Analysis of cell division in single clones of the Escherichia coli K-12 lexA mutant.

The growth of clones of lexA mutant and lexA+ cells was analyzed. During normal growth lexA mutant clones frequently divided early, producing smaller newborn cells than the lexA+ clones. Some newborn cells in the lexA clones did not elongate or divide at all, a response that was never observed in the lexA+ clones. When starved for thymidine, most of the lexA mutant clones elongated and subsequently divided. The majority of lexA+ clones also elongated but did not divide. The above results suggest that one of the functions of the lexA+ gene is coordination of DNA repair with cell division.     

Distribution of cell lengths in cultures of a lexA mutant of Escherichia coli K-12.

Distributions of cell lengths in lexA+ and lexA mutant cultures during normal growth and under thymidine starvation conditions are presented. During normal growth lexA mutant cells were slightly shorter, on the average, than were lexA+ cells. lexA mutant cells were also shorter in comparison with lexA+ cells after a period of thymidine starvation. These results are consistent with the hypothesis that the lexA gene is involved in the coordination of cell division with DNA repair.     

Isolation of recombinant plasmids and phage carrying the lexA gene of Escherichia coli K-12

The lexA gene of Escherichia coli K-12 was cloned from the plasmid pLC44-14 into pBR322. Plasmids carrying lexA+ were selected by their ability to complement a recessive tsl mutation, which is believed to be a mutation in lexA. The smallest lexA+ recombinant plasmid, pJL21, contained an EcoRI-PstI fragment 2.9 kilobases (kb) in length; two larger plasmids also contained this fragment, and genetic material to one or both sides of the EcoRI-PstI fragment. Plasmids homologous to pJL21, but carrying a dominant mutation, lexA3, or one of three recessive amber mutations in lexA, termed spr, were also isolated. To clone the EcoRI-PstI fragment onto a lambda vector, the PstI end was first converted to an EcoRI end by attachment of a 100-base pair PstI-EcoRI fragment isolated from the plasmid ColE1; the resultant EcoRI fragment was then cloned into the lambda vector lambda gt4. A restriction map of pLC44-14 was obtained for nine restriction enzymes. The orientation of this map was determined relative to the E. coli genetic map by complementation of the gene ubiA+ and by comparison with restriction enzyme digests of another plasmid, pLC11-9, which carries dnaB, a gene closely linked to lexA, but does not carry lexA.     

New role for photoreversible pyrimidine dimers in induction of prototrophic mutations in excision-deficient Escherichia coli by UV light.

UV mutagenesis to His+ in certain recA441 lexA51 bacteria was not photoreversible, indicating that pyrimidine dimers are not target lesions. Photoreversibility was observed in recA+ lexA51 bacteria, showing that pyrimidine dimers are needed to activate the recA+ protein (unlike the recA441 protein) to perform a function in UV mutagenesis distinct from cleavage of the lexA repressor.     

Production of cells without deoxyribonucleic acid during thymidine starvation of lexA- cultures of Escherichia coli K-12.

When thymidine-requiring lexA- strains were starved for thymidine, the kinetics of survival were similar to those of a nearly isogenic lexA+ strain. The size distribution of cells in the lexA- and lexA+ cultures were, however, quite different. Whereas most of the cells in the starved lexA+ cultures grew into long filamentous forms (longer than 4.0 mum), many of the lexA- cells were found to have a normal rod shape (4.0 mum or shorter). It was shown that lexA- cells undergo more divisions during thymidine starvation than lexA+ cells. Furthermore, using an autoradiographic method to analyze deoxyribonucleic acid (DNA) distribution in the starved cells, we demonstrated that cells without DNA are produced in both normal and starved lexA- cultures at a much higher frequency than in lexA+ cultures. Some of these cells may be produced by breakdown of DNA, but we favor the hypothesis that they result from an abnormal cell division process. Since lexA mutations are dominant, we conclude that a diffusible product decreases the synthesis or activity of an inhibitor of cell division in lexA- strains when DNA synthesis is blocked by thymidine starvation.     

Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities.

Salmonella typhimurium has a SOS regulon which resembles that of Escherichia coli. recA mutants of S. typhimurium have already been isolated, but no mutations in lexA have been described yet. In this work, two different lexA mutants of S. typhimurium LT2 have been constructed on a sulA background to prevent cell death and further characterized. The lexA552 and lexA11 alleles contain an insertion of the kanamycin resistance fragment into the carboxy- and amino-terminal regions of the lexA gene, respectively. SOS induction assays indicated that both lexA mutants exhibited a LexA(Def) phenotype, although SOS genes were apparently more derepressed in the lexA11 mutant than in the lexA552 mutant. Like lexA(Def) of E. coli, both lexA mutations only moderately increased the UV survival of S. typhimurium, and the lexA552 strain was as mutable as the lexA+ strain by UV in the presence of plasmids encoding MucAB or E. coli UmuDC (UmuDCEc). In contrast, a lexA11 strain carrying any of these plasmids was nonmutable by UV. This unexpected behavior was abolished when the lexA11 mutation was complemented in trans by the lexA gene of S. typhimurium. The results of UV mutagenesis correlated well with those of survival to UV irradiation, indicating that MucAB and UmuDCEc proteins participate in the error-prone repair of UV damage in lexA552 but not in lexA11. These intriguing differences between the mutagenic responses of lexA552 and lexA11 mutants to UV irradiation are discussed, taking into account the different degrees to which the SOS response is derepressed in these mutants.     

Constitutive expression of the SOS response in recA718 mutants of Escherichia coli requires amplification of RecA718 protein.

In recA718 lexA+ strains of Escherichia coli, induction of the SOS response requires DNA damage. This implies that RecA718 protein, like RecA+ protein, must be converted, by a process initiated by the damage, to an activated form (RecA) to promote cleavage of LexA, the cellular repressor of SOS genes. However, when LexA repressor activity was abolished by a lexA-defective mutation [lexA(Def)], strains carrying the recA718 gene (but not recA+) showed strong SOS mutator activity and were able to undergo stable DNA replication in the absence of DNA damage (two SOS functions known to require RecA activity even when cleavage of LexA is not necessary). lambda lysogens of recA718 lexA(Def) strains exhibited mass induction of prophage, indicative of constitutive ability to cleave lambda repressor. When the cloned recA718 allele was present in a lexA+ strain on a plasmid, SOS mutator activity and beta-galactosidase synthesis under LexA control were expressed in proportion to the plasmid copy number. We conclude that RecA718 is capable of becoming activated without DNA damage for cleavage of LexA and lambda repressor, but only if it is amplified above its base-line level in lexA+ strains. At amplified levels, RecA718 was also constitutively activated for its roles in SOS mutagenesis and stable DNA replication. The nucleotide sequence of recA718 reveals two base substitutions relative to the recA+ sequence. We propose that the first allows the protein to become activated constitutively, whereas the second partially suppresses this capability.     

Effect of tsl (thermosensitive suppressor of lex) mutation on postreplication repair in Escherichia coli K-12.

Cells of Escherichia coli K-12 carrying lexA or recA mutations are more sensitive to UV radiation than corresponding wild-type cells and are defective in postreplication repair. Supressor mutations (tsl) have been described previously which increase the UV resistance of lexA uvr+, lexA uvrA, and recAI uvr+ strains, but not the resistance of recA1 uvrA strains. We have studied the effect of the tsl-1 mutation on postreplication repair and find that the enhanced survival conferred by this mutation is correlated with an increased capacity for postreplication repair.     

On the nature of the RecBC and RecF pathways of conjugal recombination in Escherichia coli

The molecular mechanisms of the RecBC and RecF pathways for genetic recombination in E. coli were investigated by studying the kinetics of RecA protein function during conjugation. RecF recombination in recBC sbcB mutants is shown to be a much slower process than RecBC recombination in recBC+ sbcB+ strains, and is blocked by a mutation in lexA that prevents induction of RecA protein. Progress of the RecF pathway is greatly accelerated by a recAoc mutation which increases synthesis of RecA protein, but this does not restore recombination proficiency to a recBC sbcB lexA mutant. These results are interpreted to suggest that the RecF pathway directs integration of single-stranded Hfr DNA into the recipient chromosome whereas the RecBC pathway catalyses the exchange of largely double stranded DNA. This is consistent with the known stoichiometry of RecA protein catalysed heteroduplex DNA formation in vitro and with the delayed replication of RecF pathway recombinants which approximates to the time required for one round of DNA replication to generate homoduplex DNA. The regulation of the RecF pathway by lexA repressor is discussed in relation to the factors that govern the relative utilization of the two recombination pathways in wild-type cells.     

Effect of host lex, recA, recF, and uvrD genotypes on the ultraviolet light-protecting and related properties of plasmid R46 in Escherichia coli.

The ability of plasmid R46 to reduce the lethal but enhance the mutagenic effect of ultraviolet (UV) irradiation was tested in sets of Escherichia coli K-12 derivatives, wild type or with different mutations affecting DNA repair capacity, but otherwise isogenic. UV protection and enhancement of UV mutagenic effect were obtained in uvrA6, uvrB5, uvrD3, and recF143 hosts, but not in a recA56 strain. The plasmid gave some UV protection in two lexA1 and two lexA101 strains and in one lexA102 host, but produced no such effect in another lexA102 host. The plasmid restored UV mutagenic effect in a lexB30 strain, the yield of induced mutants per survivor of irradiation (10 J/m2) being about the same for the lexB30(R46) and lex+(R46) strains; by contrast the plasmid, though it reduced the UV sensitivity of the lexB30 strain, did not make it as UV-resistant as the lex+ R-strain.     

Isolation and characterization of amber mutations in the lexA gene of Escherichia coli K-12.

We describe the isolation and characterization of amber mutations in the lexA gene of Escherichia coli K-12. These mutations, designated spr(Am), were isolated and characterized in a lexA tif sfi genetic background. They abolished the sensitivity of the strain to UV light and resulted in high rates of synthesis of recA protein. Phage lambda+ failed to lysogenize the strains as observed with similar strains carrying non-amber spr mutations described previously, thereby indicating a constitutive expression of the phage induction pathway. Introduction of an amber suppressor mutation into a strain bearing the spr(Am) mutation restored expression of the LexA mutant phenotype. We conclude that spr mutations either inactivate or prevent synthesis of the lexA gene product and that loss of this product results in constitutive expression of the E. coli induction system in the tif sfi genetic background.     

Action of plasmid ColIb-P9 on the survival after ultraviolet irradiation and on the mutagenesis of the imiC, uvm, recL, uvrE and tif1 sfiA lexA spr mutants of Escherichia coli K-12 cells

To clarify the mechanisms whereby the ColIb-P9 plasmid affects DNA repair processes, its effect was studied in mutant Escherichia coli K-12 cells with altered mutagenesis and DNA repair. The plasmid was shown to protect umuC, uvm, recL and uvrE mutants after UV irradiation. The frequency of UV-induced his+ revertants increased in the presence of the plasmid in umuC, uvm and recL mutant cells. The ColIb-P9 plasmid completely restored the UV mutability and survival of umuC mutants. These results suggest that the ColIb-P9 plasmid may encode a product similar to that of the umuC gene. In the tif1 sfiA lexA spr mutant cells where SOS functions are constitutively expressed, the ColIb-P9 plasmid increased the number of his+ revertants several times. This suggests that the action of ColIb-P9 is probably brought about not via the derepression of the recA gene but at the subsequent stages of the recA+lexA+-dependent DNA error-prone repair.     

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.     

Influence of a uvrD mutation on survival and repair of X-irradiated Escherichia coli K-12 cells.

The presence of a uvrD mutation increased the X-ray sensitivities of E. coli wild-type and polA strains, but had no effect on the sensitivities of recA and recB strains, and little effect on a lexA strain. Incubation of irradiated cells in medium containing 2,4-dinitrophenol or chloramphenicol decreased the survival of wild-type and uvrD cells, but had no effect on the survival of recA, recB and lexA strains. Alkaline sucrose gradient sedimentation studies indicated that the uvrD strain is deficient in the growth-medium-dependent (Type III) repair of DNA single-strand breaks. These results indicate that the uvrD mutation inhibits certain rec+lex+-dependent repair processes, including the growth-medium-dependent (Type III) repair of X-ray-induced DNA single-strand breaks, but does not inhibit other rec+lex+-dependent processes that are sensitive to 2,4-dinitrophenol and chloramphenicol.