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.     

Mutation of recF, recJ, recO, recQ, or recR improves Hfr recombination in resolvase-deficient ruv recG strains of Escherichia coli.

The formation of recombinants in Hfr crosses was studied in Escherichia coli strains carrying combinations of genes known to affect recombination and DNA repair. Mutations in ruv and recG eliminate activities that have been shown to process Holliday junction intermediates by nuclease cleavage and/or branch migration. Strains carrying null mutations in both ruv and recG produce few recombinants in Hfr crosses and are extremely sensitive to UV light. The introduction of additional mutations in recF, recJ, recO, recQ, or recR is shown to increase the yield of recombinants by 6- to 20-fold via a mechanism that depends on recBC. The products of these genes have been linked with the initiation of recombination. We propose that mutation of recF, recJ, recO, recQ, or recR redirects recombination to events initiated by the RecBCD enzyme. The strains constructed were also tested for sensitivity to UV light. Addition of recF, recJ, recN, recO, recQ, or recR mutations had no effect on the survival of ruv recG strains. The implications of these findings are discussed in relation to molecular models for recombination and DNA repair that invoke different roles for the branch migration activities of the RuvAB and RecG proteins.     

Role of the recF gene of Escherichia coli K-12 in lambda recombination

Summary When Escherichia coli K12() lysogens are infected with heteroimmune phage, which are unable to replicate, general recombination between phage and prophage depends on the bacterial recF gene. It has been shown that in E. coli K12 postconjugational recombination, the RecF pathway only works with full efficiency if exonuclease I is absent (Clark 1973). However, results presented in this paper indicate that under conditions in which replication is blocked, the recombination pathway dependant on the recF gene is fully active in producing viral recombinants even, if the phage is Red⁺, in the presence of exonuclease I. In contrast, removal of exonuclease and protein requires elimination of exonuclease I for an efficient RecF pathway. It is concluded that the Red system cooperates with the RecF pathway and that this cooperation involves overcoming the inhibitory effects of exonuclease I. In the absence of exonuclease, protein stimulates recF-dependent recombination but does not suffice to prevent the negative effect of exonuclease I. In the presence of protein, full efficiency of the RecF pathway can be obtained either via cooperation with exonuclease I or, if the viral exonuclease is defective, via inactivation of exonuclease I. Since activity of exonuclease appears necessary to overcome the inhibitory effects of exonuclease I, it is proposed here that exonuclease diverts material from the RecF pathway in a shunt reaction which allows completion of recF-initiated recombinational intermediates via a mechanism insensitive to exonuclease I.When replication is allowed, the Rec system produces viral recombinants mainly via a recF-independent mechanism. However, a major contribution of the RecF pathway to recombination is observed after removal of the Red system and exonuclease I.Obra social de la Caja de Ahorros de Valencia (Director: S. Grisolía)     

Effect of recF, recJ, recN, recO and ruv mutations on ultraviolet survival and genetic recombination in a recD strain of Escherichia coli K12

Summary DNA repair and recombination were investigated in a recD mutant of Escherichia coli which lacked the nuclease activity of the RecBCD enzyme. The resistance of this mutant to ultraviolet (UV) light was shown to be a function of recJ. A recD recJ double mutant was found to be more sensitive to UV radiation than a recB mutant, whereas recD and recJ single mutants were resistant. Recombination in conjugational crosses with Hfr donors was also reduced in recD recJ strains, but the effect was modest in comparison with the sensitivity to UV. Within certain limits, mutations in recF, recN, recO, lexA and ruv did not affect sensitivity to UV and recombination in a recD mutant any more than in a recD ⁺ strain. The possibility that recD and recJ provide overlapping activities, either of which can promote DNA repair and recombination in the absence of the other, is discussed.     

W-reactivation of phage lambda in recF, recL, uvrA, and uvrB mutants of E. coli K-12.

Summary Assay conditions are described which permit detection of cryptic temperature sensitive RNA polymerases in vitro. RNA polymerase was prepared from fifteen different temperature sensitive mutants of Salmonella typhimurium chosen at random from a larger group isolated by localized mutagenesis and uridine suicide techniques. The dependence of enzyme activity on temperature, ionic strength and pH was studied in vitro. Assays at higher ionic strength (0.23 M) and temperature (50°C) distinguish three classes of mutants (Table 2). Activity of seven mutant RNA polymerases (called Class 1) under these conditions was 1% to 5% that of the parental RNA polymerase. Five mutant RNA polymerases (called Class 2) had 18% to 64% of the parental activity and three were not distinguishable from the parental enzyme under these conditions. Mixing experiments showed that the defect in Class 1 mutant enzymes is a property of the enzymes and not due to a diffusible inhibitor. In one case the lesion was shown to reside in the core enzyme. Class 1 mutant RNA polymerases were shown to be irreversibly inactivated during the assay at higher temperature and ionic strength. This suggests that the Class 1 enzymes may be more thermolabile than the wild type enzyme or may fail to be protected from thermal denaturation by formation of a ternary complex with template and product. We conclude that the method used to isolate these mutants (Young et al., 1976) and the assay described here (Table 2) are efficient ways to isolate and detect temperature sensitive RNA polymerase mutants of Salmonella typhimurium.     

Biochemical evidence that Escherichia coli hyi (orf b0508, gip) gene encodes hydroxypyruvate isomerase

We found a significant activity of hydroxypyruvate isomerase in Escherichia coli clone cells harboring an E. coli gene (called orf b0508 or gip), which is located downstream of the glyoxylate carboligase gene. We newly designated the gene hyi. The enzyme was purified from cell extracts of the E. coli clone. The enzyme had a molecular mass of 58 kDa and was composed of two identical subunits. The optimum pH for the isomerization of hydroxypyruvate was 6.8-7.2. The enzyme required no cofactor. It exclusively catalyzed the isomerization between hydroxypyruvate and tartronate semialdehyde. The apparent K(m) value for hydroxypyruvate was 12.5 mM. The amino acid sequence of E. coli hydroxypyruvate isomerase is highly similar to those of glyoxylate-induced proteins, Gip, found widely from prokaryotes to eukaryotes.     

Plaque assay for lambda transducing phage carrying the E. coli metB gene

A halo plaque assay has been developed for the detection of nondefective lambda transducing phage carrying functional alleles of the metB gene of Escherichia coli K12. The assay is based upon the production of phage plaques on lawns of metB- bacterial cells which are supplemented with limiting amounts of methionine and upon the subsequent transduction of methionine-starved cells in the lawn surrounding the plaques. The resulting prototrophic transductants give rise to a halo of bacterial growth surrounding the plaque. A precise genotype can be ascribed to the characteristic morphologies of selected haloes. This technique has general application for all biosynthetic markers.     

The cII-independent expression of the phage lambda int gene in RNase III-defective E. coli

This study compares the rates of lambda protein synthesis after infection of rnc- cells, which are defective in ribonuclease III (RNase III), with the analogous rates in an isogenic rnc+ host. Temporal differences in gene expression are reflected in a delay in turn-off of lambda early proteins as well as in the delayed appearance of late phage functions in rnc- host cells. Moreover, in the two hosts there is a striking difference in the regulation of gene int expression, which in wild-type cells requires the product of the lambda cII (and cIII )genes, whereas Int synthesis occurs in the absence of cII in RNase III-defective cells. These results suggest that RNase III may be a negative regulator of Int synthesis. The expression of int is also shown to be cII- and cIII-independent in rnc+ cells infected with b2-deleted phages, thus confirming previous studies on the negative regulation of int by the b2-region. Possible mechanisms of these two inhibitory effects on int expression are considered and the significance of int regulation in the control of site-specific recombination is discussed.     

The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity.

The murI gene of Escherichia coli was recently identified on the basis of its ability to complement the only mutant requiring D-glutamic acid for growth that had been described to date: strain WM335 of E. coli B/r (P. Doublet, J. van Heijenoort, and D. Mengin-Lecreulx, J. Bacteriol. 174:5772-5779, 1992). We report experiments of insertional mutagenesis of the murI gene which demonstrate that this gene is essential for the biosynthesis of D-glutamic acid, one of the specific components of cell wall peptidoglycan. A special strategy was used for the construction of strains with a disrupted copy of murI, because of a limited capability of E. coli strains grown in rich medium to internalize D-glutamic acid. The murI gene product was overproduced and identified as a glutamate racemase activity. UDP-N-acetylmuramoyl-L-alanine (UDP-MurNAc-L-Ala), which is the nucleotide substrate of the D-glutamic-acid-adding enzyme (the murD gene product) catalyzing the subsequent step in the pathway for peptidoglycan synthesis, appears to be an effector of the racemase activity.     

A new methionine locus, metR, that encodes a trans-acting protein required for activation of metE and metH in Escherichia coli and Salmonella typhimurium.

We isolated an Escherichia coli methionine auxotroph that displays a growth phenotype similar to that of known metF mutants but has elevated levels of 5,10-methylenetetrahydrofolate reductase, the metF gene product. Transduction analysis indicates that the mutant carries normal metE, metH, and metF genes; the phenotype is due to a single mutation, eliminating the possibility that the strain is a metE metH double mutant; and the new mutation is linked to the metE gene by P1 transduction. Plasmids carrying the Salmonella typhimurium metE gene and flanking regions complement the mutation, even when the plasmid-borne metE gene is inactivated. Enzyme assays show that the mutation results in a dramatic decrease in metE gene expression, a moderate decrease in metH gene expression, and a disruption of the metH-mediated vitamin B12 repression of the metE and metF genes. Our evidence suggests that the methionine auxotrophy caused by the new mutation is a result of insufficient production of both the vitamin B12-independent (metE) and vitamin B12-dependent (metH) transmethylase enzymes that are necessary for the synthesis of methionine from homocysteine. We propose that this mutation defines a positive regulatory gene, designated metR, whose product acts in trans to activate the metE and metH genes.     

Purification and properties of the Escherichia coli protein factor required for lambda integrative recombination

A purified preparation of the Escherichia coli integration host factor (IHF) displays two polypeptides of apparent molecular weight 11,000 and 9,500 when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Under nondenaturing conditions, IHF appears to exist as a 1:1 complex of these two polypeptides. Integrative recombination takes place in vitro when purified IHF and purified Int, a product of a bacteriophage lambda gene, are the only proteins added to reaction mixtures. No recombination is detected in the absence of either protein. The characteristics of the recombination reaction carried out by these two purified proteins are described. Purified IHF binds to DNA; in the presence of Int, a ternary complex is formed at one of the specific recombination sites. IHF hs no detectable endonuclease or topoisomerase activity. Several possibilities for the role of IHF in recombination are considered.     

A mutation in Escherichia coli enhancing the UV-mutability of phage lambda but not of its infectious DNA in a spheroplast assay

Summary A mutant (called mul) has been isolated from E. coli AB1157 which increases up to 13 fold the rate of clear plaque mutations of extracellularly UV-irradiated phage . The mul-mutation does not affect the UV-mutability of T4rII mutants or various bacterial markers and therefore seems to act specifically on UV-irradiated phage . However, when spheroplasts prepared from mul cells were infected with either irradiated or unirradiated -DNA equal frequencies of clear plaques were produced. As there is indirect evidence (host cell reactivation) that the spheroplasts do not significantly exclude irradiated phage DNA it seems that the mul phenotype can be expressed only in complete cells.The mutation responsible for the mul phenotype has been mapped by conjugation and transduction to be located between the markers pyrE and ilv and probably marks a new gene. The mul mutation does not increase the UV-sensitivity of the cells nor does it affect their ability to perform host cell reactivation. The presence of a recA allel in a mul mutant abolishes the UV-mutability of phage .Part of this work is from the thesis of the first author in partial fullfilment of the requirements for the doctoral degree of the University of Bochum. Some of the results have already been presented at the 32. Kongreß für Hygiene und Mikrobiologie at Münster, 1969.     

Termination of DNA replication in Escherichia coli requires a trans-acting factor.

The terminus region of the Escherichia coli chromosome contains two sites that inhibit the progression of DNA replication forks. These termination sites, designated T1 and T2, are separated by 7.5 min (350 kilobases [kb]) on the genetic map and are located at the extremities of the terminus region. They demonstrate polarity (they stop replication forks traveling in one direction but not the other) and inhibit replication forks that have passed through and are about to leave the terminus. We have used deletion mutations in the terminus region to map the locations of T1 and T2 more accurately and to initiate studies on the mechanism of replication fork inhibition. We have narrowed the boundaries of T1 and T2 to 20 and 4 kb, respectively. T1 maps between kb 80 and 100 on the physical map of the terminus region (J. P. Bouché, J. Mol. Biol. 154:1-20, 1982), and T2 maps between kb 438 and 442. In addition, we report here that deletion of the region containing the T2 termination site inactivated T1. Supplying the T2 region on a plasmid restored T1 function, demonstrating that inhibition of replication at T1 requires a trans-acting factor which maps in the vicinity of termination site T2. We have called this newly identified terminus function the termination utilization substance (tus).     

Characterization of bacteriophage lambda reverse as an Escherichia coli phage carrying a unique set of host-derived recombination functions

λ reverse (λrev) (Zissler et al., 1971a) is a recombination proficient derivative of a A phage which had lost the phage recombination genes by deletion. In this work, phages with the Rev phenotype have been obtained by a method of selection different from that of Zissler et al. (1971a). Comparison of DNA from two of our isolates and one of Zissler's by electron microscopic heteroduplex mapping shows that all three phages carry substitutions of non-λ DNA which are indistinguishable in extent, location and base sequence. Genetic and biochemical characterization of λrev strongly suggests that the substituted DNA codes for recombination functions different from the λ recombination functions which are deleted. These substituted genes apparently derive from the host chromosome or a prophage, and may be the same as the genes responsible for the SbcA and Rac phenotypes in the host.     

Mutational analysis of sequences in the recF gene of Escherichia coli K-12 that affect expression.

The level of translation of recF-lacZ fusions is reduced 20-fold by nucleotides 49 to 146 of recF. In this region of recF, we found a previously described ribosome-interactive sequence called epsilon and a hexapyrimidine tract located just upstream of the epsilon sequence. Mutational studies indicate that the hexapyrimidine sequence is involved in at least some of the reduced translation. When the hexapyrimidine sequence is mutant, mutating epsilon increases the level of translation maximally. We ruled out the possibility that ribosome frameshifting explains most of the effect of these two sequences on expression and suspect that multiple mechanisms may be responsible. In a separate report, we show that mutations in the hexapyrimidine tract and epsilon increase expression of the full-sized recF gene.     

The pheR gene of Escherichia coli encodes tRNA(Phe), not a repressor protein

Nucleotide sequence analysis and transposon 5 (Tn5) insertional mutagenesis indicate that the Escherichia coli gene pheR encodes tRNA(Phe) and not a repressor protein as previously reported. The coding region of pheR is identical to that of three other cloned tRNA(Phe) genes, pheU, pheV, and pheW. Multicopy plasmids carrying pheR, like those carrying pheU, pheV, or pheW, complement a temperature-sensitive lesion in the gene for the alpha-subunit of phenylalanyl-tRNA synthetase (pheS). The nucleotide sequences of the 5'-flanking DNA of pheR, pheU, and pheW are almost identical but are quite different from the same region of pheV. By comparison with pheV, which has two tandem promoters, pheR was found to have a single promoter. The expression of pheA (encoding chorismate mutase/prephenate dehydratase) in strains carrying the pheR374 allele was decreased to similar extents by multicopy plasmids containing either pheR or pheV. It is proposed that this decrease in pheA expression and the increase in expression of pheA previously reported for chromosomal pheR mutants are both mediated through the attenuation control mechanism that regulates pheA.