Partial purification of a dicyclohexylcarbodi-imide-sensitive membrane adenosine triphosphatase complex from the obligately anaerobic bacterium Clostridium Pasteurianum.
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ABSTRACT:
SUBMITTER: Clarke DJ
PROVIDER: S-EPMC1172775 | biostudies-other | 1976 Mar
REPOSITORIES: biostudies-other
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Project description:The inhibition of the membrane-bound adenosine triphosphatase of Escherichia coli by DCCD (dicyclohexylcarbodi-imide) is studied under conditions of varying KCl concentration. An increase in K+ concentration and in other cations causes an increase in the DCCD sensitivity of the enzyme, as well as significant changes in the kinetic parameters.
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Project description:1. Sporulation of Clostridium pasteurianum effects several changes in its proton-translocating cell-membrane H(+)-ATPase. Notable among these are the acquisition of susceptibility to activation by trypsin and a changed protein subunit composition. 2. A protein was isolated from the mother-cell membrane that inhibited the ATP phosphohydrolase activity of purified vegetative-cell-membrane H(+)-ATPase [BF(0)F(1) complex, which consists of soluble ATPase (BF(1)) and the proton-channel component (BF(0))] and rendered it susceptible to trypsin activation. 3. This trypsin-sensitive inhibitor protein had a molecular weight of 10000 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was indistinguishable from the novel protein subunit e of the mother-cell-membrane ATPase 4. In bacteriorhodopsin-containing everted membrane vesicles, the specific ATP synthetase activity of the mother-cell-membrane ATPase was significantly greater than that of the vegetative-cell-membrane ATPase. 5. Treatment with trypsin-sensitive inhibitor protein of artificial proteoliposomes containing bacteriorhodopsin and vegetative-cell-membrane H(+)-ATPase (BF(0)F(1)) significantly increased the specific ATP synthetase activity of this enzyme. 6. The ATP synthetase activity of crude cell-membrane preparations from cultures of Clostridium pasteurianum increased during that period in the course of sporulation when the membrane ATP phosphohydrolase was both most rapidly decreasing in specific activity and acquiring its susceptibility to activation by trypsin.
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Project description:The proton-translocating F1F0 ATP synthase from Clostridium thermoautotrophicum was solubilized from cholate-washed membranes with Zwittergent 3-14 at 58 degrees C and purified in the presence of octylglucoside by sucrose gradient centrifugation and ion-exchange chromatography on a DEAE-5PW column. The purified enzyme hydrolyzed ATP at a rate of 12.6 micromol min(-1) mg(-1) at 58 degrees C and pH 8.5. It was composed of six different polypeptides with molecular masses of 60, 50, 32, 19, 17, and 8 kDa. These were identified as alpha, beta, gamma, delta, epsilon, and c subunits, respectively, as their N-terminal amino acid sequences matched the deduced N-terminal amino acid sequences of the corresponding genes of the atp operon sequenced from Clostridium thermoaceticum (GenBank accession no. U64318), demonstrating the close similarity of the F1F0 complexes from C. thermoaceticum and C. thermoautotrophicum. Four of these subunits, alpha, beta, gamma, and epsilon, constituted the F1-ATPase purified from the latter bacterium. The delta subunit could not be found in the purified F1 although it was present in the F1F0 complex, indicating that the F0 moiety consisted of the delta and the c subunits and lacked the a and b subunits found in many aerobic bacteria. The c subunit was characterized as N,N'-dicyclohexylcarbodiimide reactive. The F1F0 complex of C. thermoautotrophicum consisting of subunits alpha, beta, gamma, delta, epsilon, and c was reconstituted with phospholipids into proteoliposomes which had ATP-Pi exchange, carbonylcyanide p-trifluoromethoxy-phenylhydrazone-stimulated ATPase, and ATP-dependent proton-pumping activities. Immunoblot analyses of the subunits of ATP synthases from C. thermoautotrophicum, C. thermoaceticum, and Escherichia coli revealed antigenic similarities among the F1 subunits from both clostridia and the beta subunit of F1 from E. coli.
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Project description:Here, we report the complete genome sequence of Caloramator sp. strain E03, an anaerobic thermophile that was isolated from a hot spring within the Rabbit Creek area of Yellowstone National Park. The assembly contains a single 2,984,770-bp contig with a G+C content of 31.3% and is predicted to encode 2,678 proteins.
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Project description:A strictly anaerobic Gram-stain-variable but positive by structure, non-spore-forming bacterium designated Lachnospiraceae bacterium ACC2 strain DSM 24645(T) was isolated from human subgingival dental plaque. Bacterial cells were 4-40 µm long non-motile rods, often swollen and forming curved filaments up to 200 µm. Cells contained intracellular, poorly crystalline, nanometre-sized iron- and sulfur-rich particles. The micro-organism was able to grow on yeast extract, trypticase peptone, milk, some sugars and organic acids. The major metabolic end-products of glucose fermentation were butyrate, lactate, isovalerate and acetate. The growth temperature and pH ranges were 30-42 °C and 4.9-7.5, respectively. Major fatty acids were C14?:?0, C14?:?0 DMA (dimethyl aldehyde), C16?:?0, C16?:?1?7c DMA. The whole-cell hydrolysate contained meso-diaminopimelic acid, indicating peptidoglycan type A1?. The DNA G+C content was calculated to be 55.05 mol% from the whole-genome sequence and 55.3 mol% as determined by HPLC. There were no predicted genes responsible for biosynthesis of respiratory lipoquinones, mycolic acids and lipopolysaccharides. Genes associated with synthesis of teichoic and lipoteichoic acids, diaminopimelic acid, polar lipids and polyamines were present. According to the 16S rRNA gene sequence phylogeny, strain DSM 24645(T) formed, together with several uncultured oral clones, a separate branch within the family Lachnospiraceae, with the highest sequence similarity to the type strain of Moryella indoligenes at 94.2?%. Based on distinct phenotypic and genotypic characteristics, we suggest that strain DSM 24645(T) represents a novel species in a new genus, for which the name Stomatobaculum longum gen. nov., sp. nov. is proposed. The type strain of Stomatobaculum longum is DSM 24645(T) (?=?HM-480(T); deposited in BEI Resources, an NIH collection managed by the ATCC).
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Project description:Anaerobic enrichment cultures with elemental sulfur as electron acceptor and either acetate or propionate as electron donor and carbon source at pH 10 and moderate salinity inoculated with sediments from soda lakes in Kulunda Steppe (Altai, Russia) resulted in the isolation of two novel members of the bacterial phylum Chrysiogenetes. The isolates, AHT11 and AHT19, represent the first specialized obligate anaerobic dissimilatory sulfur respirers from soda lakes. They use either elemental sulfur/polysulfide or arsenate as electron acceptor and a few simple organic compounds as electron donor and carbon source. Elemental sulfur is reduced to sulfide through intermediate polysulfide, while arsenate is reduced to arsenite. The bacteria belong to the obligate haloalkaliphiles, with a pH growth optimum from 10 to 10.2 and a salt range from 0.2 to 3.0 M Na(+) (optimum 0.4-0.6 M). According to the phylogenetic analysis, the two strains were close to each other, but distinct from the nearest relative, the haloalkaliphilic sulfur-reducing bacterium Desulfurispirillum alkaliphilum, which was isolated from a bioreactor. On the basis of distinct phenotype and phylogeny, the soda lake isolates are proposed as a new genus and species, Desulfurispira natronophila (type strain AHT11(T) = DSM22071(T) = UNIQEM U758(T)).
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