Relaxed control of RNA synthesis during nutritional shiftdowns of hisU mutant of Salmonella typhimurium.

Among the classes of histidine regulatory mutants isolated in Salmonella typhimurium, three of these mutants (hisT, hisW and hisU) exhibit pleiotrophic effects on the regulation of expression of other amino acid biosynthetic operons (1, 2). While the regulatory patterns of the hisT mutants are explained by the defective tRNA pseudouridylate synthetase (3), the exact function of the hisW and hisU loci are not as clearly defined, although both mutants exhibit reductions in the relative amino acid acceptance activity of several tRNA's (4). In studies of tRNA synthesis and processing in one such hisU mutant (hisU1820), we unexpectedly observed continued RNA synthesis during nutritional (carbon and energy source) transitions. It was also shown that this relaxed control of stable RNA formation is independent of the relA gene product.     

Mutagenic evaluation of the monocyclic aromatic amine N-methylamino-2-nitro-4-N′,N′-bis(2-hydroxyethyl)aminobenzene in the Salmonella typhimurium/mammalian microsome test

The hair-dye component N-methylamino-2-nitro-4-N′,N′-bis(2-hydroxyethyl)aminobenzene was investigated for mutagenic activity in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98. The testing was performed in the absence and in the presence of a rat-liver microsomal activation system induced by Aroclor 1254. Our results indicate that N-methylamino-2-nitro-4-N′,N′-bis(2-hydroxyethyl)aminobenzene does not induce mutations in Salmonella typhimurium strains, either in the absence or in the presence of the metabolic activation system. The purity of the compound was controlled by utilizing high-pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC).     

Frameshift lesions induced by oxazolopyridocarbazoles are recognized by the mismatch repair system in Escherichia coli

The simple reversible intercalating agent isopropyl-OPC (iPr-OPC) induces frameshift-1 mutations in Salmonella typhimurium and Escherichia coli. The mutagenic responses of S. typhimurium and E. coli wild-type strains are not proportional to the amount of drug intercalated into double-stranded nucleic acids in living bacteria; it occurs only above a minimum level of binding. The fact that mismatch-repair-deficient (mutS) as well as adenine-methylation-deficient (dam) E. coli mutants are hypermutable at low concentrations of iPr-OPC suggests that the majority of mutants induced by this intercalating drug occur as mismatch-repairable mutations (or lesions) in the newly synthesized DNA strand close to the replication fork.     

The effects of traT insertion mutations on detergent sensitivity and serum resistance of Escherichia coli and Salmonella typhimurium

The SS-A mutation of Salmonella typhimurium, which probably causes the production of a mutated TraT-like protein, sensitizes the bacteria to hydrophobic antibiotics. A similar phenotype is caused by insertion mutations in the cloned traT gene of R6-5. While the SS-A mutants are resistant to detergents and have unaltered serum resistance, the insertion mutations sensitize both S. typhimurium and Escherichia coli to detergents and abolish the increase in serum resistance caused by the wild-type traT gene product in E. coli.     

Precursors of the pyrimidine moiety of thiamine

1. A method was devised for obtaining the pyrimidine moiety of thiamine in a pure form after its excretion into the medium by de-repressed washed-cell suspensions of mutants of Salmonella typhimurium LT2. 2. By using amino acid-requiring mutants, this excretion of pyrimidine moiety was shown to be dependent on the presence of both methionine and glycine. 3. In the presence of either [Me-¹⁴C]methionine or [G-¹⁴C]methionine, methionine-requiring mutants did not incorporate radioactivity into the pyrimidine moiety. 4. In contrast, both [1-¹⁴C]glycine and [2-¹⁴C]glycine were incorporated into the pyrimidine moiety excreted by glycine-requiring mutants, and this occurred with little or no dilution of specific radioactivity. 5. The possible requirement for methionine as a cofactor and the significance of the incorporation of both carbon atoms of glycine are discussed.     

2-Deoxyribose 5-phosphate aldolase: Genetic analyses of structure

A total of 33 mutant strains of Salmonella typhimurium deficient in deoxyribose 5-phosphate activity have been isolated and characterized as missense or nonsense. Three-factor transductional analyses of the mutants were used to construct a fine structure map of the deoC gene, which codes for a peptide of 28,500 molecular weight. An unusual clustering of the missense mutants was observed, where 75% of all the missense mutants mapped in an area which corresponds to 19% of the total gene length. It is suggested that this area of the protein is particularly sensitive to amino acid replacements but that other areas of the protein are reasonably tolerant of such changes. Nonsense mutations are found scattered throughout the gene. This is expected since the carboxyl-terminal tyrosine is essential for enzymatic activity.     

Salmonella typhimurium mutants affecting establishment of lysogeny

Summary Salmonella typhimurium mutants have been isolated in which phage P22 fails to establish lysogeny. These appear to be defective in cAMP metabolism. A phage mutation overcoming the bacterial defect has been mapped between gene c ₁ and gene 12.     

The apbE Gene Encodes a Lipoprotein Involved in Thiamine Synthesis in Salmonella typhimurium

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella typhimurium. The biochemical steps and gene products involved in the conversion of aminoimidazole ribotide (AIR), a purine intermediate, to the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine have yet to be elucidated. We have isolated mutations in a new locus (Escherichia coli open reading frame designation yojK) at 49 min on the S. typhimurium chromosome. Two significant phenotypes associated with lesions in this locus (apbE) were identified. First, apbE purF double mutants require thiamine, specifically the HMP moiety. Second, in the presence of adenine, apbE single mutants require thiamine, specifically both the HMP and the thiazole moieties. Together, the phenotypes associated with apbE mutants suggest that flux through the purine pathway has a role in regulating synthesis of the thiazole moiety of thiamine and are consistent with ApbE being involved in the conversion of AIR to HMP. The product of the apbE gene was found to be a 36-kDa membrane-associated lipoprotein, making it the second membrane protein implicated in thiamine synthesis.     

Iron Transport in Salmonella typhimurium LT-2: Prevention, by Ferrichrome, of Adsorption of Bacteriophages ES18 and ES18.hl to a Common Cell Envelope Receptor

Ferrichrome prevents adsorption of phages ES18 and ES18.h1 to cells of Salmonella typhimurium. Studies of albomycin-resistant mutants defective in both ferrichrome utilization and ES18.h1 adsorption suggest a sid gene may specify a receptor for both.     

Interaction of lead nitrate and cadmium chloride withEscherichia coli K-12 andSalmonella typhimurium global regulatory mutants

Summary To investigate the interactions of heavy metals with cells, a minimal medium for the growth of enteric bacteria using glycerol-2-phosphate as the sole phosphorus source was developed that avoided precipitation of Pb²⁺ with inorganic phosphate. Using this medium, spontaneous mutants ofEscherichia coli resistant to addition of Pb(NO₃)₂ were isolated. Thirty-five independent mutants all conferred a low level of resistance. Disk diffusion assays on solid medium were used to survey the response ofE. coli andSalmonella typhimurium mutants altered in global regulatory networks to Pb(NO₃)₂) and CdCl₂. Strains bearing mutations inoxyR andrpoH were the most hypersensitive to these compounds. Based upon the response of strains completely devoid of isozymes needed to inactivate reactive oxygen species, this hypersensitity to lead and cadmium is attributable to alteration in superoxide dismutase rather than catalase levels. Similar analysis of chaperonedefective mutants suggests that these metals damage proteins in vivo.     

Involvement of the L-cysteine biosynthetic pathway in azide-induced mutagenesis in Salmonella typhimurium

L-Cystine and L-cysteine specifically reverse the mutagenic action of azide in Salmonella typhimurium and Escherichia coli. To establish whether the L-cysteine biosynthetic pathway is involved in azide-induced mutagenesis, several derivatives of a mutagen tester-strain of S. typhimurium bearing mutations in different cys genes were isolated. No mutagenic effect of azide was observed in a strain carrying mutation in the cysE gene, unless the incubation medium was supplemented with exogenous O-acetylserine. Out of 16 cysK mutants 14 were mutagenized by azide very poorly or not at all. These results indicate that the activity of O-acetylserine sulfhydrylase A, and the availability of O-acetylserine, one of the two co-substrates of the enzyme, are essential for the mutagenic action of azide in S. typhimurium     

Salmonella typhimurium Mutants with Altered Glutamate Dehydrogenase and Glutamate Synthase Activities

Although glutamate is a key compound in nitrogen metabolism, little is known about the function or regulation of its two biosynthetic enzymes, glutamate dehydrogenase and glutamate synthase. To begin the characterization of glutamate formation in Salmonella typhimurium, we isolated mutants having altered glutamate dehydrogenase and glutamate synthase activities. Mutants which failed to grow on media with glucose as the carbon source and less than 1 mM (NH₄)₂SO₄ as the nitrogen source (Asm⁻) had about one-fourth the normal glutamate synthase activity and one-half the glutamine synthetase activity. The asm mutations also prevented growth with alanine, arginine, or proline as nitrogen sources and conferred resistance to methionine sulfoximine. When a mutation (gdh-51) causing the loss of glutamate dehydrogenase activity was transferred into a strain with an asm-102 mutation, the resulting asm-102 gdh-51 mutant had a partial requirement for glutamate. A strain isolated as a complete glutamate auxotroph had a third mutation, in addition to the asm-102 gdh-51 lesions, that further decreased the glutamate synthase activities to 1/20 the normal level. Both the asm-102 and gdh-51 mutations were located on the S. typhimurium linkage map at sites distinct from those found for mutations causing similar phenotypes in Klebsiella aerogenes and Escherichia coli.     

Mutations by near-ultraviolet radiation in Escherichia coli strains lacking superoxide dismutase

Om wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic. However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O₂⁻), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant. When a sodA sodB double mutant contained a plasmid carrying katG⁺ HP-I catalase), mutation by NUV was reduced to wild-type (sodA⁺sodB⁺) levels. Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutations frequency by NUV was reduced to wild-type levels. This synergistic action of NUV and O₂⁻ suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants. Exposure to H₂O₂ induced a 2.8 fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG⁺ was introduced. These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH^. radicals, possibly by generating excess H₂O₂.     

Integration, at HAG or Elsewhere, of H2 (Phase-2 Flagellin) Genes Transduced from Salmonella to ESCHERICHIA COLI

A fla mutant of E. coli K12 was given fla⁺ and H1-i by phage P1kc cotransduction from S. typhimurium, then made Fla^- by transduction of ah1 from S. typhimurium. Motile clones expressing a Salmonella phase-2 antigen, e,n,x or 1,2, were obtained from the K12 i ah1 (therefore Fla^-) line by P1kc transduction of flagellin-specifying genes, H2-e,n,x or H2–1,2, from Salmonella donors. Of eighteen such transductants sixteen failed to show phase variation, and on transduction back to Salmonella each structural gene for a phase-2 flagellin (or at least for its antigenically determinant part) now behaved as an allele of H1, presumably in consequence of incorporation in the hag region of the K12 recipient, in place of H1-i ah1 . The e,n,x- and 1,2-specifying genes were shown to have been integrated in the K12 chromosome without the linked H1-repressor gene or the adjacent vh2 gene (controlling rate of phase-variation) and they responded to the repressing activity of an H2 allele elsewhere in the cell, in this respect resembling H1 alleles of Salmonella or hag alleles of E. coli. Two K12 e,n,x transductants had flagellin-specifying genes which when transduced back to Salmonella were integrated at H2; they are inferred to have resulted from integration of H2-e,n,x in the K12 chromosome elsewhere than the hag region. These two clones showed phase variation, between a Fla⁺ phase, with antigen e,n,x, and a Fla^- phase (with e,n,x determinant in the nonactive state and the determinant of antigen i inactivated by ah1). The two integrated e,n,x genes when in the "active" state retained the ability to repress expression of exogenote H1 alleles, which indicates that the closely linked H1-repressor gene also was integrated. One of the two exceptional transductants derived its e,n,x gene from a Salmonella donor with the linked vh2 ^- gene, which in Salmonella almost entirely prevents change of phase, and transduction of this e,n,x gene back to Salmonella recipients proved that vh2^- had been incorporated into the E. coli chromosome along with the e,n,x determinant and the H1-repressor gene. The high frequency of change of phase (Fla⁺↔Fla ^-) in the K12 e,n,x vh2^- transductant concerned suggests that vh2^- fails to prevent frequent change of state of the phase-determinant part of H2 when vh2 ^- and H2 are incorporated in the E. coli chromosome.     

his-Linked Hydrogen Sulfide Locus in Salmonella typhimurium

Investigation of strains of Salmonella typhimurium having extended his deletion mutations indicates that a genetic site (or sites) affecting H₂S production is located in the region of the chromosome adjacent to the operator end of the his operon. This site is co-transducible with the his genes. Experimental data indicate that the site is also present on an F'his factor derived from S. typhimurium, FS401. Evidence is presented for the existence of another site affecting H₂S production which is not linked to his.