Mutagenesis, by methylating and ethylating agents, in mutH, mutL, mutS, and uvrD mutants of Salmonella typhimurium LT2.

Salmonella typhimurium LT2 mutH, mutL, mutS, and uvrD mutants were especially sensitive to mutagenesis by both the recA+-dependent mutagen methyl methane sulfonate and the recA+-independent mutagen ethyl methane sulfonate, but not to mutagenesis by agents such as 4-nitroquinoline-1-oxide and UV irradiation. Similarly, these mutator strains were very sensitive to mutagenesis by the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea. The increased susceptibility to mutagenesis by small alkylating agents due to mutH, mutL, mutS, and uvrD mutations was not accompanied by an increased sensitivity to killing by these agents. Various models are discussed in an effort to explain why strains thought to be deficient in methyl-instructed mismatch repair are sensitive to mutagenesis by methylating and ethylating agents.     

Cloning and characterization of mutL and mutS genes of Vibrio cholerae: nucleotide sequence of the mutL gene.

The mutL and mutS genes of Vibrio cholerae have been identified using interspecific complementation of Escherichia coli mutL and mutS mutants with plasmids containing the gene bank of V. cholerae. The recombinant plasmid pJT470, containing a 4.7 kb fragment of V. cholerae DNA codes for a protein of molecular weight 92,000. The product of this gene reduces the spontaneous mutation frequency of the E. coli mutS mutant. The plasmid, designated pJT250, containing a 2.5 kb DNA fragment of V. cholerae and coding for a protein of molecular weight 62,000, complements the mutL gene function of E. coli mutL mutants. These gene products are involved in the repair of mismatches in DNA. The complete nucleotide sequence of mutL gene of V. cholerae has been determined.     

Structural characterization of the Salmonella typhimurium LT2 umu operon.

The umuDC operon of Escherichia coli encodes functions required for mutagenesis induced by radiation and a wide variety of chemicals. The closely related organism Salmonella typhimurium is markedly less mutable than E. coli, but a umu homolog has recently been identified and cloned from the LT2 subline. In this study the nucleotide sequence and structure of the S. typhimurium LT2 umu operon have been determined and its gene products have been identified so that the molecular basis of umu activity might be understood more fully. S. typhimurium LT2 umu consists of a smaller 417-base-pair (bp) umuD gene ending 2 bp upstream of a larger 1,266-bp umuC gene. The only apparent structural difference between the two operons is the lack of gene overlap. An SOS box identical to that found in E. coli is present in the promoter region upstream of umuD. The calculated molecular masses of the umuD and umuC gene products were 15.3 and 47.8 kilodaltons, respectively, which agree with figures determined by transpositional disruption and maxicell analysis. The S. typhimurium and E. coli umuD sequences were 68% homologous and encoded products with 71% amino acid identity; the umuC sequences were 71% homologous and encoded products with 83% amino acid identity. Furthermore, the potential UmuD cleavage site and associated catalytic sites could be identified. Thus the very different mutagenic responses of S. typhimurium LT2 and E. coli cannot be accounted for by gross differences in operon structure or gene products. Rather, the ability of the cloned S. typhimurium umuD gene to give stronger complementation of E. coli umuD77 mutants in the absence of a functional umuC gene suggests that Salmonella UmuC protein normally constrains UmuD protein activity.     

Identification of a umuDC locus in Salmonella typhimurium LT2.

The umuDC operon of Escherichia coli is required for efficient mutagenesis by UV light and many other DNA-damaging agents. The existence of a umuDC analog in Salmonella typhimurium has been questioned. With DNA probes to the E. coli umuD and umuC genes, we detected, by Southern blot hybridization, sequences similar to both of these genes in S. typhimurium LT2. We also confirmed that the presence of cloned E. coli umuD enhances the UV mutability and resistance of S. typhimurium. Our data strongly suggest that S. typhimurium contains a functional umuDC operon.     

Cloning and characterization of the L-rhamnose regulon in Salmonella typhimurium LT2.

A Salmonella typhimurium LT2 cosmid library was constructed, and a 46-kb recombinant plasmid was isolated that could complement an Escherichia coli rha mutant. Subsequent subcloning generated a 13.6-kb ClaI restriction fragment that contained a functional regulatory element. By complementation analyses using different subclones, the approximate physical locations of rhaT, rhaC1, rhaC2, rhaB, rhaA, and rhaD were determined. The nucleotide sequence spanning the rhaB and rhaC2 genes was determined.     

Isolation and characterization of the Salmonella typhimurium LT2 xylose regulon

Salmonella DNA was partially digested with EcoRI, and the digest was fractionated to obtain fragments larger than 8 kilobases (kb). These were ligated into EcoRI-cut pBR322, and the mixture was used to transform Salmonella Xyl- cells selecting for ampR xyl+ transformants. A 21- and a 27-kb plasmid were isolated, both of which contained the entire xylose regulon. The xylose regulon was localized to a 6.3-kb segment of a 13.5-kb EcoRI fragment. Subclones were constructed which contained either the genes for D-xylose isomerase and D-xylulokinase or the genes for the D-xylose transport and the D-xylose regulatory factors. The gene order determined by the subcloning experiments is consistent with that determined by genetic mapping. The spots corresponding to D-xylose isomerase and D-xylulokinase subunits were identified in two-dimensional gels of several xylose-induced strains. Each of them had a molecular weight of 45,000 and an isoelectric point of 6.2 +/- 0.1.     

Isolation and characterization of a substitution mutation in the ompR gene of Salmonella typhimurium LT2.

The expression of the genes ompC and ompF encoding major outer membrane proteins is dependent on the ompR-envZ operon. Here we describe the isolation and characterization of an ompR mutation, a single-base-pair change, that results in an Arg-to-Cys substitution. When present in multiple copies, the mutant allele conferred a dominant OmpC- OmpF+ phenotype. Furthermore, the mutant allele exhibited allele-specific negative complementation with other ompR mutations. This ability, together with its dominant character, suggested that the OmpR protein is capable of multimerization.     

Autoregulation by tandem promoters of the Salmonella typhimurium LT2 metJ gene.

Regulation of the Salmonella typhimurium metJ gene was examined by measuring beta-galactosidase activity in Escherichia coli strains lysogenic for a phage carrying a metJ-lacZ gene fusion. The results indicated that the metJ gene is regulated by its own gene product and by methionine supplementation to the growth medium. This autoregulatory mechanism involved two tandem promoters, pJ1 and pJ2, separated by approximately 65 base pairs. Deletion analysis permitted the assessment of the activity of promoters pJ1 and pJ2 individually. Promoter Pj1 was negatively regulated by the metJ gene product and by methionine. Although Pj2 regulation remained unclear, evidence is presented which suggests that it is not negatively regulated like pJ1.     

Mutations affecting the regulation of the metB gene of Salmonella typhimurium LT2.

We isolated and characterized cis-acting mutations that affect the regulation of the metB gene of Salmonella typhimurium LT2. The mutations were isolated in an Escherichia coli lac deletion strain lysogenized with lambda bacteriophage carrying a metB-lacZ gene fusion (lambda JBlac) in which beta-galactosidase production is dependent upon metB gene expression. The mutant lysogens show elevated, poorly regulated beta-galactosidase production. The altered regulation is a result of disruption of the methionine control system mediated by the metJ repressor. The mutations are located in a region of dyad symmetry centered near the -35 sequence of the metB promoter. We propose that these mutations alter the repressor binding site and define the metB operator sequence. In addition, we discuss a highly conserved, nonsymmetric DNA sequence of unknown function which occurs in the control regions of the metA, metC, metE, metF, metG, and metJB genes of both S. typhimurium and E. coli.     

Electrotransformation in Salmonella typhimurium LT2

Electroporation gives high efficiency of transformation in Salmonella typhimurium LT2, yielding 10(8)-10(9) electrotransformants per microgram of pBR322 DNA. Lipopolysaccharide (LPS) composition has little influence on electrotransformation efficiency by electroporation, unlike Ca2+ shock methods, which give ca. 10(6) transformants/microgram DNA with strains with Rc or Rd2 LPS, 10(4) transformants with most smooth and rough strains, and 10(2) transformants with strains with Re LPS. Thus cell envelope properties are less crucial in electrotransformation than in Ca2+ shock methods. The reciprocal restriction barrier between Escherichia coli K-12 and S. typhimurium LT2 reduces electrotransformation by ca. 100-fold, but host-restriction mutants reduce or eliminate the barrier.     

The Salmonella typhimurium LT2 uvrD gene is regulated by the lexA gene product.

The uvrD gene product apparently plays a role in the repair of UV damage, in mismatch repair, and in genetic recombination. A lower level of expression of the Salmonella typhimurium LT2 uvrD gene was observed in maxicells prepared from an Escherichia coli strain that contained a lexA+ plasmid than in maxicells prepared from an E. coli strain that lacked functional LexA protein. These results suggest that the uvrD+ gene is repressed by the LexA protein and is thus a member of the set of genes whose expression is increased by "SOS"-inducing treatments.     

The plasmid of Salmonella typhimurium LT2

Summary Methods of clonal analysis were applied to the study of heterogeneity of the progeny after crosses of 4 donor strains (Hfr H, Hfr C, KL 16 and KL 99) with 3 recipient strains (PC 0212, AB 712 and ECK 022). Three markers were used in each cross. The distal one was the selective marker. The inheritance of two additional proximal markers characterized the heterogeneity of clones originating from particular zygotes. In most crosses the percentage of heterogeneity exceeded 30. One of the recipient strains, obtained by conjugation of the conventional strain PC 0212 with the donor Hfr H revealed unusual properties in respect to heterogeneity. Exconjugants derived from this recipient (ECK 022) and donor Hfr H and Hfr C had a heterogeneity index of about 5%. It is shown that this unusual behavior reflects a very fast process of segregation of recombinants.In crosses with the donors KL 16 and KL 99 the same recipient revealed normal indices of heterogeneity. All these data are explained assuming that there exists a specific genetic marker which determines the process of decay of merozygotes. Tentatively it is called het. Its approximate localization was deduced from specifically designed experiments, in which the heterogeneity of the progeny was found very different, when the donor KL 16 transmitted different parts of its chromosome to the recipient ECK 022.     

Formate hydrogenlyase system in Salmonella typhimurium LT2.

Isolation from Salmonella typhimurium of mutants unable to reduce benzyl viologen under anaerobic conditions has allowed the study of the factors involved in the multienzymic formate hydrogenylase system. 1. Depending on the affected activities, different classes of mutants were found: FHL-A mutants have lost formate dehydrogenase 1 and formate dehydrogenase 2 activities; mutations in fdhA (117 min) or fdhB (33 min) lead to such a phenotype. FHL-B and FHL-C mutants have lost formate dehydrogenase 2 activity and part or all of hydrogenase activity, respectively; both types correspond to mutations in the hyd gene (approximately 90 min). FHL-D mutants have lost only formate dehydrogenase 2 activity; fhlD gene maps at 120 min. 2. In some cases, mixtures of extracts from two mutants display formate dehydrogenase 2 and formate hydrogenylase activities. Restoration studies suggest the existence of one factor sensitive to growth conditions and inactivated by oxygen or heating. This factor which is present and active in FHL-C mutants, is probably the one missing in FHL-D mutants. 3. A new scheme for the formate hydrogenylase system is proposed, in which hydrogenase transfers electrons directly to benzyl viologen.