Complex typing of Salmonella typhimurium hospital strains

A salmonellosis outbreak, caused by S. typhimurium, was investigated with the use of some microbiological and molecular-biological methods of typing. This investigation revealed that the outbreak was caused by the "outbreak" electrotype of the multi-resistant variant of the infective agent, found to have several plasmidovars. The possibilities and limitations of typing by sensitivity to antibiotics and plasmid DNA profile were shown. These methods of intraspecific typing were regarded as methods making it possible to establish the heterogeneity of S. typhimurium with the use of intraclonal markers.     

Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

The melibiose permease of Salmonella typhimurium (MelB_St) catalyzes the stoichiometric symport of galactopyranoside with a cation (H⁺, Li⁺, or Na⁺) and is a prototype for Na⁺-coupled major facilitator superfamily (MFS) transporters presenting from bacteria to mammals. X-ray crystal structures of MelB_St have revealed the molecular recognition mechanism for sugar binding; however, understanding of the cation site and symport mechanism is still vague. To further investigate the transport mechanism and conformational dynamics of MelB_St, we generated a complete single-Cys library containing 476 unique mutants by placing a Cys at each position on a functional Cys-less background. Surprisingly, 105 mutants (22%) exhibit poor transport activities (<15% of Cys-less transport), although the expression levels of most mutants were comparable to that of the control. The affected positions are distributed throughout the protein. Helices I and X and transmembrane residues Asp and Tyr are most affected by cysteine replacement, while helix IX, the cytoplasmic middle-loop, and C-terminal tail are least affected. Single-Cys replacements at the major sugar-binding positions (K18, D19, D124, W128, R149, and W342) or at positions important for cation binding (D55, N58, D59, and T121) abolished the Na⁺-coupled active transport, as expected. We mapped 50 loss-of-function mutants outside of these substrate-binding sites that suffered from defects in protein expression/stability or conformational dynamics. This complete Cys-scanning mutagenesis study indicates that MelB_St is highly susceptible to single-Cys mutations, and this library will be a useful tool for further structural and functional studies to gain insights into the cation-coupled symport mechanism for Na⁺-coupled MFS transporters.     

Loop Residues and Catalysis in OMP Synthase

Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100-109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 10(4)-fold decrease in k(cat)/K(M) for PRPP; the K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 produced 40- and 7-fold decreases in k(cat)/K(M), respectively, and E101A, D104A, and G106A were slightly faster than the wild-type (WT) in terms of k(cat), with minor effects on k(cat)/K(M). Equilibrium binding of OMP or PRPP in binary complexes was affected little by loop mutation, suggesting that the energetics of ground-state binding have little contribution from the catalytic loop, or that a favorable binding energy is offset by costs of loop reorganization. Pre-steady-state kinetics for mutants showed that K103A and E107A had lost the burst of product formation in each direction that indicated rapid on-enzyme chemistry for WT, but that the burst was retained by H105A. Δ102Δ106, a loop-shortened enzyme with Ala102 and Gly106 deleted, showed a 10(4)-fold reduction of k(cat) but almost unaltered K(D) values for all four substrate molecules. The 20% (i.e., 1.20) intrinsic [1'-(3)H]OMP kinetic isotope effect (KIE) for WT is masked because of high forward and reverse commitment factors. K103A failed to express intrinsic KIEs fully (1.095 ± 0.013). In contrast, H105A, which has a smaller catalytic lesion, gave a [1'-(3)H]OMP KIE of 1.21 ± 0.0005, and E107A (1.179 ± 0.0049) also gave high values. These results are interpreted in the context of the X-ray structure of the complete substrate complex for the enzyme [Grubmeyer, C., Hansen, M. R., Fedorov, A. A., and Almo, S. C. (2012) Biochemistry 51 (preceding paper in this issue, DOI 10.1021/bi300083p )]. The full expression of KIEs by H105A and E107A may result from a less secure closure of the catalytic loop. The lower level of expression of the KIE by K103A suggests that in these mutant proteins the major barrier to catalysis is successful closure of the catalytic loop, which when closed, produces rapid and reversible catalysis.     

Salmonella typhimurium Mutants with Altered Glutamate Dehydrogenase and Glutamate Synthase Activities

Although glutamate is a key compound in nitrogen metabolism, little is known about the function or regulation of its two biosynthetic enzymes, glutamate dehydrogenase and glutamate synthase. To begin the characterization of glutamate formation in Salmonella typhimurium, we isolated mutants having altered glutamate dehydrogenase and glutamate synthase activities. Mutants which failed to grow on media with glucose as the carbon source and less than 1 mM (NH₄)₂SO₄ as the nitrogen source (Asm⁻) had about one-fourth the normal glutamate synthase activity and one-half the glutamine synthetase activity. The asm mutations also prevented growth with alanine, arginine, or proline as nitrogen sources and conferred resistance to methionine sulfoximine. When a mutation (gdh-51) causing the loss of glutamate dehydrogenase activity was transferred into a strain with an asm-102 mutation, the resulting asm-102 gdh-51 mutant had a partial requirement for glutamate. A strain isolated as a complete glutamate auxotroph had a third mutation, in addition to the asm-102 gdh-51 lesions, that further decreased the glutamate synthase activities to 1/20 the normal level. Both the asm-102 and gdh-51 mutations were located on the S. typhimurium linkage map at sites distinct from those found for mutations causing similar phenotypes in Klebsiella aerogenes and Escherichia coli.     

Structure of peptidase T from Salmonella typhimurium.

The structure of peptidase T, or tripeptidase, was determined by multiple wavelength anomalous dispersion (MAD) methodology and refined to 2.4 A resolution. Peptidase T comprises two domains; a catalytic domain with an active site containing two metal ions, and a smaller domain formed through a long insertion into the catalytic domain. The two metal ions, presumably zinc, are separated by 3.3 A, and are coordinated by five carboxylate and histidine ligands. The molecular surface of the active site is negatively charged. Peptidase T has the same basic fold as carboxypeptidase G2. When the structures of the two enzymes are superimposed, a number of homologous residues, not evident from the sequence alone, could be identified. Comparison of the active sites of peptidase T, carboxypeptidase G2, Aeromonas proteolytica aminopeptidase, carboxypeptidase A and leucine aminopeptidase reveals a common structural framework with interesting similarities and differences in the active sites and in the zinc coordination. A putative binding site for the C-terminal end of the tripeptide substrate was found at a peptidase T specific fingerprint sequence motif.     

2-Deoxyribose Gene-Enzyme Complex in Salmonella typhimurium: Regulation of Phosphodeoxyribomutase

Phosphodeoxyribomutase, the enzyme which catalyzes the interconversion of 2-deoxyribose-1-phosphate to 2-deoxyribose-5-phosphate, has been partially purified from Salmonella typhimurium. The enzyme had an absolute requirement for manganese ion and was stimulated by glucose-1, 6-diphosphate. Phosphodeoxyribomutase was induced by deoxyribose-5-phosphate and was coordinately regulated with the enzymes thymidine phosphorylase and deoxyribose-5-phosphate aldolase, type II. Mutants deficient in these three enzymes were isolated and mapped close to the threonine locus in S. typhimurium. The three enzymes thymidine phosphorylase, deoxyribose-5-phosphate aldolase, type II, and phosphodeoxyribomutase are controlled by a series of linked genes and appear to constitute an operon.     

Substrate channeling: alpha-ketobutyrate inhibition of acetohydroxy acid synthase in Salmonella typhimurium.

Excess alpha-ketobutyrate inhibited the growth of Salmonella typhimurium LT2 by inhibiting the acetohydroxy acid synthase-catalyzed synthesis of alpha-acetolactate (a valine precursor). As a result, cells were starved for valine, and both ilvB (encoding acetohydroxy acid synthase I) and ilvGEDA (ilvG encodes acetohydroxy acid synthase II) were derepressed. The addition of valine reversed the effects of alpha-ketobutyrate.     

Complete genome sequence of Salmonella enterica serovar typhimurium bacteriophage SPN1S

To understand the interaction between the host of pathogenic Salmonella enterica serovar Typhimurium and its bacteriophage, we isolated the bacteriophage SPN1S. It is a lysogenic phage in the Podoviridae family and uses the O-antigen of lipopolysaccharides (LPS) as a host receptor. Comparative genomic analysis of phage SPN1S and the S. enterica serovar Anatum-specific phage ε15 revealed different host specificities, probably due to the low homology of host specificity-related genes. Here we report the complete circular genome sequence of S. Typhimurium-specific bacteriophage SPN1S and show the results of our analysis.     

Genetic Separation of the Inosinic Acid Cyclohydrolase-Transformylase Complex of Salmonella typhimurium

Genetic and enzymatic analyses were made with the purH mutants of Salmonella typhimurium. These mutants are purine auxotrophs which are deficient in the conversion of phosphoribosyl-aminoimidazolecarboxamide (AIC) to inosine-5'-monophosphate (IMP). Two steps are required for this process: phosphoribosyl-AIC transformylase (EC 2.1.2.3) and IMP cyclohydrolase (EC 3.5.4.10). Genetic analysis identified two complementation groups, I and II, and a third group of noncomplementing mutants (I-II). Mutations in gene I lead to complete loss of transformylase activity and no loss of cyclohydrolase activity if the mutation is of the missense type, but partial loss if it is of the chain-terminating type (nonsense or frameshift). Gene II mutants are all of the missense type and show normal transformylase activity but no cyclohydrolase activity. The noncomplementing mutants (I-II) are all of the chain-terminating type and are completely deficient in both activities. The results are explained and discussed in terms of subunit interactions of a stable enzyme complex.     

Structure of the cooperative allosteric anthranilate synthase from Salmonella typhimurium

We have determined the X-ray crystal structure of the cooperative anthranilate synthase heterotetramer from Salmonella typhimurium at 1.9 A resolution with the allosteric inhibitor l-tryptophan bound to a regulatory site in the TrpE subunit. Tryptophan binding orders a loop that in turn stabilizes the inactive T state of the enzyme by restricting closure of the active site cleft. Comparison with the structure of the unliganded, noncooperative anthranilate synthase heterotetramer from Sulfolobus solfataricus shows that the two homologs have completely different quarternary structures, even though their functional dimer pairs are structurally similar, consistent with differences in the cooperative behavior of the enzymes. The structural model rationalizes mutational and biochemical studies of the enzyme and establishes the structural differences between cooperative and noncooperative anthranilate synthase homologs.     

Substrate specificity switching of the flagellum-specific export apparatus during flagellar morphogenesis in Salmonella typhimurium.

During flagellar morphogenesis in Salmonella typhimurium, the flagellum-specific anti-sigma factor FlgM is exported out of the cells only after completion of hook assembly. In this study, we examined the export of the flagellar proteins, FlgD (hook capping protein), FlgE (hook protein), FlgK and FlgL (hook-filament junction proteins), FliD (filament capping protein), and FliC (flagellin), before and after completion of hook assembly. Like the FlgM protein, the FlgK, FlgL, FliD, and FliC proteins are exported efficiently only after completion of hook assembly. On the other hand, the FlgD and FlgE proteins are exported efficiently before, but poorly after, hook completion. These results indicate that the export properties are different between these two groups and that their export order exactly parallels the assembly order of the hook-filament structure. We propose that the substrate specificity switching occurs in the flagellum-specific export apparatus upon completion of hook assembly.     

Complex medium toxicity to some DNA repair-deficient strains of Salmonella typhimurium.

The recovery of several strains of Salmonella typhimurium LT-2 which had first been grown in minimal medium varies when the organisms are grown on minimal medium agar and complex medium agar. The strains tested included mutants with deficiencies in DNA-repair systems (uvrB-and rec-), a deep rough (rfa-) mutant, and a double mutant carrying both the uvrB- and the rfa-mutation. The uvrB- and rec-mutations imparted sensitivity to complex medium agar. The rfa-mutation suppressed the sensitivity of the uvrB-mutant to complex medium agar. Differences in colony-forming ability were not observed when the bacteria were first grown in the complex medium broth.     

Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants

Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (ΔG = −33 kcal mol⁻¹) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (K_D = 30 nm) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (K_D) and the regulation of cysteine sensitivity of SAT within the CSC.     

Chromosome Replication in Salmonella typhimurium

The replication of the Salmonella typhimurium chromosome was studied. As with E. coli 15T(-), replication was sequential. After amino acid starvation, replication proceeded from a unique and heritable region of the chromosome. 5-Bromouracil, when substituted for thymine, did not disturb the sequence of replication nor did it initiate extra replication cycles. By labeling the origin and the terminus of the chromosome with (3)H- and (14)C-thymine, respectively, it was possible to determine that the rate of chain elongation decreases as the growth rate decreases. No gap in the replication cycle could be observed.     

L-Rhamnose utilisation in Salmonella typhimurium

L-Rhamnose is degraded by strains of Salmonella typhimurium by isomerisation to L- rhamnulose , phosphorylation to L- rhamnulose -1-phosphate and cleavage to lactaldehyde and dihydroxyacetone phosphate. The enzymes involved are, respectively, rhamnose isomerase ( RhaI ), rhamnulokinase ( RhuK ) and an aldolase (Ald). Strains able to grow rapidly on L-rhamnose contained a high-affinity uptake system for 3H-L-rhamnose that was induced by L-rhamnose and repressed by D-glucose. The synthesis of RhaI and RhuK was also induced by L-rhamnose but was not repressed by D-glucose. The synthesis of Ald was constitutive. Data are presented on some strains which grow very slowly on L-rhamnose and on others which do not utilise it.