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.     

Location of Sulfate-binding Protein in Salmonella typhimurium

A method is described for location of proteins in bacteria. It depends upon two techniques. One technique is the inactivation of the protein by a reagent which is incapable of penetrating the bacterial membrane (permeability barrier). Proteins inside this membrane cannot be inactivated unless the cells are disrupted; proteins on or outside the membrane can be inactivated. The second technique depends upon inactivation of the protein by specific antibody. Antibody should not penetrate the external bacterial wall, and therefore should only inactivate proteins that are on the wall surface. Thus, proteins can be localized inside the membrane, in the wall-membrane area, or outside the wall. One reagent developed for use with the first technique is diazo-7-amino-1,3-naphthalene-disulfonate. It inactivated beta-galactoside transport, but not beta-galactosidase of intact Escherichia coli. Similarly, it inactivated sulfate binding and transport but not uridine phosphorylase activity of Salmonella typhimurium. This indicates that the sulfate-binding protein is on or outside the cell membrane, and that uridine phosphorylase is inside the cell. The organic mercurial compounds used also showed that the sensitive parts of the sulfate and alpha-methylglucoside transport systems are less reactive than the sensitive part of the beta-galactoside system. Antibody to the sulfate-binding protein inactivated the purified protein but did not inactivate this protein when intact bacteria were employed. Thus, it appears that the sulfate-binding protein does not protrude outside the cell wall. The conclusion that the binding protein is located in the wall-membrane region is supported by its release upon spheroplast formation or osmotic shock, and also by its ability to combine with sulfate in bacteria which cannot transport sulfate into the cell.     

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 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.     

Cold-sensitive Mutations in Salmonella typhimurium Which Affect Ribosome Synthesis

A number of mutations (45) expressed as cold-sensitive conditional lethal pheno-types were screened by transduction for their linkage to the streptomycin-resistance locus; 7 showed such linkage. Of these, two were studied in greater detail. The sedimentation profiles of ribosomes from cultures grown at low temperature differed from wild type and from one another. Both mutants lost ribonucleic acid control at low temperature. It is suggested that a high proportion of mutants expressing a cold-sensitive phenotype harbor mutations in genes affecting ribosome synthesis or regulation.     

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.     

Location of a mutator gene in Salmonella typhimurium by cotransduction

Kirchner, Carl E. J. (Suffolk County Community College, Selden, N.Y.), and Matthew J. Rudden. Location of a mutator gene in Salmonella typhimurium by cotransduction. J. Bacteriol. 92:1453-1456. 1966.-The LT7 strain of Salmonella typhimurium has been shown to possess a mutator gene which is responsible for an increase in mutation frequency for most loci tested. Preliminary results suggested the gene might be responsible for the production of an abnormal purine or pyrimidine base. Phage prepared on the mutator strain were used to transduce selected purine and pyrimidine LT2 mutants that do not possess this gene. A high frequency (60%) of cotransduction was observed with mutants from only one locus, purA. Transduction of additional mutants from this region gave similar results, except for one mutant (purA1) which showed no transduction of the mutator gene or the purA1 region. The results show that the mutator gene is very closely linked to the purA locus and suggest that it might be part of it.     

Mutations in Salmonella typhimurium recovered from livers and spleens of mice

Balb/c mice were inoculated intraperitoneally with TA2662, a smooth derivative of the Salmonella typhimurium Ames tester strain (TA102) which carries the mutable hisG locus on a multicopy plasmid, or TA103, which carries the same hisG gene on the chromosome. The bacteria were recovered at various times from the livers and spleens of the infected mice. Total numbers of bacteria were determined and the mutant frequency was estimated. The frequency of occurrence of histidine prototrophs in experiments using TA2662 was substantially above the frequency found with this strain grown in vitro. The mutant frequencies in experiments using TA103 recovered from mice were also highly significantly increased above background. We did not identify factors which might suggest selection in vivo for histidine prototrophs. There is sufficient histidine in body fluids of the host for the growth of His- bacteria. The His- and His+ derivatives were found to grow equally well in vitro in the presence of amounts of histidine approximating concentrations known to exist in vivo. It is probable that mutations in TA2662 are greatly underestimated, since the hisG-containing plasmid is lost at relatively high frequency during incubation in a variety of conditions.     

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 that alter the covalent modification of glutamine synthetase in Salmonella typhimurium.

glnD and glnE mutant strains of Salmonella typhimurium lack three of the four activities required for reversible covalent modification of glutamine synthetase (GS; EC 6.3.1.2). The glnD strains, which are unable to deadenylylate GS and therefore accumulate the adenylylated or less active form of the enzyme, were isolated as glutamine bradytrophs. They lack the activity of PIIA uridylyl-transferase, one of the proteins required for deadenylylation of GS; in addition, they lack PIID uridylyl-removing activity. Mutations in glnD are suppressed by second-site mutations in glnE that eliminate the activity of GS adenylyltransferase (EC 2.7.7.42) and thus prevent adenylylation of GS. The glnD and glnE strains have one-third to one-half as much total GS as the wild-type strain when they are grown in a medium containing a high concentration of NH4+. The wild-type strain derepresses synthesis of GS fourfold in response to nitrogen limitation; glnD and glnE strains derepress synthesis of the enzyme fourfold and sevenfold, respectively. Thus, mutations that alter covalent modification of GS in Salmonella do not significantly affect derepression of its synthesis. The glnD gene lies at 7 min on the Salmonella chromosome and is 50% linked to pyrH by P22-mediated transduction.     

Location on the chromosome of Salmonella typhimurium of genes governing pyrimidine metabolism

Summary Genes encoding the enzymes cytidine deaminase (cdd), uridine monophosphate pyrophosphorylase (upp), cytidine triphosphate synthetase (pyrG), and uridine phosphorylase (udp) were located on theSalmonella typhimurium chromosome at 68, 77, 90 and 122 min, respectively. Strains carrying mutations inpyrG must also carry mutations incdd in order for cytidine to be sufficiently stable metabolically to supply the cell's requirement for CTP¹.     

Two Mutations in the First Gene of the Histidine Operon of Salmonella typhimurium Affecting Control

Two strains with mutations in the first structural gene of the histidine operon of Salmonella typhimurium were characterized. (The first structural gene specifies the first enzyme of histidine biosynthesis, phosphoribosyltransferase, which is sensitive to feedback inhibition by histidine.) One mutation, hisG3934, results in a phosphoribosyltransferase which is no longer sensitive to feedback inhibition by histidine but is instead subject to inhibition by aspartic acid. The other mutation, hisG3935, allows the histidine operon to be partially repressed by several amino acids, including aspartic acid. Analysis of hisG3935 is consistent with the hypothesis that phosphoribosyltransferase is directly involved in the regulation of the histidine operon.     

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 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.     

New Class of Streptomycin-Resistant Mutants Incompatible with supX Suppressor Mutations in Salmonella typhimurium

Streptomycin-resistant colonies of Salmonella typhimurium appearing in platings of supX suppressors of strain leu-500 are less variegated in size than are those derived from strain leu-500 counterparts. Several of the streptomycin-resistant leu-500 clones, furthermore, yield suppressors and revertants of the leu-500 auxotrophy at unusually low rates, suggesting that they provide a genetic background inimicable to supX suppression. Two such "suppression-restrictive" leu-500 streptomycin-resistant (str) mutants, designated strains M(1) and M(4), were characterized as to their ability to receive the trp-supX-cysB linkage region by transduction. Coentry of a donor supX deletion mutation with the selected trp(+) marker was not observed even though these sites display more than 10% linkage in control experiments. This was demonstrably the result of nonviability of the combined supX mutant, M(1) or M(4) streptomycin-resistant genotype, rather than the lack of suppression of the leu-500 imparted auxotrophy. Both M(1)- and M(4)-type resistance was accompanied by pleiotropic effects resembling those caused by strB (nonribosomal)- rather than strA (ribosomal)-type resistance, but both restrictive mutants had a high upper limit of resistance corresponding to that of strA-type mutants. Transduction analyses indicated that the str character of neither the M(1) nor the M(4) strain was linked to the strA or the strB gene. These mutations define a previously undescribed locus, which we propose to designate strC, apparently related to streptomycin uptake rather than its intracellular action. Mutation at this locus is evidently incompatible with the inactivation or removal of the supX site, suggesting a functional association between products of the genes.