Salmonella bacteriophage glycanases: endorhamnosidases of Salmonella typhimurium bacteriophages.

Twelve bacteriphages lysing only smooth Salmonella typhimurium strains were shown to have similar morphology--an icosahedric head to which a short, noncontractile tail carrying six spikes was attached. All phages degraded their lipopolysaccharide (LPS) receptors as shown by their ability to cleave off [14C]galactosyl-containing oligosaccharides from S. typhimurium cells labeled in their LPS. The oligosaccharides inhibited the alpha-D-galactosyl-specific Bandeiraea simplicifolia lectin agglutination of human type B erythrocytes, indicating that all 12 phage glycanases were of endorhamnosidase specificity, i.e., hydrolyzed the alpha-L-rhamnopyranosyl-(1 leads to 3)-D-galactopyranosyl linkage in the S. typhimurium O-polysaccharide chain. Two of the phages, 28B and 36, were studied in more detail. Whereas the phage 28B glycanase hydrolyzed the S. typhimurium LPS into dodeca- and octasaccharides, the phage 36 glycanase in addition cleaved off tetrasaccharides. Both phage enzymes hydrolyzed the O-polysaccharide chains of LPS from Salmonella belonging to serogroups A, B, and D1, which are built up of tetrasaccharide-repeating units identical except for the nature of the 3,6-dideoxyhexopyranosyl group (R). : FORMULA:(SEE TEXT). The phage 28B and 36 endorhamnosidases hydrolyzed also an LPS from which the 3,6-dideoxyhexosyl substituents had previously been hydrolyzed off. However, neither of the enzymes was active on LPS preparations in which the C2-C3 bond of the L-rhamnopyranosyl ring had been opened by periodate oxidation. Glucosylation at O-6 of the D-galactopyranosyl residues in the S. typhimurium LPS was found to be incompatible with hydrolysis by both enzymes. However, in an LPS glucosylated at O-4 of the D-galactopyranosyl residues, the adjacent alpha-L-rhamnopyranosyl linkages were found to be perferentially cleaved.     

Deoxyribokinase from Salmonella typhimurium. Purification and properties

Deoxyribokinase, which catalyzes the ATP-dependent phosphorylation of 2-deoxy-d-ribose to 2-deoxy-d-ribose-5-P as the first step in the inducible fermentation pathway for this sugar in Salmonella typhimurium, was purified approximately 600-fold from deoxyribose-grown cells. Apparent K_m′s for 2-deoxy-d-ribose and ATP were 0.1 and 0.5 mm, respectively. The enzyme had an absolute requirement for divalent cations which was best satisfied by Mg²⁺. Optimal activity was obtained in the presence of 0.5 m NH₄⁺ or Cs⁺. Rb⁺ and K⁺ also stimulated enzyme activity whereas Na⁺ and Li⁺ inhibited. d-Ribose and 2-deoxy-d-ribitol could replace 2-deoxy-d-ribose as phosphoryl acceptor, and several ribo- and deoxyribonucleotides could replace ATP as phosphoryl donor. Molecular weight determinations gave values of 67,800 for the native enzyme and 33,500 for the dissociated enzyme, suggesting the probable existence of two subunits of similar size.     

Interference with propagation of typing bacteriophages by extrachromosomal elements in Salmonella typhimurium: bacteriophage type 505.

Samonella typhimurium bacteriophage type 505 is the most frequently encountered phage type in the Netherlands and its neighboring countries. Phage type 505 was analyzed with regard o the interference with propagation of the typing phages by the prophages and plasmids, present in the type strain S. typhimurium 505...     

Mutagenicity of nitrosodiethanolamine on Salmonella typhimurium.

Nitrosodiethanolamine was examined in the Ames assay for bacterial mutagens. In absence of S9 activation a mutagenic effect was found.     

Purification and properties of trimethylamine oxide reductase from Salmonella typhimurium.

The major inducible trimethylamine oxide reductase was purified from Salmonella typhimurium LT2. The molecular weights of the native enzyme were estimated to be 332,000 by gel filtration and 170,000 by nondenaturing disc gel electrophoresis. In sodium dodecyl sulfate-gel electrophoresis, the enzyme formed a single band of molecular weight 84,000. The isoelectric point was 4.28. Maximum activity was at pH 5.65 and 45 degrees C. Reduced flavin mononucleotide, but not reduced flavin adenine dinucleotide, served as an electron donor. The Km for trimethylamine oxide was 0.89 mM and Vmax was 1,450 U/mg of protein. The enzyme reduced chlorate with a Km of 2.2 mM and a Vmax of 350 U/mg of protein.     

Salmonella typhimurium peptidase active on carnosine.

Wild-type Salmonella typhimurium can use carnosine (beta-alanyl-L-histidine) as a source of histidine, but carnosine utilization is blocked in particular mutants defective in the constitutive enzyme peptidase D, the product of the pepD gene. Biochemical evidence for assigning carnosinase activity to peptidase D (a broad-specificity dipeptidase) includes: (i) coelution of carnosinase and dipeptidase activity from diethylaminoethyl-cellulose and Bio-Gel P-300 columns; (ii) coelectrophoresis of carnosinase and dipeptidase on polyacrylamide gels; and (iii) inactivation of carnosinase and dipeptidase activities at identical rates at both 4 and 42 degrees C. Genetic evidence indicates that mutations leading to loss of carnosinase activity map at pepD. Several independent pepD mutants have been isolated by different selection procedures, and the patterns of peptide utilization of strains carrying various pepD alleles have been studied. Many pepD mutations lead to the production of partially active peptidase D enzymes with substrate specificities that differ strikingly from those of the wild-type enzyme. The growth yields of carnosinase-deficient strains growing in Difco nutrient broth indicate that carnosine is the major utilizable source of histidine in this medium.     

Purification and kinetic properties of pyruvate kinase isoenzymes of Salmonella typhimurium.

Two forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) present in Salmonella typhimurium were purified to homogeneity from the same cultures by (NH4)2SO4 fractionation and gel filtration, anion-exchange and affinity chromatography. Mr values, subunit structure, amino acid composition and activity and stability conditions were determined for the two forms. Kinetic and regulatory properties of the two purified isoenzymes were studied.