DNA replication defect in Salmonella typhimurium mutants lacking the editing (epsilon) subunit of DNA polymerase III.

In Salmonella typhimurium, dnaQ null mutants (encoding the epsilon editing subunit of DNA polymerase III [Pol III]) exhibit a severe growth defect when the genetic background is otherwise wild type. Suppression of the growth defect requires both a mutation affecting the alpha (polymerase) subunit of DNA polymerase III and adequate levels of DNA polymerase I. In the present paper, we report on studies that clarify the nature of the physiological defect imposed by the loss of epsilon and the mechanism of its suppression. Unsuppressed dnaQ mutants exhibited chronic SOS induction, indicating exposure of single-stranded DNA in vivo, most likely as gaps in double-stranded DNA. Suppression of the growth defect was associated with suppression of SOS induction. Thus, Pol I and the mutant Pol III combined to reduce the formation of single-stranded DNA or accelerate its maturation to double-stranded DNA. Studies with mutants in major DNA repair pathways supported the view that the defect in DNA metabolism in dnaQ mutants was at the level of DNA replication rather than of repair. The requirement for Pol I was satisfied by alleles of the gene for Pol I encoding polymerase activity or by rat DNA polymerase beta (which exhibits polymerase activity only). Consequently, normal growth is restored to dnaQ mutants when sufficient polymerase activity is provided and this compensatory polymerase activity can function independently of Pol III. The high level of Pol I polymerase activity may be required to satisfy the increased demand for residual DNA synthesis at regions of single-stranded DNA generated by epsilon-minus pol III. The emphasis on adequate polymerase activity in dnaQ mutants is also observed in the purified alpha subunit containing the suppressor mutation, which exhibits a modestly elevated intrinsic polymerase activity relative to that of wild-type alpha.     

Isolation and characterization of lon mutants in Salmonella typhimurium.

In this paper we report the isolation and characterization of lon mutants in Salmonella typhimurium. The mutants were isolated by using positive selection by chlorpromazine resistance. The physiological and biochemical properties of the lon mutants in S. typhimurium are very similar to those of Escherichia coli lon mutants. Mutants altered at this locus contain little or no activity of the ATP-dependent protease La and show a number of pleiotropic phenotypes, including increased production of capsular polysaccharides, increased sensitivity to UV light and other DNA-damaging agents, and a decreased ability to degrade abnormal proteins.     

Nucleotide sequences of dnaE, the gene for the polymerase subunit of DNA polymerase III in Salmonella typhimurium, and a variant that facilitates growth in the absence of another polymerase subunit.

The dnaE gene of Salmonella typhimurium, like that of Escherichia coli, encodes the alpha subunit containing the polymerase activity of the principal replicative enzyme, DNA polymerase III. This gene, or one nearby, has been identified as the locus of suppressor mutations that promote growth by cells deleted for dnaQ, the gene for the editing subunit of this enzyme complex. Using a combination of nucleotide sequencing and marker rescue experiments, the alteration in one such suppressor was identified as a valine-to-glycine substitution at amino acid 832 of the 1,160-amino-acid alpha polypeptide. The alpha polypeptides of E. coli and S. typhimurium are identical in size and in 97% of their amino acid residues. Their identity includes the valine residue that was changed in the suppressor allele of S. typhimurium. We also localized a temperature-sensitive dnaE mutation to the 3' half of dnaE.     

Isolation and characterization of FliK-independent flagellation mutants from Salmonella typhimurium.

A flagellum of Salmonella typhimurium and Escherichia coli consists of three structural parts, a basal body, a hook, and a filament. Because the fliK mutants produce elongated hooks, called polyhooks, lacking filament portions, the fliK gene product has been believed to be involved in both the determination of hook length and the initiation of the filament assembly. In the present study, we isolated two mutants from S. typhimurium which can form flagella even in the absence of the fliK gene product. Flagellar structures were fractionated from these suppressor mutants and inspected by electron microscopy. The suppressor mutants produced polyhook-filament complexes in the fliK mutant background, while they formed flagellar structures apparently indistinguishable from those of the wild-type strain in the fliK+ background. Genetic and sequence analyses of the suppressor mutations revealed that they are located near the 3'-end of the flhB gene, which has been believed to be involved in the early process of the basal body assembly. On the basis of these results, we discuss the mechanism of suppression of the fliK defects by the flhB mutations and propose a hypothesis on the export switching machinery of the flagellar proteins.     

Isolation and characterization of catalase deficient mutants of Salmonella typhimurium.

Summary Catalase deficient mutants (kat) ofSalmonella typhimurium have been isolated. The mutationskatA1, katC6 andkatD9 appear to map at about minute 10 on theSalmonella chromosome. ThekatC6 andkatD9 lesions are complemented by theE. coli F128 (lac+ pro+) episome but thekatA1 lesion is not.KatB2 maps at about minute 100. None of the mutants are oxygen sensitive; they grow as well as wild type bacteria, even when aerated.     

Isolation and characterization of proline peptidase mutants of Salmonella typhimurium

The proline requirement of Salmonella typhimurium strain proB25 can be satisfied by either of the peptides Leu-Pro or Gly-Pro-Ala. A mutant derivative of strain proB25 isolated by penicillin selection in medium containing Leu-Pro as proline source fails to use either Leu-Pro or Gly-Pro-Ala as a source of proline. This strain is a double mutant that lacks two aminoacyl-proline-specific peptidases. One of these enzymes (peptidase Q) catalyzes the rapid hydrolysis of Leu-Pro but does not hydrolyze Gly-Pro-Ala or poly-l-proline. Mutations at a site (pepQ) near metE lead to loss of this activity. The other peptidase (peptidase P) catalyzes the hydrolysis of Gly-Pro-Ala and poly-l-proline but is only weakly active with Leu-Pro as substrate. This enzyme is similar to aminopeptidase P previously described in Escherichia coli (16). Mutations at a locus (pepP) near serA lead to loss of this enzyme.     

Isolation, sequencing and overexpression of the gene encoding the theta subunit of DNA polymerase III holoenzyme.

The gene encoding the theta subunit of DNA polymerase III holoenzyme, designated holE, was isolated using a strategy in which peptide sequence was used to derive a DNA hybridization probe. Sequencing of the gene, which maps to 41.43 centisomes of the chromosome, revealed a 76-codon open reading frame predicted to produce a protein of 8,846 Da. When placed in a tac promoter expression vector, the open reading frame directed expression of a protein, that comigrated with authentic theta subunit from purified holoenzyme, to 6% of total soluble protein.     

holE, the gene coding for the theta subunit of DNA polymerase III of Escherichia coli: characterization of a holE mutant and comparison with a dnaQ (epsilon-subunit) mutant.

DNA polymerase III holoenzyme is a multiprotein complex responsible for the bulk of chromosomal replication in Escherichia coli and Salmonella typhimurium. The catalytic core of the holoenzyme is an alpha epsilon theta heterotrimer that incorporates both a polymerase subunit (alpha; dnaE) and a proofreading subunit (epsilon; dnaQ). The role of theta is unknown. Here, we describe a null mutation of holE, the gene for theta. A strain carrying this mutation was fully viable and displayed no mutant phenotype. In contrast, a dnaQ null mutant exhibited poor growth, chronic SOS induction, and an elevated spontaneous mutation rate, like dnaQ null mutants of S. typhimurium described previously. The poor growth was suppressible by a mutation affecting alpha which was identical to a suppressor mutation identified in S. typhimurium. A double mutant null for both holE and dnaQ was indistinguishable from the dnaQ single mutant. These results show that the theta subunit is dispensable in both dnaQ+ and mutant dnaQ backgrounds, and that the phenotype of epsilon mutants cannot be explained on the basis of interference with theta function.     

Salmonella typhimurium mutants with altered expression of the pyrA gene due to changes in RNA polymerase

Rifampicin-resistant mutants of Salmonella typhimurium were isolated and tested for pleiotropic defects in the regulation of pyr gene expression. Seven per cent of all the Rifr mutants were inhibited in growth by addition of uracil (uracil-sensitive). The uracil-sensitive phenotype ( UraS ) was reversed by arginine or citrulline, but not by ornithine, and it was suppressed by mutations in either argR or pyrH , which causes increased expression of pyrA . It was shown that the basal levels of carbamoylphosphate synthase (the pyrA gene product) was reduced to approximately 60% in the mutants, and that addition of arginine and/or uracil to the growth medium caused hyperrepression of pyrA expression. The expression of other genes of the arginine and pyrimidine biosynthetic pathways was not affected significantly in the mutants. The mutations were located in the rpoB gene coding for the beta-subunit of RNA polymerase, suggesting a regulatory function of RNA polymerase in the control of pyrA expression.     

Isolation of temperature-sensitive pantothenate kinase mutants of Salmonella typhimurium and mapping of the coaA gene.

Temperature-sensitive pantothenate kinase mutants of Salmonella typhimurium LT2 were selected by using the excretion of pantothenate at the nonpermissive temperature as a screening method. Thermolability of the pathothenate kinase activity in extracts of the mutants was demonstrated. The mutations were mapped at min 89 of the Salmonella chromosome, near rpoB, by transduction. As pantothenate kinase catalyzes the first step in the biosynthesis of coenzyme A from pantothenate, the new genetic locus has been designated coaA.     

Isolation and characterization of conditional adherent and non-type 1 fimbriated Salmonella typhimurium mutants

Mutations in the genes encoding the type 1 fimbriae of Salmonella typhimurium were isolated by selecting for the deletion of Tn10 inserted adjacent to the chromosomal fim+ genes and screening for the loss of mannose-sensitive haemagglutination (HA) activity. S. typhimurium strains with Tn10 insertions in ahp were hypersensitive to peroxides, and tetracycline-sensitive derivatives of ahp::Tn10 mutants displayed two fim mutant phenotypes. The predominant class of fim mutants did not synthesize type 1 fimbriae. A second type of fim mutant synthesized type 1 fimbriae and exhibited a conditional lipoic acid requirement for HA. A fim-lip conditional mutant synthesized type 1 fimbriae when grown in Mueller-Hinton broth but the haemagglutinating activity of the fimbriae was dependent upon the addition of lipoic acid to the growth medium. Independently isolated lip mutations did not demonstrate a similar pleiotropic effect on HA. Western blots of fimbriae extracted from a fim-lip conditional mutant that was grown under permissive and restrictive conditions indicated the presence of 33 and 36.6 kDa proteins in HA+ fimbriae that were absent in HA- fimbriae. The HA+ phenotype of both conditional and non-fimbriated mutants was restored by transformation with cloned genes encoding S. typhimurium type 1 fimbriae.     

Isolation and characterization Salmonella typhimurium mutants lacking a tripeptidase (peptidase T).

Salmonella typhimurium contains an enzyme, peptidase T, that hydrolyzes a variety of tripeptides. Specificity studies with a peptidase activity stain after gel electrophoresis of crude cell extracts showed that peptidase T hydrolyzes tripeptides containing N-terminal methionine, leucine, or phenylalanine. Little or no activity could be detected against dipeptides, N-blocked or C-blocked tripeptides, and tetrapeptides. Analysis of reaction products by high-pressure liquid chromatography showed that peptidase T removes the N-terminal amino acid from tripeptides. Mutants lacking peptidase T were isolated by screening microcultures grown in the wells of plastic microtitration plates for hydrolysis of Met-Ala-Ser or Met-Gly-Gly. Mutations (pepT) that eliminate this enzyme were found to be phage P22 cotransducible with purB at approximately 25 map units on the S. typhimurium map. Comparison of the growth properties of mutant and wild-type strains suggests that peptidase T does not function in utilization of tripeptides to provide amino acids during growth.     

Identification, isolation, and characterization of the structural gene encoding the delta' subunit of Escherichia coli DNA polymerase III holoenzyme.

The gene encoding the delta' subunit of DNA polymerase III holoenzyme, designated holB, was cloned by a strategy in which peptide sequence was used to derive a DNA hybridization probe. The gene maps to 24.95 centisomes of the chromosome. Sequencing of holB revealed a 1,002-bp open reading frame predicted to produce a 36,936-Da protein. The gene has a ribosome-binding site and promoter that are highly similar to the consensus sequences and is flanked by two potential open reading frames. Protein sequence analysis of delta' revealed a high degree of similarity to the dnaX gene products of Escherichia coli and Bacillus subtilis, including one stretch of 10 identical amino acid residues. A lesser degree of similarity to the gene 44 protein of bacteriophage T4 and the 40-kDa protein of the A1 complex (replication factor C) of HeLa cells was seen. The gene, when placed into a tac promoter-based expression plasmid, directed expression of two proteins of similar size. By immunodetection with anti-holoenzyme immunoglobulin G, both proteins are judged to be products of holB.     

Temperature-sensitive ribonucleic acid polymerase mutant of Salmonella typhimurium with a defect in the beta' subunit.

Localized mutagenes of Salmonella typhimurium followed by a [3H]uridine enrichment procedure yielded a temperature-sensitive strain with a mutation in the rpo region of the chromosome. Ribonucleic acid (RNA) polymerase (EC 2.7.7.6; nucleoside triphosphate: RNA nucleotidyltransferase) purified from this mutant was considerably less active at the nonpermissive temperature than wild-type enzyme. Furthermore, the enzyme from this mutant, unlike RNA polymerase of previously isolated temperature-sensitive mutants, was as thermostable as wild-type enzyme when preincubated at 50 degrees C. Subunit reconstitution experiments have shown that the temperature sensitivity is caused by an alteration in the beta' subunit of the enzyme.     

Construction and characterization of mutants of Salmonella typhimurium deficient in DNA repair of O6-methylguanine.

Escherichia coli has two O6-methylguanine DNA methyltransferases that repair alkylation damage in DNA and are encoded by the ada and ogt genes. The ada gene of E. coli also regulates the adaptive response to alkylation damage. The closely related species Salmonella typhimurium possesses methyltransferase activities but does not exhibit an adaptive response conferring detectable resistance to mutagenic methylating agents. We have previously cloned the ada-like gene of S. typhimurium (adaST) and constructed an adaST-deletion derivative of S. typhimurium TA1535. Unexpectedly, the sensitivity of the resulting strain to the mutagenic action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was similar to that of the parent strain. In this study, we have cloned and sequenced the ogt-like gene of S. typhimurium (ogtST) and characterized ogtST-deletion derivatives of TA1535. The ogtST mutant was more sensitive than the parent strain to the mutagenicity of MNNG and other simple alkylating agents with longer alkyl groups (ethyl, propyl, and butyl). The adaST-ogtST double mutant had a level of hypersensitivity to these agents similar to that of the ogtST single mutant. The ogtST and the adaST-ogtST mutants also displayed a two to three times higher spontaneous mutation frequency than the parent strain and the adaST mutant. These results indicate that the OgtST protein, but not the AdaST protein, plays a major role in protecting S. typhimurium from the mutagenic action of endogenous as well as exogenous alkylating agents.     

Isolation of super-repressor mutants in the histidine utilization system of Salmonella typhimurium.

Two super-repressor mutations in the histidine utilization (hut) operons of Salmonella typhimurium are described. Cells bearing either of these mutations have levels of hut enzymes that do not increase above the uninduced levels when growth is in the presence of either histidine or the gratuitous inducer imidazole propionate. Both mutations lie in the region of the gene for the hut repressor, hutC, and reverse mutations of both are to the constitutive (repressor-negative) rather than to the inducible (wild type) phenotype. In hybrid merodiploid strains the super-repressor mutations are dominant over either wild-type (hutC+) or repressor-negative (hutC-) alleles. Whereas both super-repressor mutations cause the uninducible synthesis of hut enzymes, the degree of repression is different. One mutation causes repression of enzyme synthesis in one of the two hut operons to a level below the basal, uninduced level of wild-type cells. The other mutation causes repression to a lesser degree than in wild-type cells, so that the hut enzymes are present at a level above the normal basal level; this partially constitutive synthesis is greater for the enzymes of one of the hut operons than for the enzymes of the other. Thus, both mutations apparently result in repressors with altered operator-binding properties, in addition to altered inducer-binding properties.