of Escherichia coli

The recF, recO, and recR genes were originally identified as those affecting the RecF pathway of recombination in Escherichia coli cells. Several lines of evidence suggest that the recF, recO, and recR genes function at the same step of recombination and postreplication repair. In this work, we report that null mutations in recF, recO, or recR greatly reduce UV-radiation mutagenesis (UVM) in an assay for reversion from a Trp⁻ (trpE65) to a Trp⁺ phenotypes. Introduction of the defective lexA51 mutation [lexA51(Def)] and/or UmuD′ into recF, recO, and recR mutants failed to restore normal UVM in the mutants. On the other hand, the presence of recA2020, a suppressor mutation for recF, recO, and recR mutations, restored normal UVM in recF, recO, and recR mutants. These results indicate an involvement of the recF, recO, and recR genes and their products in UVM, possibly by affecting the third role of RecA in UVM.     

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.