Genetic characterization of the inducible SOS-like system of Bacillus subtilis.

The SOS-like system of Bacillus subtilis consists of several coordinately induced phenomena (e.g., cellular filamentation, prophage induction, and Weigle reactivation of UV-damaged bacteriophage) which are expressed after cellular insult such as DNA damage or inhibition of DNA replication. Mutagenesis of the bacterial chromosome and the development or maintenance of competence also appear to be involved in the SOS-like response in this bacterium. The genetic characterization of the SOS-like system has involved an analysis of (i) the effects of various DNA repair mutations on the expression of inducible phenomena and (ii) the tsi-23 mutation, which renders host strains thermally inducible for each of the SOS-like functions. Bacterial filamentation was unaffected by any of the DNA repair mutations studied. In contrast, the induction of prophage after thermal or UV pretreatment was abolished in strains carrying the recE4, recA1, recB2, or recG13 mutation. The Weigle reactivation of UV-damaged bacteriophage was also inhibited by the recE4, recA1, recB2, or recG13 mutation, whereas levels of Weigle reactivation were lower in strains which carried the uvrA42, polA5, or rec-961 mutation than in the DNA repair-proficient strain. Strains which carried the recE4 mutation were incapable of chromosomal DNA-mediated transformation, and the frequency of this event was decreased in strains carrying the recA1, recB2, or tsi-23 mutation. Plasmid DNA transformation efficiency was decreased only in strains carrying the tsi-23 mutation in addition to the recE4, recA1, or recB2 mutation. The results indicate that the SOS-like system of B. subtilis is regulated at different levels by two or more gene products. In this report, the current data regarding the genetic regulation of inducible phenomena are summarized, and a model is proposed to explain the mechanism of SOS-like induction in B. subtilis.     

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.     

Suppressibility of recA, recB, and recC mutations by nonsense suppressors.

Mutations in the recA, recB, and recC genes of Escherichia coli K-12 were surveyed to ascertain whether or not they are suppressed by nonsense suppressors. Several mutations which map in or near the recA gene, but have not been called recA mutations, were also surveyed. An amber recB mutation, recB156, and an amber recC mutation, recC155, were isolated. One recB mutation, recB95, and four recC mutations, recC22, recC38, recC82, and recC83, were found to be suppressed by a UGA suppressor. In addition to the previously isolated amber recA mutation recA99, two other recA mutations, recA52 and recA123, were found to be suppressed by amber suppressor supD32 but not by supE44.     

Genetic control of multiple pathways of post-replicational repair in uvrB strains of Escherichia coli K-12.

The effect of the recA, uvrD, exrA, and recB mutations and of post-irradiation treatment with chloramphenicol on the survival and post-replication repair after ultraviolet irradiation of uvrB strains of Escherichia coli K-12 was examined. Each of these mutations or treatments was found to decrease survival and the extent of repair. The interactions of the inhibitory effects of the uvrD, exaA, and recB mutations and chloramphenicol treatment were determined by examining the survival and repair characteristics of the several multiple mutants. The survival results suggest that the post-replication repair process in uvrB strains may be subdivided into at least five different branches. These include three branches that are blocked by the exrA, recB, or uvrD mutation, a fourth branch that is blocked by any of these mutations and is also sensitive to chloramphenicol treatment, and at least one additional branch that is not sensitive to either of these mutations or to chloramphenicol treatment. The extent of post-replicational repair observed with each of the strains is in general agreement with the pathways postulated on the basis of the survival data, although there are several apparent exceptions to this correlation.     

UV-mutagenesis in Bacillus subtilis. VII. Mutability of recA, recB and recF strains]

UV-light induces reversions to threonine-independence in BD170 Rec+ cells, and does not induce them in isogenic strain BD241 recF. Reversions to methionine-independence are induced in cells GSY1028 recB, and are not in isogenic strain GSY1025 recA. It is possible to construct, with the use of transformation, a viable strain carrying mutations recF and su, the latter imparts threinine-independence to cells. Hence, the absence of UV-induced reversions in recF (and probably in recA) cells can not be explained by the lethal effect of joining, in one genome, two mutations each of which decreases a viability of bacteria. Formation of UV-induced forward mutations which provide additional growth requirements to bacteria is not disturbed in strains recA and recF. Experimental data allow to conclude that UV-induced mutagenesis is not a function of any of two known mechanisms of recombination which function in Bacillus subtilis cells.     

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.     

W-reactivation and W-mutagenesis in UV-irradiated phage phil05 of Bacillus subtilis]

The survival of UV-irradiated phage ?105 on the lawns of several strains of Bacillus subtilis: wild type (strain 168) and 11 recombination-defficient mutants (recA1, recB2, recB3, recB19, recD27, recF15, recF18, recK4, recM13, recL16 and recO61) was investigated. All rec mutants have the phenotype Hcr+, i.e. normally host-cell reactivate UV-damaged phage. Small doses of UV-irradiation given to the wild type (rec+) cells increase the probability of survival of UV-irradiated ?105 phage (W-reactivation) and significantly enhance the frequency of c-mutants (W-mutagenesis). Maximal frequency of clear mutations in conditions of W-mutagenesis is 3-10(-3), i.e. is 100 times higher than the spontaneous background. Various rec mutations of host cells only diminish the level of W-reactivation but do not eliminate it completely. The most deficient in W-reactivation is recD27 mutant. Mutations recB2, B3, B19 and O61 have no effect on W-mutagenesis of UV-irradiated phage ?105 and on UV-induction of ?105, F15,F18 and L16 mutants. UV-irradiation of lysogenic cells of these mutants does not induce ?105 prophage.     

SOS-like induction in Bacillus subtilis: induction of the RecA protein analog and a damage-inducible operon by DNA damage in Rec+ and DNA repair-deficient strains.

We quantitated the induction of the Bacillus subtilis Rec protein (the analog of Escherichia coli RecA protein) and the B. subtilis din-22 operon (representative of a set of DNA damage-inducible operons in B. subtilis) following DNA damage in Rec+ and DNA repair-deficient strains. After exposure to mitomycin C or UV irradiation, each of four distinct rec (recA1, recB2, recE4, and recM13) mutations reduced to the same extent the rates of both Rec protein induction (determined by densitometric scanning of immunoblot transfers) and din-22 operon induction (determined by assaying beta-galactosidase activity in din-22::Tn917-lacZ fusion strains). The induction deficiencies in recA1 and recE4 strains were partially complemented by the E. coli RecA protein, which was expressed on a plasmid in B. subtilis; the E. coli RecA protein had no effect on either induction event in Rec+, recB2, or recM13 strains. These results suggest that (i) the expression of both the B. subtilis Rec protein and the din-22 operon share a common regulatory component, (ii) the recA1 and recE4 mutations affect the regulation and/or activity of the B. subtilis Rec protein, and (iii) an SOS regulatory system like the E. coli system is highly conserved in B. subtilis. We also showed that the basal level of B. subtilis Rec protein is about 4,500 molecules per cell and that maximum induction by DNA damage causes an approximately fivefold increase in the rate of Rec protein accumulation.     

Effect of recB- and recA-mutations on phage restriction in various modification-restriction plasmid systems of E. coli

The effect of recB and recA mutations on lambda vir and P1 vir restriction by different restriction-modification plasmid systems of E. coli was studied. It was shown that effect of R1 plasmid coded restriction-modification in E. coli K12 and E. coli B strains and pJA4620 plasmid coded restriction in E. coli K12 is observed only in RecB+ strain. Phenomenon of restriction-modification determined by R124, R245 plasmids does not depend of recB mutation. Effect of recA mutation has not been found in cultures harbouring R1, R245, R124 pJA4620 plasmids.     

Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli.

We isolated mutations that reduce plasmid stability in dividing cell populations and mapped these mutations to a previously undescribed gene, recD, that affects recombination frequency and consequently the formation of plasmid concatemers. Insertions of the transposable element Tn10 into recD resulted in increased concatemerization and loss of pSC101 and ColE1-like replicons during nonselective growth. Both concatemer formation and plasmid instability in recD mutants require a functional recA gene. Mutations in recD are recessive to recD+ and map to a small region of the Escherichia coli chromosome located between recB and argA. Although the recD locus is distinct from loci encoding the two previously identified subunits of the RecBC enzyme, mutations in recD appear to affect the exonuclease activity of this enzyme.     

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.     

Suppression of the UV-sensitive phenotype of Escherichia coli recF mutants by recA(Srf) and recA(Tif) mutations requires recJ+.

Mutations in recA, such as recA801(Srf) (suppressor of RecF) or recA441(Tif) (temperature-induced filamentation) partially suppress the deficiency in postreplication repair of UV damage conferred by recF mutations. We observed that spontaneous recA(Srf) mutants accumulated in cultures of recB recC sbcB sulA::Mu dX(Ap lac) lexA51 recF cells because they grew faster than the parental strain. We show that in a uvrA recB+ recC+ genetic background there are two prerequisites for the suppression by recA(Srf) of the UV-sensitive phenotype of recF mutants. (i) The recA(Srf) protein must be provided in increased amounts either by SOS derepression or by a recA operator-constitutive mutation in a lexA(Ind) (no induction of SOS functions) genetic background. (ii) The gene recJ, which has been shown previously to be involved in the recF pathway of recombination and repair, must be functional. The level of expression of recJ in a lexA(Ind) strain suffices for full suppression. Suppression by recA441 at 30 degrees C also depends on recJ+. The hampered induction by UV of the SOS gene uvrA seen in a recF mutant was improved by a recA(Srf) mutation. This improvement did not require recJ+. We suggest that recA(Srf) and recA(Tif) mutant proteins can operate in postreplication repair independent of recF by using the recJ+ function.     

Suppressors of Recb Mutations in Salmonella Typhimurium

Using a screen that directly assesses transductional proficiency, we have isolated suppressors of recB mutations in Salmonella typhimurium. The alleles of sbcB reported here are phenotypically distinct from those isolated in Escherichia coli in that they restore recombination proficiency (Rec(+)), resistance to ultraviolet light (UV(R)), and mitomycin C resistance (MC(R)) in the absence of an accompanying sbcCD mutation. In addition the sbcB alleles reported here are co-dominant to sbcB(+). We have also isolated insertion and deletion mutants of the sbcB locus. These null mutations suppress only the UV(S) phenotype of recB mutants. We have also isolated sbcCD mutations, which map near proC. These sbcCD mutations increase the viability, recombination proficiency and MC(R) of both the transductional recombination suppressors (sbcB1 & sbcB6) and the sbcB null mutations. S. typhimurium recB sbcB1 sbcCD8 strains are 15-fold more recombination proficient than wild-type strains. The increase in transductants in these strains is accompanied by a loss of abortive transductants suggesting that these fragments are accessible to the mutant recombination apparatus. Using tandem duplications, we have constructed sbcB merodiploids and found that, in a recB mutant sbcCD(+) genetic background, the sbcB(+) allele is dominant to sbcB1 for transductional recombination but co-dominant for UV(R) and MC(R). However, in a recB sbcCD8 genetic background, the sbcB1 mutation is co-dominant to sbcB(+) for all phenotypes. Our results lead us to suggest that the SbcB and SbcCD proteins have roles in RecBCD-dependent recombination.     

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.     

Escherichia coli K-12 mutants with enhanced resistance to ionizing radiation. III. The effect of rec and lexA mutations on radioresistance

Lethal action of gamma-rays on derivatives of the wild-type strain AB1157 and of two radiation-resistant mutants (Gamr444 and Gamr445) containing additional mutations dnaA46, recB21, recF143, recA56, recA430, lexA3, lexA102 or lexA3 recAo98, was studied. When the mean number of genomes per cell was reduced by means of pre-incubation at 43 degrees C, radioresistance of the strains AB1157 dnaA46 and Gamr445 dnaA46 was not changed, and that of the strain Gamr444 dnaA46 was reduced to the level of the Gamr445 dnaA46 strain. Introduction of additional mutations recB21, recA56 or lexA3 (lexA102) into the genome of the strains Gamr444 or Gamr445 made them as radiosensitive as the corresponding variants of AB1157. Additional mutations recF143 or recA430 (lexB30) significantly decreased the radioresistance of Gamr444 and Gamr445 mutants, although did not level them to corresponding derivatives of AB1157. Operator-constitutive mutation recAo98 enhanced radioresistance of all lexA3 derivatives tested but not to the level of the corresponding lexA+ strains. The role of recombinational repair and the inducible SOS system in enhanced radioresistance of Gamr mutants is discussed. The data of post-irradiation DNA degradation in various derivatives of the strains AB1157 and Gamr suggest that Gamr mutants have a constitutive inhibitor of degradation which does coincide with RecA protein.     

Effect of the<Emphasis Type="Italic"> recU</Emphasis> suppressors<Emphasis Type="Italic"> sms</Emphasis> and<Emphasis Type="Italic"> subA</Emphasis> on DNA repair and homologous recombination in<Emphasis Type="Italic"> Bacillus subtilis</Emphasis>

In Bacillus subtilis, mutant alleles of the genes sms and subA partially suppress the recombination phenotype of recU cells. When present in an otherwise Rec(+) strain, Delta sms and Delta subA alleles render cells slightly sensitive to DNA-damaging agents, and impair chromosomal transformation (3- to 10-fold reduction), but do not affect plasmid transformation (less than 1.5-fold reduction). The Delta sms and Delta subA alleles were introduced into rec-deficient strains representative of the epistatic groups alpha (recF strain), beta (addA addB), gamma (recH), epsilon (recB, Delta recU and recD strains) and zeta (Delta recS). Both the Delta sms and Delta subA mutations were found to increase sensitivity to DNA-damaging agents in recF, Delta recS and addAB cells. In contrast, the Delta sms mutations decreased the sensitivity of recB, Delta recU, recD and recH cells, and the Delta subA mutation decreased the sensitivity of recB and Delta recU cells to DNA-damaging agents. Functions classified within the epistatic groups alpha, epsilon and zeta are required for intramolecular recombination, measured as plasmid transformation. The Delta sms and Delta subA mutations, which partially suppressed the recombinational repair phenotype of mutants for functions within epistatic group epsilon, enhanced plasmid transformation of recU (recB, recD) and recS cells by 10- to 20-fold. In the absence of the proteins Sms and SubA, the recombination machinery is apparently redirected towards (an) alternative pathway(s). Furthermore, the shared ability of the Delta sms and Delta subA mutations to act as indirect suppressors of recB, recU and recD mutations supports the classification of the recBUD genes within epistatic group epsilon.     

Suppression of Escherichia coli recF mutations by recA-linked srfA mutations.

Suppressors of recF (srfA) were found by selection for resistance to mitomycin C and UV irradiation in a recB21 recC22 sbcB15 recF143 strain. srfA mutations map in recA and are dominant to srfA+. They suppress both the DNA repair and the recombination deficiencies due to recF mutations. Therefore, RecA protein which is altered by the srfA mutation can allow genetic recombination to proceed in the absence of recB, recC, and recF functions. recF is also required for induction of the SOS response after UV damage. We propose that recF+ normally functions to allow the expression of two recA activities, one that is required for the RecF pathway of recombination and another that is required for SOS induction. The two RecA activities are different and are separable by mutation since srfA mutations permit recombination to proceed but have not caused a dramatic increase in SOS induction in recF mutants. According to this hypothesis, one role for recF in DNA repair and recombination is to modulate RecA activities to allow RecA to participate in these recF-dependent processes.     

Separate Branches of the uvr Gene-Dependent Excision Repair Process in Ultraviolet-Irradiated Escherichia coli K-12 Cells; Their Dependence upon Growth Medium and the polA, recA, recB, and exrA Genes

The extent of repair of single-strand breaks (incision breaks) induced in the deoxyribonucleic acid (DNA) of Escherichia coli K-12 cells by the uvr gene-dependent excision repair process after ultraviolet (UV) radiation was determined in the wild-type, polA1, recA56, recB21, and exrA strains. The wild-type strain repaired all incision breaks after incident doses of UV radiation (254 nm) of approximately 60 J m(-2) or less when incubated in growth medium, or approximately 15 J m(-2) or less when incubated in buffer. The polA1 strain repaired the incision breaks completely after incident doses of approximately 12 J m(-2) or less when incubated in growth medium, or after approximately 4 J m(-2) when incubated in buffer. The recA13, recB21, and exrA strains showed essentially complete repair after incident doses of 10 to 15 J m(-2) whether the cells were incubated in buffer or growth medium. These results suggest that the uvr gene-dependent excision repair process may be divided into two branches, one which is dependent on the presence of growth medium and also the rec(+)exr(+) genotype, and a second which can occur in buffer (growth medium-independent) and is largely dependent on DNA polymerase I. The presence of chloramphenicol in the growth medium resulted in an inhibition of the growth medium-dependent repair occurring in wild-type and polA1 cells and had little or no effect on the extent of repair observed in recA56, recB21, or exrA cells. The similarities between the growth medium-dependent and -independent branches of excision repair and two known processes for the repair of X-ray-induced single-strand breaks are discussed.     

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.