Physiological and nutritional factors affecting synthesis of extracellular metalloproteases by Clostridium bifermentans NCTC 2914.

Extracellular protease production by Clostridium bifermentans NCTC 2914 occurred throughout the growth phase in batch culture. In both glucose-excess and -limited chemostats, protease formation was inversely related to the dilution rate, over the range D = 0.03 to 0.70 h-1. At high dilution rates (D greater than 0.25 h-1), protease activities were greatest under excess glucose conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chemostat culture effluents showed the presence of up to 18 bands of protease activity at low dilution rates, with apparent molecular masses ranging from about 36 to 125 kDa. High-performance liquid chromatography gel filtration of culture supernatants gave four peaks of activity at 34, 42, 60, and 102 kDa. Glucose, peptone, and phosphate stimulated protease formation, but ammonia concentrations up to 10 g liter-1 had little effect on the process. Culture pH in glucose-excess chemostats strongly influenced protease synthesis, which was maximal during growth at pH 6.4. The optimal pH of protease activity was 7.0. Although a wide variety of proteins were hydrolyzed by C. bifermentans proteases, none of the enzymes were collagenolytic. Of 21 different p-nitroanilide, beta-naphthylamide, and N-carbobenzoyl substrates tested, none were hydrolyzed. With the exception of Ca2+, divalent metal ions inhibited proteolysis. Experiments with protease inhibitors demonstrated that 1 mM EDTA inhibited protease activities in culture supernatants by over 90%, indicating that the enzymes were principally of the metalloprotease type.     

Activation and inhibition of rabbit muscle pyruvate kinase by transition-metal ions.

The paper reports a comparative study of the effects of Mn2+, Ni2+ and Co2+ on the reaction of ADP with phosphoenolypyruvate when catalysed by K+-activated rabbit muscle pyruvate kinase. The activation and subsequent inhibition that occurs as the bivalent ion concentration is increased is taken as evidence that the substrates of the enzyme are phosphoenolypyruvate, uncomplexed ADP and free bivalent cation. Kinetic constants for the binding of the bivalent cation to the enzyme are reported.     

Influence of pH, nutrient availability, and growth rate on amine production by Bacteroides fragilis and Clostridium perfringens

Dimethylamine, methylamine, propylamine, and pyrrolidine were the major amines formed by Bacteroides fragilis NCDO 2217 during the active phase of growth in batch culture. Production of these metabolites was strongly pH dependent and was optimal under acidic conditions (pH 6.0). Low pH also favored the formation of pyrrolidine, cadaverine, and dimethylamine by Clostridium perfringens C523, but the reverse was the case with putrescine, butylamine, and propylamine, where production was maximal at neutral pH. B. fragilis was grown in continuous culture under either starch or casein limitation. Amine formation was influenced by carbohydrate availability and was greatest when the bacteria were grown at high growth rates (dilution rate, 0.20/h) under starch limitation, where they constituted about 18% of the total fermentation products measured. Amine production was optimal and increased concomitantly with growth rate when C. perfringens was grown in glucose-limited continuous culture. Under conditions of high growth rate and glucose limitation, amines accounted for approximately 27% of the fermentation products measured. When glucose in the feed medium was increased from 5 to 15 g/liter, amine production was repressed, and under these nutritional conditions the growth rate had little effect on the process.     

Influence of pH, nutrient availability, and growth rate on amine production by Bacteroides fragilis and Clostridium perfringens.

Dimethylamine, methylamine, propylamine, and pyrrolidine were the major amines formed by Bacteroides fragilis NCDO 2217 during the active phase of growth in batch culture. Production of these metabolites was strongly pH dependent and was optimal under acidic conditions (pH 6.0). Low pH also favored the formation of pyrrolidine, cadaverine, and dimethylamine by Clostridium perfringens C523, but the reverse was the case with putrescine, butylamine, and propylamine, where production was maximal at neutral pH. B. fragilis was grown in continuous culture under either starch or casein limitation. Amine formation was influenced by carbohydrate availability and was greatest when the bacteria were grown at high growth rates (dilution rate, 0.20/h) under starch limitation, where they constituted about 18% of the total fermentation products measured. Amine production was optimal and increased concomitantly with growth rate when C. perfringens was grown in glucose-limited continuous culture. Under conditions of high growth rate and glucose limitation, amines accounted for approximately 27% of the fermentation products measured. When glucose in the feed medium was increased from 5 to 15 g/liter, amine production was repressed, and under these nutritional conditions the growth rate had little effect on the process.     

Repeated sequence sets in mitochondrial DNA molecules of root knot nematodes (Meloidogyne): nucleotide sequences, genome location and potential for host-race identification.

Within a 7 kb segment of the mtDNA molecule of the root knot nematode, Meloidogyne javanica, that lacks standard mitochondrial genes, are three sets of strictly tandemly arranged, direct repeat sequences: approximately 36 copies of a 102 ntp sequence that contains a TaqI site; 11 copies of a 63 ntp sequence, and 5 copies of an 8 ntp sequence. The 7 kb repeat-containing segment is bounded by putative tRNAasp and tRNAf-met genes and the arrangement of sequences within this segment is: the tRNAasp gene; a unique 1,528 ntp segment that contains two highly stable hairpin-forming sequences; the 102 ntp repeat set; the 8 ntp repeat set; a unique 1,068 ntp segment; the 63 ntp repeat set; and the tRNAf-met gene. The nucleotide sequences of the 102 ntp copies and the 63 ntp copies have been conserved among the species examined. Data from Southern hybridization experiments indicate that 102 ntp and 63 ntp repeats occur in the mtDNAs of three, two and two races of M.incognita, M.hapla and M.arenaria, respectively. Nucleotide sequences of the M.incognita Race-3 102 ntp repeat were found to be either identical or highly similar to those of the M.javanica 102 ntp repeat. Differences in migration distance and number of 102 ntp repeat-containing bands seen in Southern hybridization autoradiographs of restriction-digested mtDNAs of M.javanica and the different host races of M.incognita, M.hapla and M.arenaria are sufficient to distinguish the different host races of each species.     

Phorbol diester-induced phosphorylation of nuclear matrix proteins in HL60 promyelocytes. Possible role in differentiation studied by cationic detergent gel electrophoresis

Immortal HL60 promyelocytes are induced to differentiate to mortal adherent cells by a variety of agents which activate protein kinase C, including 12-O-tetradecanoylphorbol 13-acetate (TPA). In order to investigate the mechanism of this effect, we incubated HL60 cells with [32P]orthophosphate with or without TPA and extracted their proteins with the cationic detergent benzyldimethyl-n-hexadecylammonium chloride prior to electrophoresis in a discontinuous polyacrylamide gel system in the first dimension. In this system, proteins migrate toward the cathode as a function of their molecular weight, and they are separated from other radioactive components which can obscure the pattern of protein phosphorylation on sodium dodecyl sulfate (SDS) gels. SDS gel electrophoresis was used in the second dimension, resulting in the clear resolution of a large number of proteins. TPA caused many changes in the pattern of protein phosphorylation in intact cells. Two proteins which prominently increased their incorporation of 32P were investigated in particular, and they were both found to be retained in the nuclear matrix following successive extraction of cells with Triton, digestion with DNase and RNase, and extraction with 2 M NaCl. These proteins migrated with apparent molecular weights of 80,000 and 33,000 on SDS gels, and are designated NP80 and NP33, respectively. NP80 was half-maximally phosphorylated after 7 min exposure to TPA, and half-maximally phosphorylated by 10 nM TPA. NP80 co-migrated with a faint Coomassie Blue-stained protein, and NP33 co-migrated with a more prominent protein. Several proteins incorporated less 32P when the cells were exposed to TPA, including one which was extracted from nuclei with the core histones and which co-migrated with histone H2A. Further study will be needed to determine whether the differentiation of HL60 induced by TPA is mediated via phosphorylation of these nuclear matrix proteins.     

Carbohydrate utilization patterns and substrate preferences in Bacteroides thetaiotaomicron

Specific growth rates of Bacteroides thetaiotaomicron NCTC 10582 with either glucose, arabinose, mannose, galactose or xylose as sole carbon sources were 0.42/h, 0.10/h, 0.38/h, 0.38/h and 0.16/h respectively, suggesting that hexose metabolism was energetically more efficient than pentose fermentation in this bacterium. Batch culture experiments to determine whether carbohydrate utilization was controlled by substrate-induced regulatory mechanisms demonstrated that mannose inhibited uptake of glucose, galactose and arabinose, but had less effect on xylose. Arabinose and xylose were preferentially utilized at high dilution rates (D > 0.26/h) in carbon-limited continuous cultures grown on mixtures of arabinose, xylose, galactose and glucose. When mannose was also present, xylose was co-assimilated at all dilution rates. Under nitrogen-limited conditions, however, mannose repressed uptake of all sugars, showing that its effect on xylose utilization was strongly concentration dependent. Studies with individual D-ZU-14C]-labelled substrates showed that transport systems for glucose, galactose, xylose and mannose were inducible. Measurements to determine incorporation of these sugars into trichloroacetic acid-precipitable material indicated that glucose and mannose were the principal precursor monosaccharides. Xylose was only incorporated into intracellular macromolecules when it served as growth substrate. Phosphoenolpyruvate:phosphotransferase systems were not detected in preliminary experiments to elucidate the mechanisms of sugar uptake, and studies with inhibitors of carbohydrate transport showed no consistent pattern of inhibition with glucose, galactose, xylose and mannose. These results indicate the existence of a variety of different systems involved in sugar transport in B. thetaiotaomicron.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.