Binding of Bacteriophage P22 Tail Parts to Cells

Purified base-plate parts of bacteriophage P22 can bind to the host cell, Salmonella typhimurium. Although the reaction is reversible, a stable equilibrium is not formed between bound and unbound base-plate parts. This is because the binding sites on the cell, presumably the O antigens, are destroyed. The destruction of binding sites does not kill the cells, and, in fact, the binding sites are soon regenerated. The site-destroying activity reacts with P22 heads to make active phage and with antiserum made against purified phage. Therefore site-destroying activity is a characteristic of the base-plate parts and not some contaminant of the preparation.     

Phosphoenolpyruvate-dependent protein kinase enzyme I of Streptococcus faecalis: purification and properties of the enzyme and characterization of its active center

Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate- (PEP) dependent phosphotransferase system, has been purified from Streptococcus faecalis by using a large-scale preparation. Size exclusion chromatography revealed a molecular weight of 140 000. On sodium dodecyl sulfate gels, enzyme I gave one band with a molecular weight of 70 000, indicating that enzyme I consists of two identical subunits. The first 59 amino acids of the amino-terminal part of the protein have been sequenced. It showed some similarities with enzyme I of Salmonella typhimurium. The active center of enzyme I has also been determined. After phosphorylation with [32P]PEP, the enzyme was cleaved by using different proteases. Labeled peptides were isolated by high-performance liquid chromatography on a reversed-phase column. The amino acid composition or amino acid sequence of the peptides has been determined. The largest labeled peptide was obtained with Lys-C protease and had the following sequence: -Ala-Phe-Val-Thr-Asp-Ile-Gly- Gly-Arg-Thr-Ser-His*-Ser-Ala-Ile-Met-Ala-Arg-Ser-Leu-Glu-Ile-Pro-Ala- Ile-Val-Gly-Thr-Lys-. It has previously been shown that the phosphoryl group is bound to the N-3 position of a histidyl residue in phosphorylated enzyme I. The single His in position 12 of the above peptide must therefore carry the phosphoryl group.     

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.     

Existence of a Free Form of a Specific Membrane Lipoprotein in Gram-Negative Bacteria

The existence of a free form of a specific lipoprotein of molecular weight 7,200 was examined in the envelopes of several gram-negative bacteria. When the envelope proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, distinct peaks were observed in Salmonella typhimurium, Serratia marcescens, and Pseudomonas aeruginosa at the same position as the free form of the lipoprotein of Escherichia coli. However, the peak was not observed in Proteus mirabilis. The protein at the peak in S. typhimurium was shown to contain little or no histidine as expected from the amino acid composition of the lipoprotein. Furthermore, antiserum against the highly purified lipoprotein from E. coli was shown to react with the proteins from S. typhimurium and S. marcescens and to form the specific immunoprecipitates. In contrast, the protein from P. aeruginosa did not react with the antiserum at all. Thus, it is concluded that S. typhimurium and S. marcescens have the free form of the lipoprotein in their envelopes as does E. coli. P. aeruginosa contains a protein of the same size as the lipoprotein, but it is not certain whether the protein is the same structural protein as the lipoprotein from E. coli. P. mirabilis may not have any free form of the lipoprotein, may have it in a very small amount, or may have a lipoprotein of different molecular weight serving the same function.     

The primary structure of Escherichia coli K12 2-deoxyribose 5-phosphate aldolase. Nucleotide sequence of the deoC gene and the amino acid sequence of the enzyme

The sequence of the deoC gene of Escherichia coli K12 and the amino acid sequence of the corresponding protein, deoxyriboaldolase, has been established. The protein consists of 259 amino acids with a molecular weight of 27 737. The purified enzyme may exist both as a monomer and as a dimer. On the basis of amino acid composition, molecular weight and catalytic properties, the enzymes from E. coli and Salmonella typhimurium seem to be almost similar. They belong to the class I aldolases, which form Schiff base intermediates. Using data for the S. typhimurium enzyme, the lysine residue involved in the active site in the E. coli enzyme was tentatively identified.     

Purification and characterization of a tRNA methylase from Salmonella typhimurium.

A tRNA methylase, in which supK strains of Salmonella typhimurium are deficient, was purified from strain LT2 and characterized. Column chromatography of protein extracts from wild-type cells on phosphocellulose, diethylaminoethyl-Sephadex A-50, and hydroxlapatite resulted in an enzyme that was estimated to be about 50% pure. tRNA from S. typhimurium which had been incubated at pH 9.0 served as a substrate for this methylase. The enzyme has a molecular weight of about 50,000 as estimated by gel chromatography and by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The optimal assay conditions, as well as the kinetics and stability of the enzyme, were studied. As with other tRNA-methylating enzymes, S-adenosylhomocysteine is a potent inhibitor.     

Cloning, sequencing, expression and characterization of DNA photolyase from Salmonella typhimurium.

We have cloned the phr gene that encodes DNA photolyase from Salmonella typhimurium by in vivo complementation of Escherichia coli phr gene defect. The S.typhimurium phr gene is 1419 base pairs long and the deduced amino acid sequence has 80% identity with that of E. coli photolyase. We expressed the S.typhimurium phr gene in E.coli by ligating the E.coli trc promoter 5' to the gene, and purified the enzyme to near homogeneity. The apparent molecular weight of S.typhimurium photolyase is 54,000 dalton as determined by SDS-polyacrylamide gel electrophoresis, which is consistent with the calculated molecular weight of 53,932 dalton from the deduced phr gene product. S.typhimurium photolyase is purple-blue in color with near UV-visible absorption peaks at 384, 480, 580, and 625 nm and a fluorescence peak at 470 nm. From the characteristic absorption and fluorescence spectra and reconstitution experiments, S.typhimurium photolyase appears to contain flavin and methenyltetrahydrofolate as chromophore-cofactors as do the E.coli and yeast photolyases. Thus, S.typhimurium protein is the third folate class photolyase to be cloned and characterized to date. The binding constant of S.typhimurium photolyase to thymine dimer in DNA is kD = 1.6 x 10(-9) M, and the quantum yield of photorepair at 384 nm is 0.5.     

Purification and characterization of bacteriophage 9NA lysozyme

Bacteriophage 9NA is a virulent phage of Salmonella typhimurium which induces a lysozyme in host cells toward the later stages of its multiplication. 9NA lysozyme has been purified about 1000 fold starting from the lysate of 9NA infected cells. The enzyme has an optimum pH between 7 and 8 and its activity is dependent on the ionic strength of the assay medium. Salts like NaCl and KCl are inhibitory to the lysozyme. Gram-negative cells act as better substrate for the lysozyme than do Gram-positive cells. The enzyme has a molecular weight of about 2.1 X 10(4) and rapidly loses its activity at temperatures higher than 45 degrees C. The properties of 9NA lysozyme have been compared with those of T4, lambda and P22 lysozymes.     

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.     

Overproduction of the MotA protein of Escherichia coli and estimation of its wild-type level.

The motA gene of Escherichia coli was placed under the control of a high-level promoter, that of the tryptophan operon of Serratia marcescens. In the presence of the inducer beta-indoleacrylic acid, MotA was synthesized at greatly elevated levels and inserted without apparent limit into the inner membrane. Growth and motility were impaired, but not drastically so, indicating that MotA by itself does not act as a proton ionophore. Antibody raised against the overproduced protein was used to estimate that a wild-type cell contained 600 +/- 250 copies of MotA. This number is more than would be needed to surround each flagellar basal body with a single circlet of MotA protein; possible interpretations of the result are discussed. The antibody was also used to establish that the MotA protein of Salmonella typhimurium has a similar molecular weight to that of E. coli and is immunologically cross-reactive with it; functional complementation of S. typhimurium motA mutants by the E. coli gene was established.     

Ornithine transcarbamylase from Salmonella typhimurium: purification, subunit composition, kinetic analysis, and immunological cross-reactivity.

Ornithine transcarbamylase (OTCase) was purified to hemogeneity from a derepressed strain of Salmonella typhimurium. The optimal pH for enzyme activity is 8.0. The molecular weight of the enzyme was calculated to be 116,000, based on measurements of the sedimentation coefficient by sucrose gradient ultracentrifugation and the Stokes radius by gel filtration. Polyacrylamide gel electrophoresis of cross-linked OTCase in the presence of sodium dodecyl sulfate showed that the enzyme is composed of three identical subunits. The molecular weight of the monomer was determined to be 39,000. Steady-state kinetics indicate that the reaction mechanism is sequential. The limiting Michealis constants for carbamylphosphate and ornithine were determined to be 0.06 and 0.2 mM, respectively. The dissociation constant for carbamylphosphate was 0.02 mM. Product and dead-end inhibition patterns are consistent with an ordered Bi Bi mechanism, in which carbamylphosphate is the first substrate added and phosphate is the last product released. OTCase activity was inhibited by arginine, but relatively high concentrations were required for significant inhibition. The inhibition by arginine might be physiologically significant in the regulation of carbamlphosphate utilization; a single carbamylphosphate synthetase is responsible for the synthesis of carbamylphosphate for both arginine and pyrimidines in S. typhimurium and the inhibition by argine might serve to divert carbamlphosphate to the synthesis of pyrimidines when arginine is present at high concentrations. The crossreaction of OTCases from different microorganisms with purified antibodies raised against the homogeneous OTCase from S. typhimurium was investigated. The results of immunotitration and immunodiffusion experiments revealed a high degree of identity between the enzymes form S. typhimurium and Esherichia coli B and W. In these three cases, a single gen (argl) encodes OTCase. Wild-type E. coli K-12 and strain 3000 X 111, which carry two OTCase genes (argI, argF), also revealed similar cross-reactivity, supporting the hypothesis that argF is the product of a relatively recent duplication. The activity of OTCase from Bacillus subtilis was partially inhibited by antibodies against the enzyme from S. typhimurium, indicating unusual conservation of primary structure among widely different taxonomic groups. OTCase from Saccharomyces cerevisiae, whose molecular weight and primary structure are similar to those of the enzyme from S. typhimurium, was without detectable cross-reactivity.     

Location on the Salmonella typhimurium Chromosome of the Gene Encoding Nucleoside Diphosphokinase (ndk)

The gene encoding nucleosidediphosphate kinase (ndk) was located at 55 units on the Salmonella typhimurium chromosome. The ndk locus was 83% cotransducible with hisS and 2% cotransducible with glyA in phage P22-mediated crosses. A nucleosidediphosphate kinase mutant that produced only 10% of the wild-type enzyme activity (ndk-1) grew normally and produced a heat-labile enzyme.     

P22 antirepressor protein prevents in vivo recA-dependent proteolysis of P22 repressor

A method was developed to demonstrate recA-dependent P22-repressor breakdown in vivo by SDS-polyacrylamide electrophoresis of unfractionated extracts of phage-infected, lysogenic Salmonella typhimurium strains TA1530 rec+ and TA1530 recA1-. The antirepressor of P22 is not cleaved by recA protein. Under conditions of unregulated ant-overproduction (Harvey et al. 1981) antirepressor protects c2-repressor in vivo against proteolytic cleavage by recA protein.     

Periplasmic protein associated with the oligopeptide permeases of Salmonella typhimurium and Escherichia coli.

A periplasmic protein essential for the function of the oligopeptide transport system of Salmonella typhimurium was identified. This protein, encoded by the oppA gene, is one of the most abundant proteins in the periplasm and, with an apparent molecular weight of 52,000, is considerably larger than any other known periplasmic transport component. A similarly abundant periplasmic protein forms part of the oligopeptide transport system of Escherichia coli.     

Biosynthesis of bacterial glycogen: purification and structural properties of Rhodospirillum tenue adenosine diphosphate glucose synthetase.

Adenosine diphosphate glucose synthetase from the photosynthetic bacterium Rhodospirillum tenue has been purified greater than 95%. The molecular weight of the enzyme is approximately 215,000, with a subunit molecular weight of about 51,000. The enzyme appears to be composed of four similar if not identical subunits. Although the amino acid composition of the enzyme is similar to that of Escherichia coli and Salmonella typhimurium, no apparent homology has been observed between their N-terminal amino acid sequences. Antisera prepared against the R. tenue enzyme can partially inhibit the activities of adenosine diphosphate glucose synthetases from other photosynthetic bacteria.     

A 59 kiloDalton outer membrane protein of Salmonella typhimurium protects against oxidative intraleukocytic killing due to human neutrophils

We have isolated a Salmonella typhimurium (ST) mutant, JKS400, deficient in the production of a surface-exposed outer membrane protein (Omp) and phenotypically hypersensitive to the oxidative antimicrobial mechanism of polymorphonuclear leukocytes (PMNs). This Omp migrated at approximately 59 kiloDaltons (kD) in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). We found with P22 transduction that the capacities to produce the protein and to exert wild-type resistance to oxidative killing were tightly linked. Transduction of JKS400 with a P22(HT)int- bacteriophage grown on a Tn10 insertion library in LT2 yielded tetracycline-resistant isolates that had been returned to wild-type protein production. Further experiments showed that restoration of protein production was accompanied by restoration of the parental resistance phenotype to killing by PMNs and by restoration to wild-type resistance to H2O2. The map position of the Tn10 was determined to be at 96 minutes in the Salmonella chromosome. This protein appears to behave as a virulence factor, promoting the capacity of Salmonella typhimurium LT2 to survive oxygen-dependent killing mechanisms in neutrophils.     

Studies of the quaternary structure and the chemical properties of phosphoribosylpyrophosphate synthetase from Salmonella typhimurium.

Phosphoribosylpyrophosphate (PRPP) synthetase (EC 2.7.6.1) was purified to virtual homogeneity from Salmonella typhimurium cells by a modification of previously published procedures. The molecular weight of the subunit was determined to be 31,000 +/- 3,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and sedimentation equilibrium analysis of the enzyme dissolved in 6 M guanidine hydrochloride. The amino acid composition of the enzyme was determined. Proline was identified as the only NH2-terminal residue. PRPP synthetase is apparently composed of identical or nearly identical subunits. NATIVE PRPP synthetase exists in multiple states of aggregation under all conditions. However, two predominant states were demonstrated under certain conditions. A form with molecular weight of 320,000 +/- 20,000 was found at pH 7.5 in the presence of MgATP. At pH 8.2 to 8.6, with or without MgATP, the predominant form corresponded to a molecular weight of 150,000 to 200,000; sedimentation equilibrium and velocity analysis indicated 160,000 +/- 15,000 as the most reliable molecular weight. More highly aggregated forms were observed at 4 degrees and higher protein concentrations. Removal of inorganic phosphate from PRPP synthetase by dilution or dialysis resulted in disaggregation. The fundamental unit of PRPP synthetase appears to consist of five (or possibly six) subunits, which can associate to form a dimer (10 or 12 subunits) and more highly aggregated forms. A pentameric subunit structure is consistent with the multiple species resolved by electrophoresis of the native enzyme in discontinuous polyacrylamide gel systems. Visualization of PRPP synthetase by negative staining with uranyl acetate and electron microscopy revealed fields of very asymmetric molecules, the dimensions of which corresponded to the M = 160,000 form. Dimers and higher aggregates of this unit were also seen. An unusual model, in which the five subunits are asymmetrically arranged, accounts very well for the electron microscopic appearance of the enzyme. The tendency of the enzyme to aggregate is viewed as a consequence of the unsatisfied bonding regions of the fundamental asymmetric unit.     

Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus

The gene coding aspartate racemase (EC 5.1.1.13) was cloned from the lactic acid bacteria Streptococcus thermophilus IAM10064 and expressed efficiently in Escherichia coli. The 2.1 kilobase pairs long full length clone had an open reading frame of 729 nucleotides coding for 243 amino acids. The calculated molecular weight of 27,945 agreed well with the apparent molecular weight of 28,000 found in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the aspartate racemase purified from S. thermophilus. The N-terminal amino acid sequence from the purified protein exactly matches the derived sequence. In addition, the amino acid composition compiled from the derived sequence is very similar to that obtained from the purified recombinant protein. No significantly homologous proteins were found in a protein sequence data bank. Even the homology scores with alanine racemases of Salmonella typhimurium and Bacillus stearothermophilus were low. Aspartate racemase was overproduced in Escherichia coli NM522 with plasmid pAG6-2-7, which was constructed from two copies of the gene linked with a tac promoter and plasmid vector pUC18. The amount of aspartate racemase increases with the growth of E. coli and almost no degradation of the enzyme was observed. The maximum amount of the produced enzyme reached approx. 20% of the total protein of E. coli.