Cloning of the regulatory locus ompB of Salmonella typhimurium LT-2

Summary We have cloned the complete functional ompB locus of Salmonella typhimurium LT-2 into Escherichia coli K-12 using a cosmid vector and in vitro packaging into . The ompB locus of Salmonella was found to complement both envZ and ompR mutations in E. coli as well as an ompR mutation of Salmonella. The ompR part of the ompB locus was further subcloned into the multicopy plasmid pKN410 as a 1.3 kb fragment. This fragment coded for a single 28.5 kd protein corresponding to about 820 bp in length. Furthermore, the OmpR proteins of S. typhimurium and E. coli were shown to be structurally and functionally highly similar.Abbreviations SDS sedium dodecyl sulfate - kb kilobase pairs - bp base pairs - kd kilodaltons     

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.     

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.     

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.     

Compounds Which Serve as the Sole Source of Carbon or Nitrogen for Salmonella typhimurium LT-2

About 600 compounds were screened as possible carbon or nitrogen sources for Salmonella typhimurium LT-2. About 100 utilizable compounds were found.     

IlvHI locus of Salmonella typhimurium.

In Escherichia coli K-12, the ilvHI locus codes for one of two acetohydroxy acid synthase isoenzymes. A region of the Salmonella typhimurium genome adjacent to the leucine operon was cloned on plasmid pBR322, yielding plasmids pCV47 and pCV49 (a shortened version of pCV47). This region contains DNA homologous to the E. coli ilvHI locus, as judged by hybridization experiments. Plasmid pCV47 did not confer isoleucine-valine prototrophy upon either E. coli or S. typhimurium strains lacking acetohydroxy acid synthase activity, suggesting that S. typhimurium lacks a functional ilvHI locus. However, isoleucine-valine prototrophs were readily isolated from such strains after mutagenesis with nitrosoguanidine. In one case we found that the Ilv+ phenotype resulted from an alteration in bacterial DNA on the plasmid (new plasmid designated pCV50). Furthermore, a new acetohydroxy acid synthase activity was observed in Ilv+ revertants; this enzyme was similar to E. coli acetohydroxy acid synthase III in its lack of activity at low pH. This new activity was correlated with the appearance in minicells of a new polypeptide having an approximate molecular weight of 61,000. Strains carrying either pCV49 or pCV50 produced a substantial amount of ilvHI-specific mRNA. These results, together with results from other laboratories, suggest that S. typhimurium has functional ilvB and ilvG genes and a cryptic ilvHI locus. E. coli K-12, on the other hand, has functional ilvB and ilvHI genes and a cryptic ilvG locus.     

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.     

Deoxyribonucleic Acid Breaks in Heated Salmonella typhimurium LT-2 After Exposure to Nutritionally Complex Media

Minimal medium recovery of heat-treated Salmonella typhimurium LT-2 has been expressed as the reduced viability on trypticase soy agar supplemented with 0.5% yeast extract (TSY) relative to a glucose-salts (M-9) agar. Incubation of S. typhimurium LT-2 in water at 50 C for 15 min did not change the sedimentation patterns of deoxyribonucleic acid (DNA) in alkaline sucrose gradients. The same pattern was obtained if the heated cells were further incubated for 15 min at 37 C in M-9 broth. However, a marked increase in DNA single-strand breakage accompanied by a loss of viability was observed after a similar incubation of heated bacteria in TSY broth. If heated bacteria were incubated in M-9 broth before TSY broth, there was a decrease in the single-strand breakage occurring in the TSY broth. This decrease is believed to be the result of repair of heat-induced damage. We conclude that minimal medium recovery after heat treatment is due to DNA damage caused by sequential exposure to heat and TSY medium, such damage not occurring after sequential exposure to heat and M-9 medium.     

Mechanism of O-acetylserine sulfhydrylase fromSalmonella typhimurium LT-2

Summary O-Acetylserine sulfhydrylase is a pyridoxal 5-phosphate (PLP) dependent enzyme that catalyzes the final step of L-cysteine biosynthesis inSalmonella, viz. the conversion of O-acetyl-L-serine (OAS) and sulfide to L-cysteine and acetate. A spectrophotometric assay is available using 5-thio(2-nitrobenzoate) (TNB) as an analog of sulfide and monitoring the disappearance of absorbance at 412 nm. The enzyme catalyzes a ping pong mechanism with-aminoacrylate in Schiff base with the active site PLP as a covalent intermediate. Using data obtained from the pH dependence of kinetic parameters, the acid-base chemical mechanism and the optimum protonation state of enzyme and substrate functional groups necessary for binding has been determined. The Schiff base and the-amine of the substrate OAS are unprotonated for binding. There also appears to be a requirement for one active site general base to accept a proton from the-amine and to donate a proton to form cysteine. The enzyme also catalyzes an OAS hydrolase activity, and the pH dependence of this reaction suggests that the active site lysine that participated in the Schiff base linkage is protonated to start the second half reaction, and has a pK of about 8.2. The stereochemistry of³H-borohydride reduction of the Schiff base in free enzyme has been determined by degradation of the resulting pyridoxyllysine to pyridoxamine and measuring³H-release with apo-aspartate aminotransferase. The sequence around the active site lysine is AsnProSerPheSerValLysCysArg.     

Biosynthesis of bacterial glycogen: purification and properties of Salmonella typhimurium LT-2 adenosine diphosphate glucose pyrophosphorylase.

The adenosine diphosphate glucose pyrophosphorylase from a Salmonella typhimurium LT-2 mutant, JP102, derepressed in the glycogen biosynthetic enzymes was purified to homogeneity. The enzyme was found to be identical with the parent wild-type enzyme with respect to regulatory properties, immunological reactivity, and kinetic constants for the allosteric effectors and for the substrate, adenosine triphosphate. The JP102 enzyme was composed of four identical subunits, each with a molecular weight of about 48,000. This was supported by the findings that (i) gel electrophoresis under denaturing conditions showed only one component; (ii) digestion with carboxypeptidase B released stoichiometric amounts of arginine, and (iii) amino-terminal sequencing showed a single sequence for the first 27 residues. The properties of the purified S. typhimurium enzyme were compared with the properties of the previously purified Escherichia coli B enzyme.     

Salmonella typhimurium LT-2 mutants with altered glutamine synthetase levels and amino acid uptake activities.

To determine whether Salmonella typhimurium has a nitrogen control response, we have examined the regulation of nitrogen utilization in two mutants with fivefold and threefold elevations in their glutamine synthetase activities. The mutants do not require glutamine for growth on glucose--ammonia medium but do have altered growth on other nitrogen sources. They grow better than an isogenic control on media containing arginine or asparate, but more slowly with proline or alanine as nitrogen sources. This unusual growth pattern is not due to altered regulation of the ammonia assimilatory enzymes, glutamate dehydrogenase and glutamate synthase, or to changes in the enzymes for aspartate degradation. However, transport for several amino acids may be affected. Measurement of amino acid uptake show that the mutants with high glutamine synthetase levels have increased rates for glutamine, arginine, aspartate, and lysine, but a decreased rate for proline. The relationship between glutamine synthetase levels and uptake was examined in two mutants with reduced, rather than increased, glutamine synthetase production. The uptake rates for glutamine and lysine were lower in these two glutamine auxotrophs than in the Gln+ controls. These results show a correlation between the glutamine synthetase levels and the uptake rates for several amino acids. In addition, the pleiotropic growth of the mutants with elevated glutamine synthetase activities suggests that a nitrogen control response exists for S. typhimurium and that it can be altered by mutations affecting glutamine synthetase regulation.     

Identification of a umuDC locus in Salmonella typhimurium LT2.

The umuDC operon of Escherichia coli is required for efficient mutagenesis by UV light and many other DNA-damaging agents. The existence of a umuDC analog in Salmonella typhimurium has been questioned. With DNA probes to the E. coli umuD and umuC genes, we detected, by Southern blot hybridization, sequences similar to both of these genes in S. typhimurium LT2. We also confirmed that the presence of cloned E. coli umuD enhances the UV mutability and resistance of S. typhimurium. Our data strongly suggest that S. typhimurium contains a functional umuDC operon.     

Genetic mapping of the Salmonella typhimurium pepB locus.

Transposon technology has been used to map the pepB locus of Salmonella typhimurium. This locus is cotransducible by phage P22 with glyA and strB at min 56 on the Salmonella genetic map. The gene order is strB pepB glyA.     

Genetic mapping of the Salmonella typhimurium pncB locus.

The nicotinic acid phosphoribosyltransferase locus pncB was located on the Salmonella typhimurium linkage map counterclockwise relative to pyrC. P22 and P1 transductional analyses revealed linkage of pncB with aroA and pyrD, indicating a pncB map position of approximately 20 map units. The results of these cotransduction experiments also indicated that the genetic map distance between gal and pyrD is greater than the published 2.2 map units.