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.     

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.     

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.     

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.     

Altered regulation of isoleucine-valine biosynthesis in a hisW mutant of Salmonella typhimurium.

Control of isoleucine-valine biosynthesis was examined in the cold-sensitive hisW3333 mutant strain of Salmonella typhimurium. During growth at the permissive temperature (37 degrees C), the isoleucine-valine (ilv) biosynthetic enzyme levels of the hisW mutant were two- to fourfold below these levels in an isogenic hisW+ strain. Upon a reduction in growth temperature to partially permissive (30 degrees C), the synthesis of these enzymes in the hisW mutant was further reduced. However, synthesis of the ilv enzymes was responsive to the repression signal(s) caused by the addition of excess amounts of isoleucine, valine, and leucine to the hisW mutants. Such a "super-repressed" phenotype as that observed in this hisW mutant is similar to that previously shown for the hisU1820 mutant, but was different from the regulatory response of the hisT1504 mutant strain. Moreover, by the use of growth-rate-limiting amounts of the branched-chain amino acids, it was shown that this hisW mutant generally did not increase the synthesis of the ilv enzymes as did the hisW+ strain. Overall, these results are in agreement with the hypothesis that the hisW mutant is less responsive to ilv specific attenuation control than is the hisW+ strain and suggest that this limited regulatory response is due to an alteration in the amount or structure of an element essential to attenuation control of the ilv operons.     

Mutations affecting a regulated, membrane-associated esterase in Salmonella typhimurium LT2

Mutations at the apeA locus in Salmonella typhimurium lead to loss of a soluble enzyme (protease I) that hydrolyzes the chromogenic endoprotease substrate N-acetyl phenylalanine -naphthyl ester. We have isolated pseudorevertants of S. typhimurium apeA mutations that have regained the ability to hydrolyze this compound. These pseudorevertants contain mutations (apeR) that lead to overproduction of a membrane-bound esterase different from protease I. The apeR locus is phage P1 cotransducible with ilvC (83 map units) and is unlinked to apeA. Mutations at still another locus, apeE, lead to loss of the membrane-associated esterase. The apeE locus is P1 cotransducible with purE (12 map units). In an apeE-lacZ operon fusion strain, an apeR mutation increases the level of -galactosidase approximately 60-fold. We propose that apeR encodes a repressor of apeE. The evidence available suggests that the ApeE protein is not a protease.     

Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium.

Mutations in the fliK gene of Salmonella typhimurium commonly cause failure to terminate hook assembly and initiate filament assembly (polyhook phenotype). Polyhook mutants give rise to pseudorevertants which are still defective in hook termination but have recovered the ability to assemble filament (polyhook-filament phenotype). The polyhook mutations have been found to be either frameshift or nonsense, resulting in truncation of the C terminus of FliK. Intragenic suppressors of frameshift mutations were found to be ones that restored the original frame (and therefore the C-terminal sequence), but in most cases with substantial loss of natural sequence and sometimes the introduction of artificial sequence; in no cases did intragenic suppression occur when significant disruption remained within the C-terminal region. By use of a novel PCR protocol, in-frame deletions affecting the N-terminal and central regions of FliK were constructed and the resulting phenotypes were examined. Small deletions resulted in almost normal hook length control and almost wild-type swarming. Larger deletions resulted in loss of control of hook length and poor swarming. The largest deletions severely affected filament assembly as well as hook length control. Extragenic suppressors map to an unlinked gene, flhB, which encodes an integral membrane protein (T. Hirano, S. Yamaguchi, K. Oosawa, and S.-I. Aizawa, J. Bacteriol. 176:5439-5449, 1994; K. Kutsukake, T. Minamino, and T. Yokoseki, J. Bacteriol. 176:7625-7629, 1994). They were either point mutations in the C-terminal cytoplasmic region of FlhB or frameshift or nonsense mutations close to the C terminus. The processes of hook and filament assembly and the roles of FliK and FlhB in these processes are discussed in light of these and other available data. We suggest that FliK measures hook length and, at the appropriate point, sends a signal to FlhB to switch the substrate specificity of export from hook protein to late proteins such as flagellin.     

Influence of methionine biosynthesis on serine transhydroxymethylase regulation in Salmonella typhimurium LT2.

The enzyme serine transhydroxymethylase (EC 2.1.2.1; L-serine:tetrahydrofolate-5,10-hydroxymethyltransferase) is responsible both for the synthesis of glycine from serine and production of the 5,10-methylenetetrahydrofolate necessary as a methyl donor for methionine synthesis. Two mutants selected for alteration in serine transhydroxymethylase regulation also have phenotypes characteristic of metK (methionine regulatory) mutants, including ethionine, norleucine, and alpha-methylmethionine resistance and reduced levels of S-adenosylmethionine synthetase (EC 2.5.1.6; adenosine 5'-triphosphate:L-methionine S-adenosyltransferase) activity. Because this suggested the existence of a common regulatory component, the regulation of serine transhydroxymethylase was examined in other methionine regulatory mutants (metK and metJ mutants). Normally, serine transhydroxymethylase levels are repressed three- to sixfold in cells grown in the presence of serine, glycine, methionine, adenine, guanine, and thymine. This does not occur in metK and metJ mutants; thus, these mutations do affect the regulation of both serine transhydroxymethylase and the methionine biosynthetic enzymes. Lesions in the metK gene have been reported to reduce S-adenosylmethionine synthetase levels. To determine whether the metK gene actually encodes for S-adenosylmethionine synthetase, a mutant was characterized in which this enzyme has a 26-fold increased apparent Km for methionine. This mutation causes a phenotype associated with metK mutants and is cotransducible with the serA locus at the same frequency as metK lesions. Thus, the affect of metK mutations on the regulation of glycine and methionine synthesis in Salmonella typhimurium appears to be due to either an altered S-adenosylmethionine synthetase or altered S-adenosylmethionine pools.     

UGA Nonsense Mutations in Salmonella typhimurium

Salmonella typhimurium strain LT-2 carries a weak UGA suppressor activity. This activity prevents the detection of some UGA mutants as auxotrophs and probably accounts for the rarity of his UGA mutants in this strain. A selection method is described which permits the isolation of these rare his UGA mutants. Map distribution of his UGA mutations is normal, and their polarity effects are indistinguishable from the polarity effects of amber and ochre mutations at similar locations. Isolation and properties of a prototrophic his UGA mutant are described. UGA mutants are common among lac mutants isolated from Salmonella strains carrying an F'lac episome. Apparently the suppressor activity is insufficient to prevent detection of lac UGA mutants. It is not yet clear whether the suppressor activity plays an important role in normal cell physiology.     

Expression of 9 Salmonella typhimurium enzymes for cobinamide synthesis. Identification of the 11-methyl and 20-methyl transferases of corrin biosynthesis.

Nine of the cbi genes from the 17.5 kb cob operon of Salmonella typhimurium previously shown by genetic studies to be involved in the biosynthesis of cobinamide from precorrin-2, have been subcloned and expressed in Escherichia coli. Seven of the gene products were found in the soluble fraction of cell lysates and have been purified. The gene products corresponding to cbi E, F, H and L were shown by SAM binding and by homology with other SAM-binding proteins to be candidates for the methyltransferases of vitamin B12 biosynthesis. The enzymatic functions of the gene products of cbiL and cbiF are associated with C-methylation at C-20 of precorrin-2 and C-11 of precorrin-3.     

A locus affecting nucleoid segregation in Salmonella typhimurium.

Thirteen temperature-sensitive lethal mutations of Salmonella typhimurium map near metC at 65 min and form the clmF (conditional lethal mutation) locus. The mutations in this region were ordered by three-point transduction crosses. After a shift to the nonpermissive temperature, many of these clmF mutants failed to complete the segregation of nucleoids into daughter cells; daughter nucleoids appeared incompletely separated and asymmetrically positioned within cells. Some clmF mutants showed instability of F' episomes at permissive growth temperatures yet showed no detectable defect with smaller multicopy plasmids such as pSC101 or pBR322. In addition, many of the clmF mutants rapidly lost viability yet continued DNA replication at the nonpermissive temperature. These results suggest that the clmF locus encodes at least one indispensable gene product that is required for faithful partitioning of the bacterial nucleoid and F-plasmid replicons.     

Oxygen regulation in Salmonella typhimurium

Regulation by oxygen of the peptidase T (pepT) locus of Salmonella typhimurium was studied by measuring beta-galactosidase levels in strains containing a pepT::Mu d1(Apr lac) operon fusion. beta-Galactosidase was induced in anaerobic cultures and late-exponential and stationary-phase aerated cultures. Peptidase T activity also was induced under these growth conditions. pepT+ but not pepT strains will utilize as amino acid sources the tripeptides Leu-Leu-Leu and Leu-Gly-Gly only when grown anaerobically. Mutations at two loci, oxrA and oxrB (oxygen regulation) prevent induction of the pepT locus. The oxrA locus is homologous to the fnr locus of Escherichia coli. We have isolated 12 independent Mu d1 insertions (oxd::Mu d1, oxygen dependent) that show induction of beta-galactosidase in anaerobic cultures and stationary-phase aerated cultures. These insertions fall into nine classes based on map location. All of the oxd::Mu d1 insertions are regulated by oxrA and oxrB and therefore define a global regulon that responds to oxygen limitation.     

鼠伤寒沙门氏菌维生素B2生物合成的调节

7 independent rib genes fusions with MudJ (lacZ, Kanr) were isolated by transposon MudJ mutagenesis in Salmonella typhimurium. 5 of them are blue on the X-gal plate, and the beta-galactosidase activity of the cells grown in E medium containing various concentration of riboflavin were assayed. The results showed that the expression of rib gene are not repressed by riboflavin. It appears to be synthesized constitutively in Salmonella typhimurium.     

Interference of azide with cysteine biosynthesis in Salmonella typhimurium.

The growth inhibition of Salmonella typhimurium aziA mutants by sodium azide is reversed by cystine and related compounds. NADPH-sulphite reductase (hydrogen-sulphide:NADP+ oxidoreductase; EC 1.8.1.2), an enzyme of cysteine biosynthesis, is inhibited in cell extracts by sodium azide. AziB mutants which are able to grow in the presence of the inhibitor without cystine were isolated. About half of them were mapped in the cysK gene and have only residual activity of its product, O-acetylserine sulphydrylase A [O-acetyl-L-serine acetate-lyase (adding hydrogen-sulphide); EC 4.2.99.8]. Sensitivity of wild type and aziA mutants to azide was also reversed by a constitutive mutation in cysB, the regulatory gene of cysteine biosynthesis. CysK and cysB mutants showed cross-resistance to azide and 1,2,4-triazole. It is suggested that the resistance of these mutants to azide is due to an increased activity of NADPH-sulphite reductase.     

An indispensable gene for NAD biosynthesis in Salmonella typhimurium.

We have located the nadD locus between lip and leuS at 14 min on the Salmonella typhimurium chromosome, and we have shown it to be the structural gene for nicotinic acid mononucleotide adenylyltransferase. This is the first indispensable gene of pyridine nucleotide metabolism that has been identified. Mutants altered at this locus, isolated by their 6-aminonicotinamide resistance phenotype, accumulate abnormally large pools of nicotinic acid mononucleotide in vivo; many exhibit a temperature-sensitive lethal phenotype. Enzyme assays reveal markedly lower transferase activity in mutant extracts than in nadD+ extracts. The partial dominance of nadD mutants when placed in a nadD+/nadD diploid suggests that nicotinic acid mononucleotide adenylyltransferase is a multimeric enzyme.     

Identification of a repressor gene involved in the regulation of NAD de novo biosynthesis in Salmonella typhimurium.

Mutations at the nadI locus affect expression of the first two genes of NAD synthesis, nadA and nadB, which are unlinked. Genetic data imply that the regulatory effects of nadI mutations are not due to indirect consequences of physiological alterations. Two types of mutations map in the nadI region. Common null mutations (nadI) show constitutive high-level expression of the nadB and nadA genes. Rare nadIs mutations cause constitutive low-level expression of nadB and nadA. Some nadIs mutations shut off the expression of the biosynthetic genes sufficiently to cause a nicotinic acid auxotrophy. Spontaneous revertants of auxotrophic nadIs mutants have a NadI- phenotype, including some with deletions of the nadI locus. The nadI locus encodes a repressor protein acting on the unlinked nadA and nadB genes.