L-Methionine SR-sulfoximine-resistant glutamine synthetase from mutants of Salmonella typhimurium

Two mutants of Salmonella typhimurium resistant to growth inhibition by the glutamine synthetase transition state analog, L-methionine SR-sulfoximine, were isolated and characterized. These mutants are glutamine bradytrophs and cannot use growth rate-limiting nitrogen sources. Although this phenotype resembles that of mutants with lesions in the regulatory gene for glutamine synthetase, glnG, these mutations do not lie in the glnG gene. Purification and characterization of the glutamine synthetase from one of the mutants and a control strain demonstrated that the mutant enzyme is defective in the reverse gamma-glutamyltransferase activity but has biosynthetic activity that is resistant to inhibition by L-methionine SR-sulfoximine. The mutant enzyme also has a 4.4-fold higher apparent Km for glutamate (0.2 mM versus 2.1 mM, respectively) and a 13.8-fold higher Km for NH3 (6.4 mM versus 0.46 mM) than the enzyme from the control. These data show that the glutamine synthetase protein has been altered by this mutation, designated as glnA982, and suggest that the L-methionine SR-sulfoximine resistance is conferred by a change in the NH3 binding domain of the enzyme.     

Studies on ribosomal mutants of Salmonella typhimurium LT-2

Summary Mutations conferring resistance to spectinomycin, resistance to neomycinkanamycin and cold-sensitivity of ribosome biosynthesis were located on the Salmonella typhimurium chromosome in relation to the genes cysG, strA, and aroC. The effect of temperature and growth medium on the expression of these mutations, and the interactions between the mutations was determined as a prerequisite to the mapping. On the basis of chromosomal location and dominance, it is concluded that cold-sensitivity of ribosome synthesis can result from mutations in several distinct genes.This work was supported by Public Health Service research grant AI-05526.     

Derepressed levels of glutamate synthase and glutamine synthetase in Escherichia coli mutants altered in glutamyl-transfer ribonucleic acid synthetase.

The levels of glutamate synthase and of glutamine synthetase are both derepressed 10-fold in strain JP1449 of Escherichia coli carrying a thermosensitive mutation in the glutamyl-transfer ribonucleic acid (tRNA) synthetase and growing exponentially but at a reduced rate at a partially restrictive temperature, compared with the levels in strain AB347 isogenic with strain JP1449 except for this thermosensitive mutation and the marker aro. These two enzymes catalyze one of the two pathways for glutamate biosynthesis in E. coli, the other being defined by the glutamate dehydrogenase. We observed a correlation between the percentage of charged tRNAGlu and the level of glutamate synthase in various mutants reported to have an altered glutamyl-tRNA synthetase activity. These results suggest that a glutamyl-tRNA might be involved in the repression of the biosynthesis of the glutamate synthase and of the glutamine synthetase and would couple the regulation of the biosynthesis of these two enzymes, which can work in tandem to synthesize glutamate when the ammonia concentration is low in E. coli but whose structural genes are quite distant from each other. No derepression of the level of the glutamate dehydrogenase was observed in mutant strain JP1449 under the conditions where the levels of the glutamine synthetase and of the glutamate synthase were derepressed. This result indicates that the two pathways for glutamate biosynthesis in E. coli are under different regulatory controls. The glutamate has been reported to be probably the key regulatory element of the biosynthesis of the glutamate dehydrogenase. Our results indicate that the cell has chosen the level of glutamyl-tRNA as a more sensitive probe to regulate the biosynthesis of the enzymes of the other pathway, which must be energized at a low ammonia concentration.     

Nitrogen control of Salmonella typhimurium: co-regulation of synthesis of glutamine synthetase and amino acid transport systems.

Nitrogen control in Salmonella typhimurium is not limited to glutamine synthetase but affects, in addition, transport systems for histidine, glutamine, lysine-arginine-ornithine, and glutamate-aspartate. Synthesis of both glutamine synthetase and transport proteins is elevated by limitation of nitrogen in the growth medium or as a result of nitrogen (N)-regulatory mutations. Increases in the amounts of these proteins were demonstrated by direct measurements of their activities, by immunological techniques, and by visual inspection of cell fractions after gel electrophoresis. The N-regulatory mutations are closely linked on the chromosome to the structural gene for glutamine synthetase, glnA: we discuss the possibility that they lie in a regulatory gene, glnR, which is distinct from glnA. Increases in amino acid transport in N-regulatory mutant strains were indicated by increased activity in direct transport assays, improved growth on substrates of the transport systems, and increased sensitivity to inhibitory analogs that are trnasported by these systems. Mutations to loss of function of individual transport components (hisJ, hisP, glnH, argT) were introduced into N-regulatory mutant strains to determine the roles of these components in the phenotype and transport behavior of the strains. The structural gene for the periplasmic glutamine-binding protein, glnH, was identified, as was a gene argT that probably encodes the structure of the lysine-arginine-ornithine-binding protein. Genes encoding the structures of the histidine- and glutamine-binding proteins are not linked to glnA or to each other by P22-mediated transduction; thus, nitrogen control is exerted on several unlinked genes.     

The regulation of glutamine transport and glutamine synthetase in Salmonella typhimurium.

Transport of glutamine by the high-affinity transport system is regulated by the nitrogen status of the medium. With high concentrations of ammonia, transport is repressed; whereas with Casamino acids, transport is elevated, showing behaviour similar to glutamine synthetase. A glutamine auxotroph, lacking glutamine synthetase activity, had elevated transport activity even in the presence of high concentrations of ammonia (and glutamine). This suggests that glutamine synthetase is involved in the regulation of the transport system. A mutant with low glutamate synthase activity had low glutamine transport and glutamine synthetase activities, which could not be derepressed. A mutant in the high-affinity glutamine transport system showed normal regulation of glutamate synthase and glutamine synthetase. Possible mechanisms for this regulation are discussed.     

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.     

Biochemical and genetic characterization of l-glutamate transport and utilization in Salmonella typhimurium LT-2 mutants

Two systems for l-glutamate transport were found in Salmonella typhimurium LT-2 GltU⁺ (glutamate utilization) mutants. The first one is similar to the glt system previously described in Escherichia coli; by transductional analysis the structural gene, gltS, coding for the transport protein was located at minute 80 of the chromosome as part of the operon gltC-gltS, and its regulator, the gltR gene, near minute 90; the gltS gene product transports both l-glutamate and l-aspartate, is sodium independent, and is -hydroxyaspartate sensitive. The second transport system, whose structural gene was called gltF and is located at minute 0, was l-glutamate specific, sodium independent, and -methylglutamate sensitive. Two aspartase activities occurred in S. typhimurium LT-2: the first one was present only in the GltU⁺ mutants, had a pH 6.4 optimum, was essential for both l-glutamate and l-aspartate metabolism, and mapped at minute 94, close to the ampC gene. The second one had a pH 7.2 optimum, could be induced by several amino acids, and thus may have a general role in nitrogen metabolism.     

New Salmonella typhimurium mutants with altered outer membrane permeability

We describe three new classes of Salmonella typhimurium mutants with increased sensitivity to hydrophobic agents. In contrast to many previously described mutants, the phage sensitivity pattern of these mutants did not give any indication of defective lipopolysaccharide. Furthermore, they had no detectable changes in their phospholipid or outer membrane protein composition, and their growth rate and cell morphology were normal. Class B mutants were nearly as sensitive to novobiocin, fusidic acid, erythromycin, rifampin, and clindamycin as are deep rough (heptoseless) mutants; in addition they were sensitive to methicillin, penicillin (to which heptoseless mutants are resistant), gentian violet, and anionic and cationic detergents. Class A and C mutants had less sensitive, but characteristic phenotypes. None of the three classes were sensitive to serum bactericidal action. The class B mutation mapped between map positions 7 and 11 on the S. typhimurium chromosome, and the class C mutation mapped between positions 5 and 7. The map position for the class A mutation remained undefined, but it was separate from the class B and C mutations and, like those, did not correspond to any gene loci known to participate in the synthesis of major outer membrane constituents.     

Isolation and crystallization of unadenylylated glutamine synthetase from Salmonella typhimurium

The enzyme glutamine synthetase (GS) has been isolated from a mutant strain of Salmonella typhimurium, constructed by Kustu, which lacks the enzymatic activity for adenylylation of glutamine synthetase. Thus the purified GS is uniformly unadenylylated, as confirmed by gel electrophoresis and enzyme assays. It crystallizes readily in many morphologies, at least six of which are distinct polymorphs. The most favorable crystal form for structural studies belongs to space group C2, with unit cell dimensions a = 235.5 A, b = 134.5 A, c = 200.1 A, beta = 102.8 degrees, and with one GS molecule per asymmetric unit. The crystals diffract to about 2.8 A resolution in rotation X-ray photographs and thus appear suitable for structural studies at moderate resolution. These crystals are isomorphous with crystalline GS from Escherichia coli in both adenylylated and unadenylylated states, suggesting that the enzymes from the two bacteria are similar molecules, and that adenylylation does not greatly affect the conformation of the molecule.     

Mapping and characterization of the nad genes in Salmonella typhimurium LT-2.

An ampicillin enrichment technique was used to isolate 39 nicotinic acid-requiring mutants of Salmonella typhimurium LT-2. Using interrupted-mating and transductional mapping procedures, three loci, designated nadA, nadB, and nadC, were identified. These loci mapped at 33, 82, and 6 min, respectively, on the S. typhimurium linkage map. The arrangement of the loci on the Salmonella linkage map corresponded closely to the nadA, nadB, and nadC loci on the Escherichia coli K-12 linkage map, indicating that the de novo pathway to nicotinamide adenine dinucleotide and the genes governing the enzymes involved in this pathway in S. typhimurium are very similar to those in E. coli. Evidence is also presented which indicates that the product of the nadC locus in S. typhimurium LT-2 is the enzyme quinolinic acid phosphoribosyltransferase. All nadC mutants of S. typhimurium secreted between 2 and 8 mumol of quinolinic acid per 100 ml of secretion medium. In addition, none of the nadC mutants isolated were able to grow in 10(-3) M quinolinic acid, whereas all nadA and nadB mutants of S. typhimurium grew well in the presence of quinolinic acid. Transductional crosses between nadB mutants provided evidence suggestive of more than one locus in the nadB region.     

Bacillus subtilis glutamine synthetase mutants pleiotropically altered in glucose catabolite repression.

Strain SF22, a glutamine-requiring (Gln-) mutant of Bacillus subtilis SMY, is likely to have a mutation in the structural gene for glutamine synthetase, since this strain synthesized 22 to 55% as much glutamine synthetase antigen as did wild-type cells in a 10-min period but had less than 3% of wild-type glutamine synthetase enzymatic activity. The expression of several genes subject to glucose catabolite repression was altered in the Gln- mutant. The induced levels of alpha-glucosidase, histidase, and aconitase were 3.5- to 4-fold higher in SF22 cells than in wild-type cells grown in glucose-glutamine medium, and citrate synthase levels were 8-fold higher in the Gln- mutant than in wild-type cells. The relief of glucose catabolite repression in the Gln- mutant may result from poor utilization of glucose. Examination of the intracellular metabolite pools of cells grown in glucose-glutamine medium showed that the glucose-6-phosphate pool was 2.5-fold lower, the pyruvate pool was 4-fold lower, and the 2-ketoglutarate pool was 2.5-fold lower in the Gln- cells than they were in wild-type cells. Intracellular levels of glutamine were sixfold higher in the Gln- mutant than in wild-type cells. Measurements of enzymes involved in glutamine transport and utilization showed that the elevated pools of glutamine in the Gln- mutant resulted from a threefold increase in glutamine permease and a fivefold decrease in glutamate synthase. The pleiotropic effect of the gln-22 mutation on the expression of several genes suggests that either the glutamine synthetase protein or its enzymatic product, glutamine, is involved in the regulation of several metabolic pathways in B. subtilis.     

A degradation pathway of propionate in Salmonella typhimurium LT-2

Salmonella typhimurium LT-2 can utilize propionate as its sole carbon source. Studies on growth, oxidation by resting cell suspensions and by permeabilized cells, suggest that the propionate is transported by the acetate system. This result was confirmed using labeled propionate and acetate. ATP-monocarboxylate phosphotransferase, acyl-CoA orthophosphate acyl-transferase, propionyl-CoA dehydrogenase, acrylyl-CoA hydratase, lactate dehydrogenase, phosphoenolpyruvate (PEP) synthase and PEP-carboxylase activities have been identified in extracts of cells grown on propionate. Mutants deficient in PEP-carboxylase and synthase are unable to utilize propionate. On the basis of results obtained, it seems that the propionate degradation pathway occurs via acrylate and that PEP-synthase and PEP-carboxylase are essential enzymes.     

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.     

Proline transport in Salmonella typhimurium: putP permease mutants with altered substrate specificity.

The putP gene encodes a proline permease required for Salmonella typhimurium LT2 to grow on proline as the sole source of nitrogen. The wild-type strain is sensitive to two toxic proline analogs (azetidine-2-carboxylic acid and 3,4-dehydroproline) also transported by the putP permease. Most mutations in putP prevent transport of all three substrates. Such mutants are unable to grow on proline and are resistant to both of the analogs. To define domains of the putP gene that specify the substrate binding site, we used localized mutagenesis to isolate rare mutants with altered substrate specificity. The position of the mutations in the putP gene was determined by deletion mapping. Most of the mutations are located in three small (approximately 100-base-pair) deletion intervals of the putP gene. The sensitivity of the mutants to the proline analogs was quantitated by radial streaking to determine the affinity of the mutant permeases for the substrates. Some of the mutants showed apparent changes in the kinetics of the substrates transported. These results indicate that the substrate specificity mutations are probably due to amino acid substitutions at or near the active site of proline permease.