Genetic Analysis of Recombination-Deficient Mutants of Escherichia coli K-12 Carrying rec Mutations Cotransducible with thyA

The rec mutations carried by 20 strains of Escherichia coli K-12 which are defective in genetic recombination and sensitive to ultraviolet light and X rays, and whose lambda lysogens show spontaneous phage production, have been mapped near thyA. In 15 of the strains, the rec mutation fails to complement recB21 but complements rec-22. The other five strains carry a rec mutation which complements recB21 but not rec-22. These mutations map closer to thyA than those which fail to complement recB21. They therefore appear to be defective in a different recombination gene, denoted recC. The order of recB and recC on the linkage map of E. coli K-12 is thyA-recC-recB-argA.     

Dark Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light II. Effect of uvr Genes on Liquid Holding Recovery

The uvr mutations of Escherichia coli K-12 decrease the ability of cells to survive ultraviolet light (UV), to excise pyrimidine dimers from their deoxyribonucleic acid and to reactivate bacteriophage exposed to UV. The rec mutations decrease the ability of the cells to survive UV and to undergo genetic recombination. Certain rec mutations, including recA1, rec-12, recA13, and rec-56, are necessary for the expression of liquid-holding recovery (LHR), observed as an increase in colony-forming ability when irradiated cells are held in buffer in the dark. These rec mutations appear to act indirectly to permit the detection of LHR rather than to affect its occurrence directly. We have tested the effect of uvr markers on LHR in cells containing one of these rec mutations. Recombinants containing rec-56 together with a uvr marker were constructed and tested for LHR. None of the 39 recombinants examined, carrying uvrA6, uvrB5, or uvrC34, showed LHR. Three rec(-)uvr(-) strains were also tested for photoreactivation. In all three, photoreactivation was observed, indicating that they contained detectable amounts of pyrimidine dimers. Our results are consistent with the idea that uvr mutations inactivate LHR, and suggest that LHR reflects excision-dependent repair of pyrimidine dimers.     

Dark Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light I. Effect of rec− Mutations on Liquid Holding Recovery

We have examined various derivatives of Escherichia coli K-12 for liquid holding recovery, a type of recovery originally observed in E. coli B irradiated with ultraviolet light. Although most of the K-12 derivatives tested showed relatively little or no recovery under our conditions, four of the six independent rec(-) mutants examined, those carrying recA1, rec-12, recA13, and rec-56, respectively, displayed marked recovery. These mutants are distinguished from rec(+) strains by their increased sensitivity to ultraviolet radiation and decreased ability to undergo genetic recombination. Two of them have also been reported to release large amounts of their deoxyribonucleic acid as acid-soluble material, especially after irradiation. None of the three uvr(-) mutants examined, containing uvrA6, uvrB5, or uvrC34, showed comparable liquid holding recovery. The one rec(-) uvr(-) derivative tested, carrying recA13 and uvrA6, did not appear to undergo liquid holding recovery, although recA13 uvr(+) strains did. Genetic analysis of one strain, a recA13 mutant, indicated that all the rec(+) derivatives obtained from it by conjugation, transduction and reversion, had lost the property of showing liquid holding recovery. From these results, we conclude that in E. coli K-12 the expression of liquid holding recovery depends upon certain rec(-) mutations.     

Escherichia coli K-12 mutants in which viability is dependent on recA function.

A gene required for growth and viability in recA mutants of Escherichia coli K-12 was identified. This gene, rdgB (for Rec-dependent growth), mapped near 64 min on the E. coli genetic map. In a strain carrying a temperature-sensitive recA allele, recA200, and an rdgB mutation, DNA synthesis but not protein synthesis ceased after 80 min of incubation at 42 degrees C, and there was extensive DNA degradation. The rdgB mutation alone had no apparent effect on DNA synthesis or growth; however, mutant strains did show enhanced intrachromosomal recombination and induction of the SOS regulon. The rdgB gene was cloned and its-gene product identified through the construction and analysis of deletion and insertion mutations of rdgB-containing plasmids. The ability of a plasmid to complement an rdgB recA mutant was correlated with its ability to produce a 25-kilodalton polypeptide as detected by the maxicell technique.     

Genetic analysis of the RecE pathway of genetic recombination in Escherichia coli K-12.

The RecE pathway of genetic recombination in Escherichia coli K-12 was defined to be the pathway that is utilized in deoxyribonucleic acid exonuclease V (ExoV)-defective cells which express constitutively recE+, the structural gene for deoxyribonucleic acid exonuclease VIII. Dependence on ExoVIII was shown by the occurrence in a recB21 sbcA23 strain of recombination deficiency mutations in recE, the structural gene for ExoVIII. Point mutations in recE were found as well as deletion mutations in which the entire Rac prophage, carrying recE, was lost. In addition, strain construction and mutagenesis revealed the dependence of the RecE pathway on recA+ and on recF+. Dependence on a fourth gene was shown by a mutation (rec-77) which does not map near the other genes. The problem of distinguishing the RecE pathway from that previously called RecF is discussed.     

Defective regulation of the phenylalanine biosynthetic operon in mutants of the phenylalanyl-tRNA synthetase operon.

Among mutants of Escherichia coli resistant to p-fluorophenylalanine (PFP) were some with constitutive expression of the phenylalanine biosynthetic operon (the pheA operon). This operon is repressed in the wild type by phenylalanine. The mutation in three of these mutants mapped in the aroH-aroD region of the E. coli chromosome at 37 min. A plasmid bearing wild-type DNA from this region restored p-fluorophenylalanine sensitivity and wild-type repression of the pheA operon. Analysis of subclones of this plasmid and comparison of its restriction map with published maps indicated that the mutations affecting regulation of the pheA operon lie in the structural genes for phenylalanyl-tRNA synthetase, pheST, probably in pheS. Thus, the pheST operon has a role in the regulation of phenylalanine biosynthesis, the most likely being that wild-type phenylalanyl-tRNA synthetase maintains a sufficient intracellular concentration of Phe-tRNA(Phe) for attenuation of the pheA operon in the presence of phenylalanine. A revised gene order for the 37-min region of the chromosome is reported. Read clockwise, the order is aroD, aroH, pheT, and pheS.     

Cloning and characterization of the Haemophilus influenzae Rd rec-1+ gene.

The Haemophilus influenzae Rd rec-1+ gene was cloned from a partial chromosomal digest into a plasmid vector as a 20-kilobase-pair (kbp) BstEII fragment and then subcloned. The smallest subclone with rec-1+ activity carried a 3.1-kbp EcoRI fragment. The identity of the rec-I gene in these clones was confirmed by transforming an Rd strain carrying a leaky rec-1 mutation (recA4) to resistance to methyl methanesulfonate (MMS) by using whole or digested plasmids. It was demonstrated that the Rec+ phenotype of the MMSr transformants was linked to the strA, novAB, and mmsA loci, as expected if the recA4 allele had been replaced by rec-1+. In growing cultures (rec-1 or rec+), all rec-1+-carrying plasmids induced near-maximal levels of transformability when their hosts reached stationary phase; these levels are 100 to 1,000 times higher than the values seen with strains not carrying a Rec plasmid. Transfer of the 3.1-kbp subclone was greatly reduced compared with transfer of similarly sized vector plasmids, and the resulting transformants grew slowly; this suggests an explanation of my failure to directly clone this fragment from chromosomal DNA digests. Transfer of a rec-1+ plasmid to a very poorly genetically transformable H. influenzae Rb strain resulted in greatly increased transformability. Transfer of such plasmids to a noncompetent H. influenzae Rc strain did not render this strain competent. It is suggested that transformability of Rd and Rb strains is limited by rec-1 expression but that the noncompetence of Rc has some other basis.     

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.     

Recombination-deficient mutants of Bacillus subtilis.

Two mutant strains of Bacillus subtilis Marburg, NIG43 and NIG45, were isolated. They showed high sensitivities to gamma rays, ultraviolet light (UV), and chemicals. Deficiencies in genetic recombination of these two mutants were shown by the experiments on their capacity in transformation. SPO2 transfection, and PBS1 phage transduction, as well as on their radiation and drug sensitivities and their Hcr+ capacity for UV-exposed phage M2. Some of these characteristics were compared with those of the known strains possessing the recA1 or recB2 alleles. Mapping studies revealed that the mutation rec-43 of strain NIG43 lies in the region of chromosome replication origin. The order was purA dna-8132 rec-43. Another mutation, rec-45, of strain NIG45 was found to be tightly linked to recA1. The mutation rec-43 reduced mainly the frequency of PBS1 transduction. On the other hand, the mutation rec-45 reduced the frequency of recombination involved both in transformation and PBS1 transduction. The mutation rec-43 of strain NIG43 is conditional, but rec-45 of strain NIG45 is not. The UV impairment in cellular survival of strain NIG43 was gradually reverted at higher salt or sucrose concentrations, suggesting cellular possession of a mutated gene produce whose function is conditional. In contrast to several other recombination-deficient strains, SPO2 lysogens of strain NIG43 and NIG45 were not inducible, indicating involvement of rec-43+ or rec-45+ gene product in the development of SPO2 prophage to a vegetative form. The UV-induced deoxyribonucleic acid degradation in vegetative cells was higher in rec-43 and rec-45 strains.     

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.     

Identification of a new sporulation locus, spoIIIF, in Bacillus subtilis

We have isolated a mutant of Bacillus subtilis, strain 590, which is blocked at stage III of sporulation. The spo mutation which is carried by this strain is linked to pheA by transformation and defines a previously unidentified locus, spoIIIF. The spoIIIF locus is contiguous with the spoVB locus, in which a mutation causes a block at stage V of sporulation. We also give a detailed genetic map of the pheA region of the chromosome.     

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

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

Isolation and Characterization of an Escherichia coli K-12 Mutant with a Temperature-Sensitive RecA− Phenotype

A mutation that causes a temperature-sensitive RecA(-) phenotype was identified in a derivative of a PolA(-) strain that failed to grow at high temperature. The mutant allele (recA200) was shown to be linked to cysC, conferred a sharply temperature-sensitive, ultraviolet-sensitive Rec(-) phenotype in the range 35 to 42 C, and in crosses failed to show complementation at 42 C with Hfr's that transferred recA(-). Double mutants that carried both recA200 and polA were examined for ability to grow and synthesize DNA at restrictive temperatures.     

Chromosomal Location and Properties of Radiation Sensitivity Mutations in Bacillus subtilis

Transformation and transduction crosses involving recA1, recB2, and urv-1 mutations have shown that these mutations belong to three distinct unlinked genetic loci. The precise position of these loci on the Bacillus subtilis chromosome map has been determined. The behavior of recB2 strains in transformation studies suggested a dominance of recB2(+) function over recB2 and an early expression of this phenotype during transformation. Strains bearing two ultraviolet sensitivity markers possess a phenotype characteristic of the marker with the most adverse effect on recombination. The possibility that the effects of the two mutations are additive was also considered. Results are also presented which show that a phage-induced enzyme is not responsible for the high transducibility of recA1 strains.     

Temperature-sensitive recA mutant of Escherichia coli K-12: deoxyribonucleic acid metabolism after ultraviolet irradiation.

A mutant of Escherichia coli K-12 temperature sensitive for genetic recombination was investigated and found to carry a mutation that could be cotransduced with cysC and hence could be in the recA gene. To determine whether recA+ can complement this mutation, matings were carried out at 35 and 40 C between Hfr donors that transfer recA+ or recA1 early and recipients carrying wild-type or mutant alleles. It was found that recA+ but not recA1 complements this mutation in zygotic temporary partial diploids. The mutant allele was accordingly designated recA44. A transductant carrying recA44 behaved normally at low temperatures but more like recA- strains at high temperatures with respect to recombinant colony formation in Hfr matings, cell survival, and deoxyribonucleic acid (DNA) synthesis after ultraviolet irradiation, cellular DNA breakdown, and prophage induction when lysogenic for lambda. Alkaline sucrose sedimentation studies of DNA from recA44 cells showed that short DNA molecules synthesized immediately after ultraviolet irradiation increased in molecular weight during subsequent incubation at 32 C but not at 45 C. Hence, recA+ is required for this molecular weight increase. Cells exposed to ultraviolet light synthesized DNA that remained of low molecular weight during a 40-min incubation at 32 C. This material increased in molecular weight in recArut not in recA44 cells during subsequent incubation at 45 C. Thus, the availability of recA+ during the first 40 min at 32 C after irradiation did not obviate the need for recA+ in the subsequent phases of this post-replication repair process.     

recA-genes of Erwinia chrysanthemi ENA49

The recA gene of Erwinia chrysanthemi ENA49 has been cloned in vivo in Escherichia coli K12, recA13 cells using the plasmid pULB113. On the basis or DNA repair and recombination deficiencies complementation, of restoration of the inducible "SOS"-response functions the functional identity of the cloned gene with the recA gene was concluded. The recA gene was localized in the 18th min region of the chromosomal genetical map of Erwinia chrysanthemi ENA49 between the genes proA and pheA.     

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.     

Repair of Radiation-Induced Damage in Escherichia coli II. Effect of rec and uvr Mutations on Radiosensitivity, and Repair of X-Ray-Induced Single-Strand Breaks in Deoxyribonucleic Acid

Strains of Escherichia coli K-12 mutant in the genes controlling excision repair (uvr) and genetic recombination (rec) have been studied with reference to their radiosensitivity and their ability to repair X-ray-induced single-strand breaks in deoxyribonucleic acid (DNA). Mutations in the rec genes appreciably increase the radiosensitivity of E. coli K-12, whereas uvr mutations produce little if any increase in radiosensitivity. For a given dose of X-rays, the yield of single-strand breaks has been shown by alkaline sucrose gradient studies to be largely independent of the presence of rec or uvr mutations. The rec(+) cells (including those carrying the uvrB5 mutation) could efficiently rejoin X-ray-induced single-strand breaks in DNA, whereas recA56 mutants could not repair these breaks to any great extent. The recB21 and recC22 mutants showed some indication of repair capacity. From these studies, it is concluded that a correlation exists between the inability to repair single-strand breaks and the radiosensitivity of the rec mutants of E. coli K-12. This suggests that unrepaired single-strand breaks may be lethal lesions in E. coli.