Mutations that affect the regulation of phs in Salmonella typhimurium.

The regulation of phs [production of hydrogen sulphide (H2S)] in Salmonella typhimurium is complex. Previous studies have shown that expression is dependent upon the presence of reduced sulphur and anaerobiosis and is modulated by carbon source and growth stage. Transposon mutagenesis failed to find any potential trans-acting factors effective in the regulation of phs in relation to oxygen. Spontaneous mutants capable of expressing phs-lac aerobically were isolated and characterized. These mutations are closely linked to phs and affect not only oxygen regulation but also the requirement for cyclic AMP and reduced sulphur. Analysis of merodiploid strains indicates that these mutations cis-acting and that phs is not subject to autoregulation.     

Salmonella typhimurium mutants defective in the formate dehydrogenase linked to nitrate reductase.

Six fdn mutants of Salmonella typhimurium defective in the formation of nitrate reductase-linked formate dehydrogenase (FDHN) but capable of producing both the hydrogenase-linked formate dehydrogenase (FDHH) and nitrate reductase were characterized. Results of phage P22 transduction experiments indicated that there may be three fdn genes located on the metE-metB chromosomal segment and distinct from all previously identified fdh and chl loci. All six FDHH+ FDHN- mutants were found to make FDHN enzyme protein which was indistinguishable from that of the wild type in electrophoretic studies. However, the results of the spectral studies indicated that all six mutants were defective in the anaerobic cytochrome b559 associated with FDHN. All contained the cytochrome b559 associated with nitrate reductase in amounts equal to or greater than the wild type. The results of the transduction experiments also indicated that the metE- metB segment of the Salmonella chromosome resembles that of Escherichia coli more than was originally thought.     

Genetic analysis of mutants of Salmonella typhimurium deficient in formate dehydrogenase activity

Summary A genetical study of mutants of Salmonella typhimurium deficient in formate dehydrogenase activity was performed. The affected gene was designated fdh A and mapped at 116 min, the order of genes in that region being xyl-fdh A-mtl-cys E.Abbreviations FHL formate hydrogenylase - FDH (PMS) formate dehydrogenase (phenazine methosulfate) - FDH (BV) formate dehydrogenase (benzyl viologen) - HYD hydrogenase - NR nitrate-reductase - TTR tetrathionate-reductase     

Genetic characterization of the glutamate dehydrogenase gene (gdhA) of Salmonella typhimurium.

Salmonella typhimurium mutants, either devoid or glutamate dehydrogenase activity or having a thermolabile glutamate dehydrogenase protein, were used to identify the structural gene (gdhA) for this enzyme. Transductions showed that the mutations producing these phenotypes were linked to both the pncA and nit genes, placing the gdhA locus between 23 and 30 U on the S. typhimurium chromosome. Additional transductions with several Tn10 insertions established the gene order as pncA-gdhA-nit. Since few genetic markers exist in this region of the chromosome, Hfr strains were constructed to orient the pncA-gdhA-nit cluster with outside genes. Conjugation experiments provided evidence for the gene order pyrD-pncA-gdhA-nit-trp. To further characterize gdhA, we used Mu cts d1 (Apr lac) insertions in this gene to select numerous strains containing deletions with various endpoints. Transductions of these deletions with strains containing different gdh mutations and with a mutant having a thermolabile glutamate dehydrogenase protein permitted us to construct a deletion map of the gdhA region.     

Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression.

The spoT gene of Salmonella typhimurium has been identified. Mutations in spoT map between gltC and pyrE at 79 min. The spoT1 mutant has elevated levels of guanosine 5'-diphosphate-3'-diphosphate (ppGpp) during steady-state growth and exhibits a slower than normal decay of ppGpp after reversal of amino acid starvation. The spoT1 mutation elevates his operon expression but is distinct from known his regulatory mutations. Elevated his operon expression in spoT mutants causes resistance to the histidine analogs, 1,2,4-triazole-3-alanine and 3-amino-1,2,4-triazole. These properties of spoT mutants allowed us to identify and characterize additional spoT mutants. Approximately 40% of these mutants are temperature sensitive for growth on minimal medium, suggesting that the spoT function is essential or that excessive accumulation of ppGpp is lethal.     

Regulation of the fdhF gene encoding the selenopolypeptide for benzyl viologen-linked formate dehydrogenase in Escherichia coli

Summary In Podospora anserina a chromosome walk near the mating type locus was made possible through isolation of genomic sequences linked to a plasmid integrated in this part of the genome. Genetic analysis of 86 transformants obtained from the 5 first cosmids of this walk was performed. These data and those reported elsewhere for cosmids resulting from another chromosome walk allow us to draw two clear-cut rules for transformation with cosmids. First, the large majority of transformants arise from integration at the resident locus, contrasting with the heterologous process which predominates for plasmids. Second, all homologous integrations are highly unstable while all non-homologous integrations are stable. Analysis of the timing of the instability reveals that loss of the selective marker is probably limited to the fruiting body.     

Histidine regulation in Salmonella typhimurium. 8. Mutations of the hisT gene

The hisT gene, one of six genes in which mutation causes derepression of the histidine operon in Salmonella typhimurium, is shown to code for a protein that is not essential for the growth of the bacteria. This is indicated by the characterization of particular classes of mutations in the hisT gene: amber mutations, frame-shift mutations, and temperature-sensitive mutations that affect repression but not growth. In addition, the class of semilethal mutations was selected for but not found.     

Mutations of putP that alter the lithium sensitivity of Salmonella typhimurium

The putP gene encodes the major proline permease in Salmonella typhimurium that couples transport of proline to the sodium electrochemical gradient. To identify residues involved in the cation binding site, we have isolated putP mutants that confer resistance to lithium during growth on proline. Wild-type S. typhimurium can grow well on proline as the sole carbon source in media supplemented with NaCl, but grows poorly when LiCl is substituted for NaCl. In contrast to the growth phenotype, proline permease is capable of transporting proline via Na+/proline or Li+/proline symport. Therefore, we selected mutants that grow well on media containing proline as the sole carbon source in the presence of lithium ions. All of the mutants assayed exhibit decreased rates of Li+/proline and Na+/proline cotransport relative to wild type. The location of each mutation was determined by deletion mapping: the mutations cluster in two small deletion intervals at the 5' and 3' termini of the putP gene. The map positions of these lithium resistance mutations are different from the locations of the previously isolated substrate specificity mutations. These results suggest that Lir mutations may define domains of the protein that fold to form the cation binding site of proline permease.     

Mutations affecting glutamine synthetase activity in Salmonella typhimurium.

A positive selection procedure has been devised for isolating mutant strains of Salmonella typhimurium with altered glutamine synthetase activity. Mutants are derived from a histidine auxotroph by selecting for ability to grow on D-histidine as the sole histidine source. We hypothesize that the phenotype may be based on a regulatory increase in the activities of the D-histidine racemizing enzymes, but this has not been established. Spontaneous glutamine-requiring mutants isolated by the above selection procedure have two types of alterations in glutamine synthetase activity. Some have less than 10% of parent activity. Others have significant glutamine synthetase activity, but the enzyme have an altered response to divalent cations. Activity in mutants of the second type mimics that of highly adenylylated wild-type enzyme, which is believed to be in-active in vivo. Glutamine synthetase from one such mutant is more heat labile than wild-type enzyme, indicating that it is structurally altered. Mutations in all strains are probably in the glutamine synthetase structural gene (glnA). They are closely linked on the Salmonella chromosome and lie at about min 125. The mutants have normal glutamate dehydrogenase activity.     

Differential gene expression in culturable and non-culturable Salmonella typhimurium

Differential gene expression in culturable and non-culturable forms of Salmonella typhimurium was studied by the molecular display method. Six fragments of differentially expressed gene cDNA, depending on culturable or non-culturable state of the cultures, were isolated, cloned, and sequenced. Identification of corresponding S. typhimurium differentially expressed genes was carried out by comparing the sequences of cDNA fragments with the bacterial genome data base.     

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.     

Regulatory gene of phenylalanine biosynthesis (pheR) in Salmonella typhimurium

4-Fluorophenylalanine-resistant mutants of Salmonella typhimurium were isolated in which synthesis of chorismate mutase P-prephenate dehydratase (specified by pheA) was highly elevated. Transduction analysis showed that the mutation affecting pheA activity was not linked to pheA, and conjugation and merodiploid analysis indicated that it was in the 95- to 100-min region of the Salmonella chromosome. Evidence is presented for the hypothesis that the mutation responsible for constitutivity of chorismate mutase P-prephenate dehydratase occurred in pheR, a gene specifying a cytoplasmic product that affected pheA. pheR mutants were found to carry a second mutation, tyrO. The tyrO mutation acts cis to cause increased levels of the tyrosine biosynthetic enzymes 3-deoxy-d-arabinoheptulosonate 7-phosphate synthetase (tyr) and prephenate dehydrogenase, but it has no effect on regulation of pheA.