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the genes for butanol and acetone formation in clostridium acetobutylicum atcc 824 reside on a large plasmid whose loss leads to degeneration of the strain.degeneration is the process whereby clostridium acetobutylicum atcc 824 loses the capacity to produce acetone and butanol after repeated vegetative transfers or in continuous culture. two degenerate mutants (m5 and dg1) of c. acetobutylicum atcc 824 do not contain the four genes (ctfa, ctfb, adc, and aad) for acetone and butanol formation. strain atcc 824 contains a 210-kb plasmid (psol1) which is absent in m5 and dg1. psol1 carries the four acetone and butanol formation genes. a restriction map ...19979286999
cloning, sequencing, and expression of dnak-operon proteins from the thermophilic bacterium thermus thermophilus.we propose that the dnak operon of thermus thermophilus hb8 is composed of three functionally linked genes: dnak, grpe, and dnaj. the dnak and dnaj gene products are most closely related to their cyanobacterial homologs. the dnak protein sequence places t. thermophilus in the plastid hsp70 subfamily. in contrast, the grpe translated sequence is most similar to grpe from clostridium acetobutylicum, a gram-positive anaerobic bacterium. a single promoter region, with homology to the escherichia col ...19979349721
acetate kinase from clostridium acetobutylicum: a highly specific enzyme that is actively transcribed during acidogenesis and solventogenesis.acetate kinase (atp:phosphotransferase, ec 2.7.2.1) has been purified 294-fold from acid-producing cells of clostridium acetobutylicum dsm 1731 to a specific activity of 1087 u mg-1 (adp-forming direction). the dimeric enzyme consisted of subunits with a molecular mass of 43 kda. the molecular mass of the native acetate kinase was in the range 87-94 kda as judged by gel filtration and native gel electrophoresis. the enzyme showed high specificity for the substrates acetate and atp, and maximal a ...19979353928
physical and genetic map of the clostridium acetobutylicum atcc 824 chromosome.a physical and genetic map of the clostridium acetobutylicum atcc 824 chromosome was constructed. the macrorestriction map for ceui, eagi, and sstii was created by ordering the 38 restriction sites by one- and two-dimensional pulsed-field gel electrophoresis (pfge) and by using an original strategy based on the ceui enzyme and indirect end labelling by hybridization on both sides of the ceui sites with rrs (16s rna) and 3' rrl (23s rna) probes. the circular chromosome was estimated to be 4.15 mb ...19979393708
characterisation of a transposon-induced pleiotropic mutant of clostridium acetobutylicum p262.transposon-induced metronidazole resistance was used as a selection system for the isolation of clostridium acetobutylicum p262 mutants with altered electron transport pathways. the metronidazole resistant transconjugant of interest, mutant 3r, displayed resistance to dna damaging agents, uv and bleomycin, and harboured a single transposon insertion within a structural gene, designated sum(susceptibility to metronidazole). the sum gene encoded a 334 amino-acid protein, with 36% identity and 57-5 ...199716887617
electroporation of, plasmid isolation from and plasmid conservation in clostridium acetobutylicum dsm 792.procedures have been developed allowing recombinant dna work with clostridium acetobutylicum dsm 792. electroporation was used to introduce plasmid dna into exponentially growing clostridial cells and 6 x 10(2) transformants/microgram dna could be obtained at a time constant of 5.5 ms, 1.8 kv, 50 microf, and 600 omega. the method also allowed the taxonomic group iv strain ni-4082 to be transformed (10(1) transformants/microgram dna). plasmid preparation from recombinant clostridia was optimal wh ...19989866174
expression of clostridium acetobutylicum atcc 824 genes in escherichia coli for acetone production and acetate detoxification.a synthetic acetone operon (ace4) composed of four clostridium acetobutylicum atcc 824 genes (adc, ctfab, and thl, coding for the acetoacetate decarboxylase, coenzyme a transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into escherichia coli on vector pact. acetone production demonstrated that ace4 is expressed in e. coli and resulted in the reduction of acetic acid levels in the fermentation broth. since different e. coli strains va ...19989501448
complementation of an escherichia coli polypeptide deformylase mutant with a gene from clostridium acetobutylicum atcc 824.the clostridium acetobutylicum atcc 824 dna containing the 3' end of a pria homolog, deformylase (def), and the 5' end of formyltransferase (fmt) has been cloned, sequenced, and used to complement an escherichia coli mutant. while def and fmt have been found sharing an operon in other organisms, the presence of a third gene within a putative operon has not previously been found.19989504995
endogenous cellulases in animals: isolation of beta-1, 4-endoglucanase genes from two species of plant-parasitic cyst nematodes.beta-1,4-endoglucanases (egases, ec 3.2.1.4) degrade polysaccharides possessing beta-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce egases endogenously. so far, all previously identified egase genes involved in the digestive system of animals originate from symbiotic microorganisms. here we report ...19989560201
sporulation and time course expression of sigma-factor homologous genes in clostridium acetobutylicum.the gene for the vegetative sigma factor a of clostridium acetobutylicum was constitutively transcribed during growth and formed an operon together with dnae. sporulation-specific sigma factors e, g, and k were sequentially induced shortly before mature endospores could be detected. maximal transcription in the course of spore formation was found to be in the order sige-sigg-sigk, thus matching the pattern described for bacillus subtilis. from primer extension experiments promoter structures cou ...19989561744
cloning, sequence, and expression of the phosphofructokinase gene of clostridium acetobutylicum atcc 824 in escherichia coli.the pfk gene encoding phosphofructokinase (pfk) from the anaerobic bacterium clostridium acetobutylicum atcc 824 was cloned and sequenced. the gene was identified in a plasmid library by complementation of an e. coli pfk mutant and by the ability to amplify a fragment by pcr using primers based on homologous regions of pfk from other microorganisms. nucleotide sequence analysis revealed a coding region for a 319-aa protein homologous to pfks from other organisms. enzyme assay and ability to comp ...19989625784
cloning of an ef-p homologue from bacteroides fragilis that increases b. fragilis glutamine synthetase activity in escherichia coli.investigations of possible regulators of bacteroides fragilis glutamine synthetase (gs) activity were done in escherichia coli using a compatible dual-plasmid system. the b. fragilis glna gene, together with upstream and downstream flanking regions, was cloned onto the low copy number plasmid pacyc184 and expressed in the e. coli glna ntrb ntrc deletion strain, ymc11. gs activity was monitored following co-transformation with a b. fragilis genomic library carried on the compatible plasmid pecor2 ...19989648740
the hyperthermophilic bacterium thermotoga maritima has two different classes of family c dna polymerases: evolutionary implications.bacterial dna polymerase iii (family c dna polymerase), the principal chromosomal replicative enzyme, is known to occur in at least three distinct forms which have provisionally been classified as class i ( escherichia coli dna pol c-type), class ii ( bacillus subtilis dna pol c-type) and class iii (cyanobacteria dna pol c-type). we have identified two family c dna polymerase sequences in the hyperthermophilic bacterium thermotoga maritima. one dna polymerase consisting of 842 amino acid residue ...19989826752
the s box regulon: a new global transcription termination control system for methionine and cysteine biosynthesis genes in gram-positive bacteria.the molecular mechanisms for regulation of the genes involved in the biosynthesis of methionine and cysteine are poorly characterized in bacillus subtilis. analyses of the recently completed b. subtilis genome revealed 11 copies of a highly conserved motif. in all cases, this motif was located in the leader region of putative transcriptional units, upstream of coding sequences that included genes involved in methionine or cysteine biosynthesis. additional copies were identified in clostridium ac ...199810094622
metabolism analysis and on-line physiological state diagnosis of acetone-butanol fermentation.fermentation equations for acetone-butanol (ab) were applied in a metabolic analysis of the reaction network under various conditions; that is, at different phs and a high nadh2 turnover rate using methyl viologen, in a clostridium acetobutylicum culture. the results disclosed variations in the pattern of rate changes that reflected changes in the physiological state. a linear relationship was found to exist between nadh2 generation and butanol production rate. by coupling an automated measureme ...199810099293
genetic manipulation of acid and solvent formation in clostridium acetobutylicum atcc 824the genes coding for enzymes involved in butanol or butyrate formation were subcloned into a novel escherichia coli-clostridium acetobutylicum shuttle vector constructed from pimp1 and a chloramphenicol acetyl transferase gene. the resulting replicative plasmids, referred to as pthaad (aldehyde/alcohol dehydrogenase) and pthbut (butyrate operon), were used to complement c. acetobutylicum mutant strains, in which genes encoding aldehyde/alcohol dehydrogenase (aad) or butyrate kinase (buk) had bee ...199810191392
clpe, a novel type of hsp100 atpase, is part of the ctsr heat shock regulon of bacillus subtilis.clp atpases, which include the ubiquitous hsp100 family, are classified according to their structural features and sequence similarities. during the course of the bacillus subtilis genome sequencing project, we identified a gene encoding a new member of the hsp100 family. we designated this protein clpe, as it is the prototype of a novel subfamily among the clp atpases, and have identified homologues in several bacteria, including listeria monocytogenes, enterococcus faecalis, streptococcus pyog ...199910320580
characterization of methylglyoxal synthase from clostridium acetobutylicum atcc 824 and its use in the formation of 1, 2-propanediol.a gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in clostridium acetobutylicum atcc 824. the enzyme was overexpressed in escherichia coli and purified. methylglyoxal synthase has a native molecular mass of 60 kda and an optimum ph of 7.5. the km and vmax values for the substrate dihydroxyacetone phosphate were 0.53 mm and 1.56 mmol min(-1) microgram(-1), respectively. when e. coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in e. coli bl21(de ...199910388730
the glyceraldehyde-3-phosphate dehydrogenase of clostridium acetobutylicum: isolation and purification of the enzyme, and sequencing and localization of the gap gene within a cluster of other glycolytic genes.glyceraldehyde-3-phosphate dehydrogenase was purified from clostridium acetobutylicum by sequential ammonium sulfate precipitation, gel filtration and anion-exchange chromatography (to a specific activity of 27 u mg-1). the enzyme had a molecular mass of 40 kda as determined by sds-page and a native molecular mass of 160 kda as determined by nondenaturing page, indicating that it has a homotetrameric composition. its ph optimum was between 8.5 and 9.3. the corresponding gene (gap) was cloned and ...199910463150
catabolism of branched-chain alpha-keto acids in enterococcus faecalis: the bkd gene cluster, enzymes, and metabolic route.genes encoding a branched-chain alpha-keto acid dehydrogenase from enterococcus faecalis 10c1, e1alpha (bkda), e1beta (bkdb), e2 (bkdc), and e3 (bkdd), were found to reside in the gene cluster ptb-buk-bkddabc. the predicted products of ptb and buk exhibited significant homology to the phosphotransbutyrylase and butyrate kinase, respectively, from clostridium acetobutylicum. activity and redox properties of the purified recombinant enzyme encoded by bkdd indicate that e. faecalis has a lipoamide ...199910464218
development and characterization of a gene expression reporter system for clostridium acetobutylicum atcc 824.a gene expression reporter system (pht3) for clostridium acetobutylicum atcc 824 was developed by using the lacz gene from thermoanaerobacterium thermosulfurogenes em1 as the reporter gene. in order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pht3 in order to construct vectors pht4, pht5, and phta, respe ...199910473377
stoichiometric modeling of clostridium acetobutylicum fermentations with non-linear constraints.a stoichiometric model of clostridium acetobutylicum and related strains has been previously derived. the stoichiometric matrix of the model contains a singularity which has prevented the calculation of a unique set of fluxes which describe the primary metabolic activity. to resolve the singularity, we have developed a non-linear constraint relating the acetate and butyrate uptake fluxes. subsequently, we developed a software package utilizing a model independent heuristic global optimization ap ...199910483106
stable escherichia coli-clostridium acetobutylicum shuttle vector for secretion of murine tumor necrosis factor alpha.recombinant plasmids were constructed to secrete mouse tumor necrosis factor alpha (mtnf-alpha) from clostridium acetobutylicum. the shuttle plasmids contained the clostridial endo-beta1, 4-glucanase (egla) promoter and signal sequence that was fused in frame to the mtnf-alpha cdna. the construction was first tested in escherichia coli and then introduced in c. acetobutylicum dsm792 by electroporation. controls confirmed the presence and stability of the recombinant plasmids in this organism. so ...199910508051
molecular cloning of the dnak gene region from bacillus sphaericus in the context of genomic comparisons.the dnak gene region of bacillus sphaericus was cloned as a 3.8 kb hindiii fragment and an overlapping 1.7 kb ecori fragment by using an internal b. sphaericus specific dnak gene probe generated by polymerase chain reaction (pcr). complete dna sequencing of the two fragments revealed three complete open reading frames (orfs). these orfs exhibited a high degree of identity to the grpe dnak, and dnaj heat shock genes from other gram-positive bacteria. the order of the genes was found to be grpe-dn ...199910518301
the ald gene, encoding a coenzyme a-acylating aldehyde dehydrogenase, distinguishes clostridium beijerinckii and two other solvent-producing clostridia from clostridium acetobutylicum.the coenzyme a (coa)-acylating aldehyde dehydrogenase (aldh) catalyzes a key reaction in the acetone- and butanol (solvent)-producing clostridia. it reduces acetyl-coa and butyryl-coa to the corresponding aldehydes, which are then reduced by alcohol dehydrogenase (adh) to form ethanol and 1-butanol. the aldh of clostridium beijerinckii nrrl b593 was purified. it had no adh activity, was nad(h) specific, and was more active with butyraldehyde than with acetaldehyde. the n-terminal amino acid sequ ...199910543811
regulation of the sol locus genes for butanol and acetone formation in clostridium acetobutylicum atcc 824 by a putative transcriptional repressor.a gene (orf1, now designated solr) previously identified upstream of the aldehyde/alcohol dehydrogenase gene aad (r. v. nair, g. n. bennett, and e. t. papoutsakis, j. bacteriol. 176:871-885, 1994) was found to encode a repressor of the sol locus (aad, ctfa, ctfb and adc) genes for butanol and acetone formation in clostridium acetobutylicum atcc 824. primer extension analysis identified a transcriptional start site 35 bp upstream of the solr start codon. amino acid comparisons of solr identified ...19999864345
metabolic flux analysis elucidates the importance of the acid-formation pathways in regulating solvent production by clostridium acetobutylicum.metabolic flux analysis was used to investigate the roles of the acid formation pathways in clostridium acetobutylicum. the acid formation pathways were revealed to serve different roles in wildtype fermentations than previously expected. specifically, enzymes known to catalyze butyrate formation were found to uptake butyrate without concomitant production of acetone. this role was further corroborated by flux analysis of a recombinant strain overexpressing the butyrate formation enzymes. analys ...199910937935
functional characterisation of the chaperones dnak, dnaj, and grpe from clostridium acetobutylicum.the dnak chaperone system is involved in various cellular processes such as the control of the folded and oligomeric state of proteins under stress and non-stress conditions. in this study we functionally characterised the homologues of the dnak system from clostridium acetobutylicum dnak, dnaj, grpe and orfa were heterologously synthesised in escherichia coli and affinity purified via a his-tag. by optimising the stoichiometry, we were able to refold guanidinium hydrochloride-denatured firefly ...19999919660
a four-dimensional view of assembly of a morphogenetic protein during sporulation in bacillus subtilis.we report the use of a fusion to the green fluorescent protein to visualize the assembly of the morphogenetic protein spoiva around the developing forespore during the process of sporulation in the bacterium bacillus subtilis. using a deconvolution algorithm to process digitally-collected optical sections, we show that spoiva, which is synthesized in the mother cell chamber of the sporangium, assembled into a spherical shell around the outer surface of the forespore. time-lapse fluorescence micr ...19999922240
ctsr, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in gram-positive bacteria.clpp and clpc of bacillus subtillis encode subunits of the clp atp-dependent protease and are required for stress survival, including growth at high temperature. they play essential roles in stationary phase adaptive responses such as the competence and sporulation developmental pathways, and belong to the so-called class iii group of heat shock genes, whose mode of regulation is unknown and whose expression is induced by heat shock or general stress conditions. the product of ctsr, the first ge ...19999987115
antisense rna strategies for metabolic engineering of clostridium acetobutylicum.we examined the effectiveness of antisense rna (as rna) strategies for metabolic engineering of clostridium acetobutylicum. strain atcc 824(prd4) was developed to produce a 102-nucleotide asrna with 87% complementarity to the butyrate kinase (bk) gene. strain atcc 824(prd4) exhibited 85 to 90% lower bk and acetate kinase specific activities than the control strain. strain atcc 824(prd4) also exhibited 45 to 50% lower phosphotransbutyrylase (ptb) and phosphotransacetylase specific activities than ...199910049845
purification and characterization of two extremely thermostable enzymes, phosphate acetyltransferase and acetate kinase, from the hyperthermophilic eubacterium thermotoga maritima.phosphate acetyltransferase (pta) and acetate kinase (ak) of the hyperthermophilic eubacterium thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. pta had an apparent molecular mass of 170 kda and was composed of one subunit with a molecular mass of 34 kda, suggesting a homotetramer (alpha4) structure. the n-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from clostridium acetobutylicum rather than t ...199910074080
utilisation of saccharides in extruded domestic organic waste by clostridium acetobutylicum atcc 824 for production of acetone, butanol and ethanol.domestic organic waste (dow) collected in the netherlands was analysed and used as substrate for acetone, butanol and ethanol (abe) production. two different samples of dow, referred to as fresh dow and dried dow, were treated by extrusion in order to expand the polymer fibres present and to obtain a homogeneous mixture. the extruded material was analysed with respect to solvent and hot water extractives, uronic acids, lignin, sugars and ash. the total sugar content in the polymeric fractions of ...200010968627
spo0a directly controls the switch from acid to solvent production in solvent-forming clostridia.the spo0a genes of clostridium beijerinckii ncimb 8052 and clostridium cellulolyticum atcc 35319 were isolated and characterized. the c-terminal dna-binding domains of the predicted products of spo0a from these two organisms, as well as 16 other taxonomically diverse species of bacillus and clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues). a 12-amino-acid motif (srverairhaie) that forms the putative dna recognition helix is particularl ...200010972834
nucleotide sequence, expression and transcriptional analysis of the bifidobacterium longum mb 219 lacz gene.the gene encoding beta-galactosidase was isolated by functional complementation of escherichia coli from bifidobacterium longum mb219, which exhibited the highest activity among ten bifidobacterium strains tested of the species b. longum, b. breve, b. adolescentis, b. indicum, b. animalis and b. cuniculi. the nucleotide sequence of the 5.0-kb fragment conferring the positive beta-galactosidase phenotype to e. coli revealed the presence of a lacz-type gene encoding a 1023-amino-acid protein that ...200010985745
cloning and nucleotide sequence of the dna gyrase (gyra) gene from mycoplasma hominis and characterization of quinolone-resistant mutants selected in vitro with trovafloxacin.we report the cloning and characterization of the gyra gene of the mycoplasma hominis dna gyrase, which was previously shown to be associated with quinolone resistance in this organism. the 2,733-bp gyra gene encodes a protein of 911 amino acids with a calculated molecular mass of 102.5 kda. as expected, m. hominis gyra exhibits higher homology with the gyra subunits of the gram-positive bacteria clostridium acetobutylicum, bacillus subtilis, streptococcus pneumoniae, and staphylococcus aureus t ...200010991851
toxins, butyric acid, and other short-chain fatty acids are coordinately expressed and down-regulated by cysteine in clostridium difficile.it was recently found that a mixture of nine amino acids down-regulate clostridium difficile toxin production when added to peptone yeast extract (py) cultures of strain vpi 10463 (s. karlsson, l. g. burman, and t. akerlund, microbiology 145:1683-1693, 1999). in the present study, seven of these amino acids were found to exhibit a moderate suppression of toxin production, whereas proline and particularly cysteine had the greatest impact, on both reference strains (n = 6) and clinical isolates (n ...200010992498
differential regulation of two thiolase genes from clostridium acetobutylicum dsm 792.thiolase of clostridium acetobutylicum is an important enzyme involved in both, acid and solvent fermentation. two thiolase genes (thla and thib) have been cloned and sequenced from clostridium acetobutylicum dsm 792, showing high homology to each other and to thiolases of pha-synthesizing bacteria. the thla gene is identical to the gene already cloned and sequenced from strain atcc 824 (stim-herndon et al., 1995, gene 154: 81-85). using primer extension and s1 nuclease analysis a transcriptiona ...200011075929
characterization of recombinant strains of the clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition?two metabolic engineering tools, namely gene inactivation and gene overexpression, were employed to examine the effects of two genetic modifications on the fermentation characteristics of clostridium acetobutylicum. inactivation of the butyrate kinase gene (buk) was examined using strain pjc4bk, while the combined effect of buk inactivation and overexpression of the aad gene-encoding the alcohol aldehyde dehydrogense (aad) used in butanol formation-was examined using strain pjc4bk(ptaad). the tw ...200010581430
broad-host-range shuttle vectors for screening of regulated promoter activity in viridans group streptococci: isolation of a ph-regulated promoter.viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. we therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group stre ...200010653715
a novel genetically engineered pathway for synthesis of poly(hydroxyalkanoic acids) in escherichia coli.a new pathway to synthesize poly(hydroxyalkanoic acids) (pha) was constructed by simultaneously expressing butyrate kinase (buk) and phosphotransbutyrylase (ptb) genes of clostridium acetobutylicum and the two pha synthase genes (phae and phac) of thiocapsa pfennigii in escherichia coli. the four genes were cloned into the bamhi and ecori sites of pbr322, and the resulting hybrid plasmid, pbpp1, conferred activities of all three enzymes to e. coli jm109. cells of this recombinant strain accumula ...200010653745
electrotransformation of clostridium acetobutylicum atcc 824 using high-voltage radio frequency modulated square pulses.molecular biological improvement of industrial solventogenic clostridia could be enhanced by a higher efficiency of electrotransformation. in this research, we used a new approach to determine the frequency spontaneously generated by clostridium acetobutylicum atcc 824 cells during the application of a square high-voltage pulse. once the frequency of 100 khz was determined we transformed clostridial cells with psos84 plasmid dna using radio-frequency modulated high-voltage square pulses (electri ...200010735989
phosphine generation by mixed- and monoseptic-cultures of anaerobic bacteria.a microbial basis for bioreductive generation of phosphine is proposed, which could account at least in part for the presence of this toxic gas in natural anaerobic environments and in sewage and landfill gases. phosphine generation under anaerobic growth conditions was dependent upon both the culture inoculum source (animal faeces) and enrichment culture conditions. phosphine was detected in headspace gases from mixed cultures under conditions promoting fermentative growth of mixed acid and but ...200010811253
isolation of mesophilic solvent-producing clostridia from colombian sources: physiological characterization, solvent production and polysaccharide hydrolysis.one hundred and seventy-eight new butanol-acetone producing bacteria related to saccharolytic clostridia were isolated from agricultural sources in colombia and their fermentation potential was evaluated. thirteen isolates produced more total solvents from glucose than clostridium acetobutylicum atcc 824. the isolates with the highest single solvent production were ibun 125c and ibun 18a with 0.46 mol butanol and 0.96 mol ethanol formed from 1 mol glucose, yielding 25. 2 and 29.1 g l(-1) total s ...200010812180
exploitation of butyrate kinase and phosphotransbutyrylase from clostridium acetobutylicum for the in vitro biosynthesis of poly(hydroxyalkanoic acid).active butyrate kinase (buk) and phosphotransbutyrylase (ptb) were purified in three steps: ammonium sulfate precipitation, hydrophobic chromatography on phenyl-sepharose and affinity chromatography on matrex red a from recombinant escherichia coli k2006 (pjc7). they were then successfully exploited for in vitro synthesis of 3-hydroxybutyryl-coa (3hbcoa), 4-hydroxybutyryl-coa (4hbcoa), 4-hydroxyvaleryl-coa (4hvcoa) and poly(hydroxyalkanoic acid) (pha). in addition, the ability of the pha synthas ...200010855714
phylogeny and functional conservation of sigma(e) in endospore-forming bacteria.conservation of the sporulation processes between bacillus spp. and clostridium spp. was investigated through evolutionary and complementation analyses of sigma(e). alignment of partial predicted sigma(e) amino acid sequences from three bacillus spp., paenibacillus polymyxa and five clostridium spp. revealed that amino acid residues previously reported to be involved in promoter utilization (m124, e119 and n120) and strand opening (c117) are conserved among all these species. phylogenetic analys ...200010878124
sequence analysis of the atp operon of clostridium acetobutylicum dsm 792 encoding the f0f1 atp synthase.the atp gene region of clostridium acetobutylicum dsm 792 has been fully sequenced. it contains the f0f1 atpase genes in the order atpibefhagdc, whose products share high sequence homology to the respective proteins of a variety of other bacteria. it is the first such sequence available for a mesophilic clostridium. significant differences to other reported atp operons are a distal transcription start point 219 bp upstream of the translation start point and a second transcription initiation site ...200010902917
identification of two genes encoding putative new members of the ecf subfamily of eubacterial rna polymerase sigma factors in clostridium acetobutylicum.two genes from clostridium acetobutylicum dsm 792 were identified which are predicted to encode new members of the ecf subfamily of eubacterial rna polymerase sigma factors. the sigx gene has the potential to encode a 184-amino acid protein with a molecular mass of 21,870 da and with the highest overall similarity to fecl of escherichia coli (27 % identical residues). the second gene, which is predicted to encode an alternative sigma factor of the ecf subfamily, is the previously described orf2 ...200010937434
identification and characterization of a second butyrate kinase from clostridium acetobutylicum atcc 824.a gene encoding a new butyrate kinase isozyme (bkii) was identified from the c. acetobutylicum atcc 824 dna database. the enzyme was expressed in escherichia coli, purified, and characterized. the purified enzyme exhibited a subunit molecular mass of 43 kda by sds-page, and a native molecular mass of 80 kda by gel filtration suggesting it functions as a dimer. in the butyryl phosphate-forming direction the optimal ph of bkii was 8.5. the enzyme had a km of 0.62 m and a turn over rate of 2.2 x 10 ...200010937485
acetone, butanol and ethanol production from domestic organic waste by solventogenic clostridia.domestic organic waste (dow) was washed and dried to 85 % dryness by vam (the netherlands). this material contained 25.1 g glucose, 8.4 g xylose and 5.8 g other monosaccharides/100 g dry matter. using mansonite steam explosion and enzymatic hydrolysis, a hydrolysate containing 15.4 g glucose, 2.2 g xylose and 0.8 g other monosaccharides per l was made. clostridium acetobutylicum dsm 1731 produced 1.5 and c. beijerinckii b-592 0.9 g/l abe and clostridium lmd 84.48 1.9 g/l ibe, respectively, from ...200010937486
kdpe of clostridium acetobutylicum is a highly specific response regulator controlling only the expression of the kdp operon.kdpe from clostridium acetobutylicum was enriched in form of its strep-tag-derivative to allow easy immunodetection. it could be artificially phosphorylated by acetyl phosphate or carbamyl phosphate. only phosphorylated clostridial kdpe was able to bind to a region upstream of the clostridial kdp structural genes. the minimal sequence requirements for binding were determined and found to share significant similarity with the escherichia coli kdpe binding motif. however, the clostridial protein p ...200010937487
analysis of a catabolic operon for sucrose transport and metabolism in clostridium acetobutylicum atcc 824.the utilization of sucrose by clostridium acetobutylicum atcc 824 was investigated. sucrose was found to be transported via a phosphoenol-pyruvate (pep)-dependent phosphotransferase system (pts) and a metabolic pathway identical to that previously identified in c. beijerinckii, was established. the genes encoding the proteins of this pathway were identified from the c. acetobutylicum genome sequence, in the order scrakb encoding enzyme ii of the sucrose pts, fructokinase and sucrose 6-phosphate ...200010937490
comparative fermentation studies of industrial strains belonging to four species of solvent-producing clostridia.industrial and culture collection strains of solvent-producing clostridia, classified as clostridium acetobutylicum, clostridium beijerinckii, clostridium saccharobutylicum, and clostridium saccharoperbutylacetonicum were utilised in a comparative study of fermentation performance in a laboratory fermentation medium, a molasses fermentation medium, and a maize fermentation medium under standardised culture conditions. at least one representative strain was selected from each of the sub-groups wi ...200010937496
regulation of carbon and electron flow in clostridium butyricum vpi 3266 grown on glucose-glycerol mixtures.the metabolism of clostridium butyricum was manipulated at ph 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. in contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. ...200111160107
molecular analysis of the mannitol operon of clostridium acetobutylicum encoding a phosphotransferase system and a putative pts-modulated regulator.clostridium acetobutylicum dsm 792 accumulates and phosphorylates mannitol via a phosphoenolpyruvate (pep)-dependent phosphotransferase system (pts). pep-dependent mannitol phosphorylation by extracts of cells grown on mannitol required both soluble and membrane fractions. neither the soluble nor the membrane fraction could be complemented by the opposite fraction prepared from glucose-grown cells, indicating that the mannitol-specific pts consists of both a soluble (iia) and a membrane-bound (i ...200111160802
analysis of kdpc of the k(+)-transporting kdpfabc complex of escherichia coli.the kdp complex, a high affinity atp-driven k(+) transport system of escherichia coli, is composed of the four membrane-bound subunits kdpf, kdpa, kdpb and kdpc. whereas the role of kdpb (catalytical subunit), kdpa (k(+)-translocating subunit) and kdpf (stabilizing peptide) is well understood, the function of kdpc is still unknown. therefore, a kdpc deletion strain was constructed and complementation experiments were performed using different kdpc constructs. truncations of the kdpc gene reveale ...200111248697
specific targeting of cytosine deaminase to solid tumors by engineered clostridium acetobutylicum.the presence of severe hypoxia and necrosis in solid tumors offers the potential to apply an anaerobic bacterial enzyme/prodrug approach in cancer treatment. in this context the apathogenic c. acetobutylicum was genetically engineered to express and secrete e. coli cytosine deaminase (cdase). considerable levels of functional cytosine deaminase were detected in lysates and supernatants of recombinant c acetobutylicum cultures. after administration of the recombinant clostridium to rhabdomyosarco ...200111393282
clostridium uliginosum sp. nov., a novel acid-tolerant, anaerobic bacterium with connecting filaments.an anaerobic, acid-tolerant bacterium, ck55t, was isolated from an acidic forest bog. cells of ck55t stained gram-negative but did not have an outer membrane. cells were spore-forming, motile rods with peritrichous flagella, formed chains or aggregates and were linked by connecting filaments that were composed of a core and outer sheath. cellobiose, glucose, xylose, mannose, mannitol, sucrose and peptone supported growth. arabinose, lactose, raffinose, h2/co2, co/co2, vanillate, casamino acids a ...200111411680
clostridium felsineum and clostridium acetobutylicum are two distinct species that are phylogenetically closely related.the gene sequences encoding the 16s rrna of clostridium felsineum dsm 794t and ncimb 10690t were determined. both sequences exhibited a relatively very low degree of similarity to the previously determined 16s rrna gene sequence from c. felsineum dsm 794t. c. felsineum is a member of the major clostridium cluster, cluster i, and is phylogenetically closely related to clostridium acetobutylicum. dna-dna hybridization results clearly indicated that c. felsineum and c. acetobutylicum belong to dist ...200111411721
physical and genetic map of the clostridium saccharobutylicum (formerly clostridium acetobutylicum) ncp 262 chromosome.a physical and genetic map of the clostridium saccharobutylicum ncp 262 chromosome was constructed. the order of macrorestriction fragments was determined by analysing fragments generated after single and double digestion with the restriction enzymes bsshii, i-ceui, sse8387i, rsrii and sfii and separation by pfge. the i-ceui backbone of c. saccharobutylicum was constructed by indirect end-labelling with rrs- and 3' rrl-specific probes located on either side of the i-ceui site in the rrn operon, ...200111429467
identification of the gene encoding nadh-rubredoxin oxidoreductase in clostridium acetobutylicum.nadh-rubredoxin oxidoreductase (nror), a flavoprotein from the obligately anaerobe clostridium acetobutylicum is encoded by an orf (nror) of 1140 nucleotides. whereas primary structure analysis reveals that nror has amino acid sequence patterns homologous with those involved in fad and nad-binding, the enzyme is distantly related to other flavoproteins in the databank. nror is highly active for reducing clostridial rubredoxin (rd) especially against c. acetobutylicum rd with an efficiency (k(cat ...200111444870
genome sequence and comparative analysis of the solvent-producing bacterium clostridium acetobutylicum.the genome sequence of the solvent-producing bacterium clostridium acetobutylicum atcc 824 has been determined by the shotgun approach. the genome consists of a 3.94-mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. comparison of c. acetobutylicum to bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximi ...200111466286
a case of anaerobic abscessed hydroxyapatite orbital implants.the purpose of this report is to document an unusual case of implant infection in a patient who had undergone enucleation and hydroxyapatite orbital implant surgery. a 32-year-old woman presented with chronic orbital discomfort and discharge following a history of hydroxyapatite orbital implant surgery at another hospital 4 years previous. she exhibited profuse discharge with a yellow, creamy color and marked conjunctival chemosis. granulation tissue was noted on the central conjunctival surface ...200111530822
expression of the klebsiella pneumoniae cg21 acetoin reductase gene in clostridium acetobutylicum atcc 824.acetoin reductase catalyzes the production of 2,3-butanediol from acetoin. the gene encoding the acetoin reductase of klebsiella pneumoniae cg21 was cloned and expressed in escherichia coli and clostridium acetobutylicum atcc 824. the nucleotide sequence of the gene encoding the enzyme was determined to be 768 bp long. expression of the k. pneumoniae acetoin reductase gene in e. coli revealed that the enzyme has a molecular mass of about 31,000 da based on sodium dodecyl sulfate polyacrylamide g ...200111687934
anaerobic fermentation of gelatinized sago starch-derived sugars to acetone-1-butanol-ethanol solvent by clostridium acetobutylicum.a study of the kinetics and performance of solvent-yielding batch fermentation of individual sugars and their mixture derived from enzymic hydrolysis of sago starch by clostridium acetobutylicum showed that the use of 30 g/l gelatinized sago starch as the sole carbon source produced 11.2 g/l total solvent, i.e. 1.5-2 times more than with pure maltose or glucose used as carbon sources. enzymic pretreatment of gelatinized sago starch yielding maltose and glucose hydrolyzates prior to the fermentat ...200111702403
transcriptional regulation of pentose utilisation systems in the bacillus/clostridium group of bacteria.in bacillus subtilis, utilisation of xylose, arabinose and ribose is controlled by the transcriptional factors xylr, arar and rbsr, respectively. here we apply the comparative approach to the analysis of these regulons in the bacillus/clostridium group. evolutionary variability of operon structures is demonstrated and operator sites for the main transcription factors are predicted. the consensus sequences for the xylr and rbsr binding sites vary in different subgroups of genomes. the functional ...200111750820
emended descriptions of clostridium acetobutylicum and clostridium beijerinckii, and descriptions of clostridium saccharoperbutylacetonicum sp. nov. and clostridium saccharobutylicum sp. nov.on the basis of 16s rrna gene sequencing and dna-dna reassociation, industrial solvent-producing clostridia have been assigned to four species. in this study, the phenotypic characteristics of clostridium acetobutylicum, clostridium beijerinckii, 'clostridium saccharoperbutylacetonicum', and an unnamed clostridium sp. represented by the strains ncp 262t and nrrl b643 are compared. in addition, a further 40 strains of solvent-producing clostridia have been classified by biotyping, dna fingerprint ...200111760952
nitrogen-fixation genes and nitrogenase activity in clostridium acetobutylicum and clostridium beijerinckii.several solvent-producing clostridia, including clostridium acetobutylicum and c. beijerinckii, were previously shown to be nitrogen-fixing organisms based on the incorporation of 15n2 into cellular material. the key nitrogen-fixation (nif) genes, including nifh, nifd, and nifk for nitrogenase component proteins as well as nife, nifn, nifb and nifv for synthesis of the iron-molybdenum cofactor (femoco) of nitrogenase, have now been identified in c. acetobutylicum or c. beijerinckii or both. the ...200111781802
characterization of a maltose transport system in clostridium acetobutylicum atcc 824.the utilization of maltose by clostridium acetobutylicum atcc 824 was investigated. glucose was used preferentially to maltose, when both substrates were present in the medium. maltose phosphoenolpyruvate (pep)-dependent phosphotransferase system (pts) activity was detected in extracts prepared from cultures grown on maltose, but not glucose or sucrose, as the sole carbon source. extract fractionation and pts reconstitution experiments revealed that the specificity for maltose is contained entir ...200111781805
orf5/solr: a transcriptional repressor of the sol operon of clostridium acetobutylicum?the gene of orf5 (solr) of clostridium acetobutylicum dsm 792 was subcloned and overexpressed in escherichia coli. the protein was purified with ni-nta agarose and used for dna binding assays. no dna binding of orf5 to regions upstream of the sol operon from c. acetobutylicum was observed. overexpression of orf5 in c. acetobutylicum led to a change in the organism's pattern of glycosylated exoproteins. the orf5 protein was localized in the cell membrane fraction and to a small extent in the supe ...200111781806
degeneration of solventogenic clostridium strains monitored by fourier transform infrared spectroscopy of bacterial cells.strain degeneration in solventogenic clostridia is a known problem in the technical acetone-butanol fermentation bioprocess, especially in the continuous process mode. clostridial strain degeneration was studied by fourier transform infrared (ft-ir) spectroscopy of the bacterial cells. degenerative variant formation in two strains, clostridium beijerinckii ncimb 8052 and clostridium species aa332, was detected spectroscopically. colonies on solid media were sampled, or assayed directly in situ b ...200111781807
fermentation characterization and flux analysis of recombinant strains of clostridium acetobutylicum with an inactivated solr gene.the effect of solr inactivation on the metabolism of clostridium acetobutylicum was examined using fermentation characterization and metabolic flux analysis. the solr-inactivated strain (solrh) of this study had a higher rate of glucose utilization and produced higher solvent concentrations (by 25%, 14%, and 81%, respectively, for butanol, acetone, and ethanol) compared to the wild type. strain solrh(ptaad), carrying a plasmid-encoded copy of the bifunctional alcohol/aldehyde dehydrogenase gene ...200111781808
molecular characterization and transcriptional analysis of adhe2, the gene encoding the nadh-dependent aldehyde/alcohol dehydrogenase responsible for butanol production in alcohologenic cultures of clostridium acetobutylicum atcc 824.the adhe2 gene of clostridium acetobutylicum atcc 824, coding for an aldehyde/alcohol dehydrogenase (aadh), was characterized from molecular and biochemical points of view. the 2,577-bp adhe2 codes for a 94.4-kda protein. adhe2 is expressed, as a monocistronic operon, in alcohologenic cultures and not in solventogenic cultures. primer extension analysis identified two transcriptional start sites 160 and 215 bp upstream of the adhe2 start codon. the expression of adhe2 from a plasmid in the dg1 m ...200211790753
changes in protein synthesis and identification of proteins specifically induced during solventogenesis in clostridium acetobutylicum.at the end of the exponential growth phase the gram-positive bacterium clostridium acetobutylicum performs a metabolic switch from classical sugar fermentation accompanied by the production of acetate and butyrate to reinternalization and oxidation of these acids to acetone and butanol. protein synthesis in acidogenic and solventogenic c. acetobutylicum cells was compared by two-dimensional gel electrophoresis of radioactively labeled proteins. the results show that the switch from acid to solve ...200211824611
control of butanol formation in clostridium acetobutylicum by transcriptional activation.the sol operon of clostridium acetobutylicum is the essential transcription unit for formation of the solvents butanol and acetone. the recent proposal that transcriptional regulation of this operon is controlled by the repressor orf5/solr (r. v. nair, e. m. green, d. e. watson, g. n. bennett, and e. t. papoutsakis, j. bacteriol. 181:319-330, 1999) was found to be incorrect. instead, regulation depends on activation, most probably by the multivalent transcription factor spo0a. the operon is tran ...200211889105
transcriptional regulation of solventogenesis in clostridium acetobutylicum.solvent synthesis in clostridium acetobutylicum is induced in concert with sporulation to counteract the dangerous effects of produced butyric and acetic acids and to provide the cell with sufficient time to complete endospore formation. cardinal transcription units for butanol and acetone production are the sol and adc operons encoding butyraldehyde/butanol dehydrogenase and coenzyme a transferase as well as acetoacetate decarboxylase. induction is achieved by a decreased level of dna supercoil ...200211931561
the esat-6/wxg100 superfamily -- and a new gram-positive secretion system?esat-6 is a small secreted protein of unknown function from mycobacterium tuberculosis that is of fundamental importance in virulence and protective immunity. a psi-blast search has identified distant homologues of esat-6 in more tractable bacteria, including bacillus subtilis, bacillus anthracis, staphylococcus aureus and clostridium acetobutylicum. the genes for esat-6-like proteins often cluster with genes encoding homologues of b. subtilis yuka. i speculate that the esat-6-like and yuka-like ...200211973144
amyp, a reporter gene to study strain degeneration in clostridium acetobutylicum atcc 824.clostridium acetobutylicum produces an extracellular alpha-amylase when grown on glucose as the sole carbon source. this enzyme was previously characterized from a biochemical point of view but its encoding gene was never identified. the 2283-bp amyp gene encodes a 83013-da mature protein with an n-terminal domain that exhibits strong identity to the family 13 glycosyl hydrolases such as the bacillus alpha-amylases. transcriptional analysis revealed that amyp is transcribed in solventogenic but ...200212023083
northern, morphological, and fermentation analysis of spo0a inactivation and overexpression in clostridium acetobutylicum atcc 824.the clostridium acetobutylicum atcc 824 spo0a gene was cloned, and two recombinant strains were generated, an spo0a inactivation strain (sko1) and an spo0a overexpression strain [824(pmpsoa)]. sko1 was developed by targeted gene inactivation with a replicative plasmid capable of double-crossover chromosomal integration--a technique never used before with solventogenic clostridia. sko1 was severely deficient in solvent formation: it produced only 2 mm acetone and 13 mm butanol, compared to the 92 ...200212057953
molecular characterization and utilization of the cak1 filamentous viruslike particle derived from clostridium beijerinckii.an examination of the replication origin and stability determinant associated with the cak1 filamentous viruslike particle recovered from clostridium beijerinckii ncimb 6444 was carried out. seven deletion derivatives, pcke, pcep1, pdt5, pckp, pdth102, pyl102e and pyl102, were constructed and transformed into c. beijerinckii ncimb 8052. the successful transformation of pcke, pdt5, pckp, pdth102, pyl102e and pyl102 into c. beijerinckii 8052, together with the corresponding recovery of single-stra ...200212074052
expression, purification, and characterization of recombinant nonphosphorylating nadp-dependent glyceraldehyde-3-phosphate dehydrogenase from clostridium acetobutylicum.clostridium acetobutylicum gapn was cloned and expressed in escherichia coli bl-21. the iptg-induced nonphosphorylating nadp-dependent gapdh (gapn) has been purified about 34-fold from e. coli cells and its physical and kinetic properties were investigated. the purification method consisted of a rapid and straightforward procedure involving anion-exchange and hydroxyapatite chromatographies. the purified protein is an homotetrameric of 204kda exhibiting absolute specificity for nadp. chromatofoc ...200212182834
cloning and molecular characterization of an immunogenic liga protein of leptospira interrogans.a clone expressing a novel immunoreactive leptospiral immunoglobulin-like protein a of 130 kda (liga) from leptospira interrogans serovar pomona type kennewicki was isolated by screening a genomic dna library with serum from a mare that had recently aborted due to leptospiral infection. liga is encoded by an open reading frame of 3,675 bp, and the deduced amino acid sequence consists of a series of 90-amino-acid tandem repeats. a search of the ncbi database found that homology of the liga repeat ...200212379666
reactivity of partially reduced arylhydroxylamine and nitrosoarene metabolites of 2,4,6-trinitrotoluene (tnt) toward biomass and humic acids.sequential anaerobic/aerobic treatment of 2,4,6-trinitrotoluene (tnt) generally results in the incorporation of residues into biomass and natural organic matter fractions of a system. to better understand the potential contribution of hydroxylamine and nitroso moieties in these reactions, studies were conducted using model systems taking advantage of the biocatalytic-activity of clostridium acetobutylicum that does not produce aminated tnt derivatives. to evaluate binding to biomass only, system ...200212387411
characterization of the cellulolytic complex (cellulosome) of clostridium acetobutylicum.a large cellulosomal gene cluster was identified in the recently sequenced genome of clostridium acetobutylicum atcc 824. sequence analysis revealed that this cluster contains the genes for the scaffolding protein cipa, the processive endocellulase cel48a, several endoglucanases of families 5 and 9, the mannanase man5g, and a hydrophobic protein, orfxp. surprisingly, genetic organization of this large cluster is very similar to that of clostridium cellulolyticum, the model of mesophilic clostrid ...200212445640
functional organization of a single nif cluster in the mesophilic archaeon methanosarcina mazei strain gö1.the mesophilic methanogenic archaeon methanosarcina mazei strain gö1 is able to utilize molecular nitrogen (n2) as its sole nitrogen source. we have identified and characterized a single nitrogen fixation (nif) gene cluster in m. mazei gö1 with an approximate length of 9 kbp. sequence analysis revealed seven genes with sequence similarities to nifh, nifi1, nifi2, nifd, nifk, nife and nifn, similar to other diazotrophic methanogens and certain bacteria such as clostridium acetobutylicum, with the ...200215803652
cloning, characterization, and functional expression of the klebsiella oxytoca xylodextrin utilization operon (xyntb) in escherichia coli.escherichia coli is being developed as a biocatalyst for bulk chemical production from inexpensive carbohydrates derived from lignocellulose. potential substrates include the soluble xylodextrins (xyloside, xylooligosaccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis. adjacent genes encoding xylobiose uptake and hydrolysis were cloned from klebsiella oxytoca m5a1 and are functionally expressed in ethanologenic e. coli. the xy ...200314532050
analysis of the elements of catabolite repression in clostridium acetobutylicum atcc 824.the ptsh gene, encoding the phosphotransferase protein hpr, from clostridium acetobutylicum atcc 824 was identified from the genome sequence, cloned and shown to complement a ptsh mutant of escherichia coli. the deduced protein sequence shares significant homology with hpr proteins from other low-gc gram-positive bacteria, although the highly conserved sequence surrounding the ser-46 phosphorylation site is not well preserved in the clostridial protein. nevertheless, the hpr was phosphorylated i ...200314593248
mutagenicity of nitroaromatic degradation compounds.the mutagenicity of 2,4-dinitrotoluene (24dnt), and 2,6-dinitrotoluene (26dnt), and their related transformation products such as hydroxylamine and amine derivatives, which are formed by clostridium acetobutylicum, were tested in crude cell extracts using salmonella typhimurium ta100. a previous publication already reported the mutagenic activities of 2,4,6-trinitrotoluene (tnt) and its related hydroxylamine derivatives in this test system. a time course of the mutagenicity during the anaerobic ...200314551991
phylogenetic reconstruction of gram-positive organisms based on comparative sequence analysis of molecular chaperones from the ruminal microorganism ruminococcus flavefaciens fd-1.primers designed on the basis of nucleotide sequences conserved in dnak and groel from gram-positive organisms were used to pcr amplify internal regions of the cognate genes from the anaerobic ruminal cellulolytic bacterium ruminococcus flavefaciens fd-1. genome walking was then utilized to elucidate the remainder of the sequences in addition to upstream and downstream regions. the full sequence of the gene encoding the groes protein (groes) was found directly upstream from groel. the deduced am ...200314568141
biosynthesis of poly (3-mercaptopropionate) and poly (3-mercaptopropionate-co-3-hydroxybutyrate) with recombinant escherichia coli.polythioesters newly emerged as a type of novel polymer and they have showed great potential for application in industries. in this study, genes of butyrate kinase (buk) and phosphotransbutyrylase (ptb) from clostridium acetobutylicum, and poly (3-hydroxybutyrate) (phb) synthase gene from thiocapsa pfennigii were used for construction of a metabolic pathway to synthesize the polythioesters. when 3-mercaptopropionate and 3-hydroxybutyrate were fed, poly (3-mercaptopropoinate) [poly (3mp)] and pol ...200315966321
electrotransformation of clostridium paraputrificum m-21 with some plasmids.clostridium paraputrificum m-21 was transformed with several shuttle plasmids constructed for clostridium acetobutylicum-escherichia coli and clostridium perfringens-e. coli by electroporation. the clostridium stercorarium xylanase gene xyn10b was successfully expressed in c. paraputrificum m-21 and the expressed protein did not suffer from proteolysis by host protease(s). this system will provide us with a genetic tool for genetic and metabolic engineering of this bacterium.200316233526
a segmental nearest neighbor normalization and gene identification method gives superior results for dna-array analysis.an intuitive normalization and gene identification method is proposed. after segmentation of the entire expression range into intensity intervals, the mean and standard deviation of the logarithm of expression ratios are calculated for each interval using the nearest neighbor genes. genes with high differential expression are excluded from these calculations. for glass arrays, normalization is performed for each interval by using the mean of the logarithm of expression ratios in the interval. fo ...200312529501
characterization of the cipa scaffolding protein and in vivo production of a minicellulosome in clostridium acetobutylicum.the cipa gene encoding the clostridium acetobutylicum scaffolding protein cipa was cloned and expressed in escherichia coli. cipa contains an n-terminal signal peptide, a family 3a cellulose-binding domain (cbd), five type i cohesin domains, and six hydrophilic domains. the uniqueness of cipa lies in the enchainment of cohesin domains that are all separated by a hydrophilic domain. affinity-purified cipa was used in equilibrium-binding experiments to characterize the interaction of cipa with cry ...200312533485
the effect of carbon sources and lactate dehydrogenase deletion on 1,2-propanediol production in escherichia coli.in previous studies, we showed that cofactor manipulations can potentially be used as a tool in metabolic engineering. in this study, sugars similar to glucose, that can feed into glycolysis and pyruvate production, but with different oxidation states, were used as substrates. this provided a simple way of testing the effect of manipulating the nadh/nad+ ratio or the availability of nadh on the metabolic patterns of escherichia coli under anaerobic conditions and on the production of 1,2-propane ...200312545384
the genome sequence of clostridium tetani, the causative agent of tetanus disease.tetanus disease is one of the most dramatic and globally prevalent diseases of humans and vertebrate animals, and has been reported for over 24 centuries. the manifestation of the disease, spastic paralysis, is caused by the second most poisonous substance known, the tetanus toxin, with a human lethal dose of approximately 1 ng/kg. fortunately, this disease is successfully controlled through immunization with tetanus toxoid; nevertheless, according to the world health organization, an estimated ...200312552129
production by clostridium acetobutylicum atcc 824 of celg, a cellulosomal glycoside hydrolase belonging to family 9.the genome sequence of clostridium acetobutylicum atcc 824, a noncellulolytic solvent-producing strain, predicts the production of various proteins with domains typical for cellulosomal subunits. most of the genes coding for these proteins are grouped in a cluster similar to that found in cellulolytic clostridial species, such as clostridium cellulovorans. cac0916, one of the open reading frames present in the putative cellulosome gene cluster, codes for celg, a putative endoglucanase belonging ...200312571006
molecular cloning of the gene for 2,6-beta-d-fructan 6-levanbiohydrolase from streptomyces exfoliatus f3-2.the gene encoding a 2,6-beta-d-fructan 6-levanbiohydrolase (lf2ase) (ec 3.2.1.64) that converts levan into levanbiose was cloned from the genomic dna of streptomyces exfoliatus f3-2. the gene encoded a signal peptide of 37 amino acids and a mature protein of 482 amino acids with a total length of 1560 bp and was successfully expressed in escherichia coli. the similarities of primary structure were observed with levanases from clostridium acetobutylicum, bacillus subtilis, b. stearothermophilus ( ...200312586402
design of antisense rna constructs for downregulation of the acetone formation pathway of clostridium acetobutylicum.we investigated the effect of antisense rna (asrna) structural properties on the downregulation efficacy of enzymes in the acetone-formation pathway (acetoacetate decarboxylase [aadc] and coenzyme a-transferase [coat]) of clostridium acetobutylicum strain atcc 824. first, we generated three strains, c. acetobutylicum atcc 824 (padc38as), 824(padc68as), and 824(padc100as), which contain plasmids that produce asrnas of various lengths against the aadc (adc) transcript. western analysis showed that ...200312618456
2,4,6-trinitrotoluene reduction by an fe-only hydrogenase in clostridium acetobutylicum.the role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (tnt) in clostridium acetobutylicum was evaluated. an fe-only hydrogenase was isolated and identified by using tnt reduction activity as the selection basis. the formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a k(m) of 152 micro m. comparisons between the wild type and a mutant strain lacking the region encoding an al ...200312620841
fedbatch operation using clostridium acetobutylicum suspension culture as biocatalyst for enhancing hydrogen production.we demonstrated the feasibility of fedbatch operation using clostridium acetobutylicum suspension culture as a biocatalyst for the continuous production of hydrogen. the optimum operating ph and temperature of the current cultivation system for hydrogen production were ph 6.0 and 37 degrees c, respectively. the volumetric loading of the bioreactor for hydrogen production can be as high as 650 mmol hydrogen/l culture with a yield at approximately 2.0 mol hydrogen/mol glucose. acetate and butyrate ...200312675576
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