Characterization of selenium-containing tRNAGlu from Clostridium sticklandii

A selenium-containing tRNA from Clostridium sticklandii has been shown to be an isoaccepting tRNAGlu (W.-M. Ching and T. C. Stadtman (1982) Proc. Natl. Acad. Sci. USA 79, 374-377). Not only is this tRNAGlu one of the most abundant selenium-containing tRNA species but it is also the major glutamate isoacceptor in this organism. The selenonucleoside, which is located at the first position of the anticodon, was identified as 5-methylaminomethyl-2-selenouridine (A. J. Wittwer, L. Tsai, W.-M. Ching, and T. C. Stadt (1984) Biochemistry 23, 4650-4655). Other modified nucleosides present in this tRNA include 4-thiouridine, pseudouridine, ribothymidine, modified guanosine, and two different modified adenosines. When this seleno-tRNAGlu is incubated in 1.0 M Tris X HCl, pH 8.5, partial deselenization occurs. Moreover, treatment with cyanogen bromide almost completely removes the selenium. The presence of selenium in this tRNAGlu is essential for its enzymatic acylation with glutamate. This seleno-tRNAGlu recognizes both GAA and GAG codons. However, at 10 mM magnesium, which is near the physiological range, the GAA codon is slightly favored. In a cell free translation system, the acylated seleno-tRNAGlu is a very active glutamate donor.     

Degradation of pyrimidine bases in Clostridium sticklandii

Resting cells of Clostridium sticklandii took up thymine or uracil, when grown in a medium containing 40 mM serine and 20 mM thymine or uracil. The uptake was much lower, when the cells had been grown in a complex medium. Cell-free extracts from cells grown in the complex medium reduced the two bases to the dihydro compounds and decomposed dihydrothymine to -ureidoisobutyrate, as indicated by thin-layer chromatography. Uptake and degradation were stimulated by both NADH and NADPH. Further breakdown did not occur, as ¹⁴CO₂ was not evolved from C-2-labelled thymine or uracil. The rates of pyrimidine uptake and breakdown of C. sticklandii were lower than those reported for C. sporogenes (Hilton et al., 1975).     

Sheathed Cells in Cultures of Clostridium sporogenes

Many strains of Clostridium sporogenes were shown to contain two types of cells which exhibited strikingly different growth habits. Over 99% of the population of most strains were motile bacilli which occurred singly or in short chains. Infection by any of several C. sporogenes bacteriophages lysed most of these cells and revealed a minority population component consisting of cells which grew in extremely long chains. Each chain was surrounded by and contained in a long tubular polysaccharide sheath which was ultrastructurally quite separate and distinct from the cell walls of the enclosed cells. The sheathed cells were identical to "normal" cells of C. sporogenes in anaerobiosis, Gram reaction, sporulation, deoxyribonucleic base composition, general morphology, and ultrastructure. They differed from the "normal" cells in having a sheath, in being nonmotile, and in that they were infected by C. sporogenes bacteriophages but not usually lysed by them. The sheathed cells appeared spontaneously in cultures cloned from single colonies and were demonstrably present in cultures before bacteriophage infection. Thus, they were not contaminants but were normal, although inconspicuous, growth forms of C. sporogenes which were selected but not induced by bacteriophage infection.     

Regulation of protease production in Clostridium sporogenes

The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions.     

Purification and characterization of threonine dehydrogenase from Clostridium sticklandii

Threonine dehydrogenase from Clostridium sticklandii has been purified 76-fold from cells grown in a defined medium to a homogeneous preparation of 234 units · mg^-1 protein. Purification was obtained by chromatography on Q-Sepharose fast flow and Reactive green 19-Agarose. The native enzyme had a molecular mass of 67 kDa and consisted of two identical subunits (33 kDa each). The optimum pH for catalytic activity was 9.0. Only l-threo-threo-nine, dl--hydroxynorvaline and acetoin were substrates; only NAD was used as the natural electron acceptor. The apparent K _m values for l-threonine and NAD were 18 mM and 0.1 mM, respectively. Zn²⁺, Co²⁺ and Cu²⁺ ions (0.9 mM) inhibited enzyme activity. The N-terminal amino acid sequence revealed similarities to the class of non-metal short-chain alcohol dehydrogenases, whereas the threonine dehydrogenase from Escherichia coli belongs to the class of medium chain, zinc-containing alcohol dehydrogenases.Abbreviations PMSF phenylmethylsulfonyl fluoride - Dea diethanolamine - Tris tris-(hydroxy-methyl)-aminomethane - Nbs ₂ 5,5-dithiobis-(2-nitrobenzoic acid) - ApADN 3-acetylpyridine adenine diucleotide - thio-NAD thionicotinamide adenine dinucleotide - NBT nitro blue tetrazolium chloride     

Purification and properties of ornithine racemase from Clostridium sticklandii

Ornithine racemase has been purified to homogeneity from Clostridium sticklandii, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is the first racemase known to be highly specific to ornithine. This PLP-dependent enzyme has an M(r) of 92, 000, with a K(m) for L-ornithine of 0.77 +/- 0.05 mM and a k(cat) of 980 +/- 20 s(-1).     

Thiaminase Activity Amongst Strains of Clostridium sporogenes

All the 28 strains of Clostridium sporogenes type I tested produced thiaminase. Only 2 of the 16 strains of Cl. sporogenes type II tested were positive for the enzyme; these gave a weak positive reaction. The single strain of Cl. sporogenes type III behaved in a manner similar to the strains of type I, giving a strong positive thiaminase reaction. Thiaminase production amongst the strains of Cl. sporogenes does in the main support the cultural, biochemical and immunological properties described earlier.     

Purification and properties of proline reductase from Clostridium sticklandii.

Proline reductase of Clostridium sticklandii is a membrane-bound protein and is released by treatment with detergents. The enzyme has been purified to homogeneity and is estimated by gel filtration and sedimentation equilibrium centrifugation to have a molecular weight of 298,000 to 327,000. A minimum molecular weight of 30,000 to 31,000 was calculated on the basis of sodium dodecyl sulfate-acrylamide gel electrophoresis and amino acid composition. Amino acid analysis showed a preponderance of acidic amino acids. No tryptophan was detected in the protein either spectrophotometrically or by amino acid analysis. A total of 20 sulfhydryl groups measured by titration of the reduced protein with 5,5'-dithiobis(2-nitrobenzoic acid) is in agreement with 20 cystic acid residues determined in hydrolysates of performic acid-oxidized protein. No molybdenum, iron, or selenium was found in the pure protein. Although NADH is the physiological electron donor for the proline reductase complex, the purified 300,000 molecular weight reductase component is inactive in the presence of NADH in vitro. Dithiothreitol, in contrast, can serve as electron donor both for unpurified (putative proline reductase complex) and purified proline reductase in vitro.     

Regulation of protease production in Clostridium sporogenes.

The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions.     

Purification and properties of a quinone-dependent p-nitrophenylphosphatase from Clostridium sticklandii

A highly specialized phosphatase that depends on both a quinone (e.g., 2-methyl-1,4-napthoquinone) and a sulfhydryl compound for activity was purified to homogeneity from extracts of Clostridium sticklandii. Selective adsorption to Cibacron Blue-Sepharose 4B followed by elution with p-nitrophenylphosphate was an effective enrichment procedure. An affinity matrix containing vitamin K5 (4-amino-2-methyl-1-naphthol) covalently attached to Sepharose 4B selectively retained the enzyme and was also used in its purification. The only known substate for the enzyme, p-nitrophenylphosphate, is hydrolyzed to equivalent amounts of orthophosphate and p-nitrophenol. Although a protein phosphotyrosine residue seemed a likely candidate as the natural substrate, the enzyme failed to hydrolyze 32P-labeled phosphotyrosine residues in casein, in vinculin, or in denatured glutamine synthetase. Also, free O-phosphotyrosine and numerous phosphate esters that serve as substrates for common phosphomonoesterases were not hydrolyzed. The molecular weight of the native enzyme, estimated by Sephacryl-S-200 gel chromatography, is 27,600. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed a single component with a molecular weight of 28,600. From the amino acid composition, a minimum molecular weight of 28,000 was calculated.     

Purification and characterization of RNase P from Clostridium sporogenes

RNase P is a multi-subunit enzyme responsible for the accurate processing of the 5' terminus of all tRNAs. The RNA subunit from Clostridium sporogenes has been partially purified and characterized. The RNA is approximately 400 nucleotides long and makes a precise endonucleolytic cleavage at the mature 5' terminus of tRNA. The RNA requires moderate concentrations of Mg2+ (20 mM) and relatively high concentrations of NH4Cl (800 mM) for optimal activity. Mn2+ effectively substitutes for Mg2+ at 2 mM. Zn2+, Ni2+, Ca2+, and Co2+ are ineffective at stimulating activity. Monovalent ions are, in general, more effective the greater the ionic radius (NH+4 greater than Cs greater than Rb greater than K greater than Na). In contrast to the activity of Bacillus subtilis, C. sporogenes RNase P RNA is significant more active in (NH4)2SO4 than in NH4Cl.