David R. Woods

Initiation of solvent production, clostridial stage and endospore formation in Clostridium acetobutylicum P262

SummaryA minimal medium was used to investigate the triggers regulating the initiation of solvent production and differentiation in Clostridium acetobutylicum P262. The accumulation of acid end-products caused the inhibition of cell division and the initiation of solvent production and cell differentiation. Initiation only occurred with a narrow pH range. Glucose or ammonium limited cultures failed to achieve the necessary threshold of acid end-products and solvent production and differentiation were not initiated. The addition of acid end-products or ammonium to cultures containing suboptimal levels of glucose or nitrogen respectively, enhanced solvent production. Resuspension of cells in media containing the threshold level of acid end-products and residual glucose induced endospore formation. Glucose or ammonium limitation did not induce sporulation and there was a requirement for glucose and ammonium during solventogenesis and endospore formation. Initiation of solvent production and clostridial stage formation were essential for sporulation. The induction of endospore formation in C. acetobutylicum P262 differs from that in the aerobic endospore forming bacteria where sporulation is initiated by nutrient starvation.

Molecular analysis and nucleotide sequence of the adh1 gene encoding an NADPH-dependent butanol dehydrogenase in the Gram-positive anaerobe Clostridium acetobutylicum

The nucleotide sequence of a 2081-bp fragment of Clostridium acetobutylicum DNA containing the adh1 gene was determined. The butanol dehydrogenase gene is referred to as the adh1 gene since it was shown to have activity using butanol and ethanol as substrates. The adh1 gene consisted of 1164 bp and encoded an alcohol dehydrogenase (ADH) enzyme of 388 aa residues with an Mr of 43,274. The adh1 gene was separated from an upstream open reading frame by an intergenic region of 354 bp. No promoter consensus sequences were identified in the intergenic upstream region and the adh1 gene did not appear to be expressed off its own promoter in Escherichia coli. Three separate types of ADH have been recognized. The ADH1 from C. acetobutylicum exhibited 39% homology with the Fe-containing ADH2 from Zymomonas mobilis and 37% homology with the ADH4 from Saccharomyces cerevisiae, but showed little or no homology with the other characterised types of ADH.

An efficient cyanide-degrading Bacillus pumilus strain

A Gram-positive, aerobic, endospore-forming bacterium was isolated by an enrichment technique for the ability to degrade cyanide and was identified as a Bacillus pumilus strain. The bacterium rapidly degraded 100 mg l-1 of free cyanide in the absence of added inorganic and organic substances. The ability to degrade cyanide was linked to the growth phase and was not exhibited before late exponential/early stationary phase. Cyanide-degrading activity could not be induced before this time by the addition of 20 mg cyanide l-1. Production of the cyanide-degrading activity required 0.01 mg Mn2+ l-1 and did not occur at Mn2+ concentrations below 0.002 mg l-1. Cyanide-degrading activity was intracellular and cell-free extracts rapidly degraded cyanide.

Molecular analysis of a novel glutamine synthetase of the anaerobe Bacteroides fragilis

The nucleotide sequence of a 2777 bp DNA segment containing the Bacteroides fragilis glnA gene was determined. The B. fragilis glnA open reading frame of 2187 bp encoded a glutamine synthetase (GS) subunit of 729 amino acid residues with a calculated Mr of 82,827. The apparent Mr of the GS subunit determined by SDS-PAGE was approximately 75,000. A single mRNA transcription start point was identified upstream of the B. fragilis glnA open reading frame. The B. fragilis GS subunit is approximately 270 and 400 amino acids longer than the GSI and GSII subunits, respectively, of other prokaryotes and eukaryotes. The GSI and GSII holoenzymes are dodecamers and octamers respectively, whereas the GS of B. fragilis is a hexamer. Although GSI and GSII subunits show amino acid similarity in five conserved regions, this similarity is not strongly conserved in the B. fragilis GS. The GS of B. fragilis is not regulated by adenylylation and lacks the adenylylation site. It also lacks the Trp residue associated with the active site in GSI and GSII enzymes from other prokaryotes and eukaryotes.

Characterization of Extracellular Alkaline Proteases and Collagenase Induction in Vibrio alginolyticus

The number and approximate molecular weights of extracellular alkaline proteases produced by Vibrio alginolyticus were determined by gelatin-PAGE. Three major bands of protease activity with apparent molecular weights of approximately 28 000, 22 500 and 19 500 (proteases 1, 2 and 3, respectively) and two minor bands of protease activity with apparent molecular weights of approximately 15 500 and 14 500 (proteases 4 and 5, respectively) were obtained after gelatin-PAGE. The activities of the five proteases were inhibited by serine protease inhibitors but their activities were not affected by inhibitors of trypsin-like enzymes. Histidine, which inhibited V. alginolyticus collagenase, did not inhibit the activities of the alkaline serine proteases. The production of protease 1, however, was enhanced by histidine. Protease 1 production was also affected by temperature and production was depressed at 37 degrees C. Gelatin-PAGE of a commercial V. alginolyticus collagenase preparation revealed four bands of activity which were identified as collagenases with apparent molecular weights of approximately 45 000, 38 500, 33 500 and 31 000. The collagenase preparation was contaminated with two serine proteases. The release of [3H]proline from collagen matrices produced by smooth muscle cells was shown to be a sensitive assay for bacterial collagenases and was used to show that V. alginolyticus produced a basal constitutive level of extracellular collagenase. The constitutive levels of collagenase were affected by aeration.

Cloning and expression of Clostridium acetobutylicum endoglucanase, cellobiase and amino acid biosynthesis genes in Escherichia coli.

Clostridium acetobutylicum P262 endoglucanase and cellobiase genes, cloned on a 4.9 kb DNA fragment in the recombinant plasmid pHZ100, were expressed from their own promoter in Escherichia coli. Active carboxymethylcellulase and cellobiase enzymes were produced, but there was no degradation of Avicel. The endoglucanase activities observed in cell extracts of E. coli HB101(pHZ100) differed in their pH and temperature optima from those previously reported for C. acetobutylicum P270. Complementation of E. coli arg and his mutations by cloned C. acetobutylicum DNA was also observed.

Cloning, sequencing and analysis of expression of a Butyrivibrio fibrisolvens gene encoding a beta-glucosidase.

The cloning, expression and nucleotide sequence of a 3.74 kb DNA segment on pLS215 containing a beta-glucosidase gene (bglA) from Butyrivibrio fibrisolvens H17c was investigated. The B. fibrisolvens bglA open reading frame (ORF) of 2490 bp encoded a beta-glucosidase of 830 amino acid residues with a calculated Mr of 91,800. In Escherichia coli C600(pLS215) cells the beta-glucosidase was localized in the cytoplasm and these cells produced an additional protein with an apparent Mr of approximately 94,000. The bglA gene was expressed from its own regulatory region in E. coli and a single mRNA initiation point was identified upstream of the bglA ORF and adjacent to a promoter consensus sequence. The primary structure of the beta-glucosidase showed greater than 40% similarity with a domain of 237 amino acids present in the beta-glucosidases of Kluyveromyces fragilis and Clostridium thermocellum. The B. fibrisolvens beta-glucosidase hydrolysed cellobiose to a limited extent, cellotriose to cellobiose and glucose, and cellotetraose and cellopentaose to predominantly glucose.

Nucleotide sequence of the Vibrio alginolyticus calcium-dependent, detergent-resistant alkaline serine exoprotease A

The nucleotide sequence of the Vibrio alginolyticus alkaline serine exoprotease A (ProA) gene cloned in Escherichia coli was determined. The exoprotease A gene (proA) consisted of 1602 bp which encoded a protein of 534 amino acids (aa) with an Mr of 55,900. The region upstream from the gene was characterized by a putative promoter consensus region (-10 -35), a ribosome-binding site and ATG start codon. The proA gene encodes a typical 21-aa N-terminal signal sequence which, when fused to alkaline phosphatase by means of transposon TnphoA, was able to mediate transport of the alkaline phosphatase to the periplasm in E. coli. Deletions of up to 106 aa from the C terminus of ProA did not result in the loss of extracellular protease activity. Additional V. alginolyticus genes were not involved in the secretion into the medium of the cloned ProA in E. coli. The amino acid sequence of ProA showed low overall homology to a Serratia marcescens serine exoprotease but significant homology was detected with other subtilisin family exoproteases. The fungal proteinase K, another sodium dodecyl sulfate-resistant protease, had 44% aa homology with ProA.

Sequencing and expression of a cellodextrinase (ced1) gene from Butyrivibrio fibrisolvens H17c cloned in Escherichia coli.

SummaryThe nucleotide sequence of a 2.314 kb DNA segment containing a gene (cedl) expressing cellodextrinase activity from Butyrivibrio fibrisolvens H17c was determined. The B. fibrisolvens H17c gene was expressed from a weak internal promoter in Escherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a GTG start codon. The complete amino acid sequence (547 residues) was deduced and homology was demonstrated with the Clostridium thermocellum endoglucanase D (EGD), Pseudomonas fluorescens var. cellulose endoglucanase (EG), and a cellulase from the avocado fruit (Persea americana). The ced1 gene product Cedl showed cellodextrinase activity and rapidly hydrolysed short-chain cellodextrins to yield either cellobiose or cellobiose and glucose as end products. The Cedl enzyme released cellobiose from p-nitrophenyl-β-d-cellobioside and the enzyme was not inhibited by methylcellulose, an inhibitor of endoglucanase activity. Although the major activity of the Cedl enzyme was that of a cellodextrinase it also showed limited activity against endoglucanase specific substrates [carboxymethylcellulose (CMC), lichenan, laminarin and xylan]. Analysis by SDS-polyacrylamide gel electrophoresis with incorporated CMC showed a major activity band with an apparent Mr of approximately 61000. The calculated Mr of the ced1 gene product was 61023.

The relationship between sporulation and solvent production in clostridium acetobutylicum P262

SummarySingle and multiple Clostridium acetobutylicum P262 sporulation, clostridial stage, granulose, capsule and solvent producing mutants were isolated. Although common regulatory components were involved in the regulation of these events, each individual pathway was able to function independently of each other. Inhibitors of DNA replication inhibited spore formation but did not affect solvent, clostridial stage, granulose and capsule production.