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measurement and regulation of the culture reduction state in clostridium acetobutylicum.with a constant glucose feed concentration, the change in the continuous culture dillution rate resulted in an altered fermentation profile and the cellular nadh content. the cultures growing at high dillution rates demonstrated an oxidative metabolism low nadh and butanol concentrations. the low specific nadh flourescence (f/x) at high butanol production rates suggested that a rapid regeneration of nadh to nad is essential for a high solventogenic culture activity. the culture florescence and b ...199118600748
expression of cloned homologous fermentative genes in clostridium acetobutylicum atcc 824.we have previously cloned the acetone-formation pathway gene, encoding acetoacetate decarboxylase (adc), and butyrate-formation pathway gene, encoding phosphotransbutyrylase (ptb), of clostridium acetobutylicum atcc 824 in escherichia coli. here we report their subcloning in bacillus subtilis and transfer to strain atcc 824 via electrotransformation, where the corresponding enzyme activities were expressed at elevated levels, using pfnk1, a new b. subtilis/c. acetobutylicum shuttle vector. plasm ...19921368230
mrna analysis of the adc gene region of clostridium acetobutylicum during the shift to solventogenesis.by using primer extension analysis, we located the transcription start point of the acetoacetate decarboxylase (adc) gene of clostridium acetobutylicum 90 nucleotides upstream from the initiation codon with a as the first transcribed nucleotide. from this site the promoter structure tttact(18 bp)tataat was identified; it shows high homology to the consensus sequences of gram-positive bacteria and escherichia coli. northern blot experiments revealed a length of 850 bases for the transcript of the ...19921370288
molecular characterization of two clostridium acetobutylicum atcc 824 butanol dehydrogenase isozyme genes.a 4-kb segment of dna containing two previously cloned butanol dehydrogenase (bdh) isozyme genes (d. petersen, r. welch, f. rudolph, and g. bennett, j. bacteriol. 173:1831-1834, 1991) was sequenced. two complete open reading frames (orfs) were identified (bdha and bdhb), along with a third truncated orf (orf1). the translation products of bdha and bdhb corresponded to the n-terminal sequences of the purified bdh i and bdh ii proteins, respectively. the two isozymes had a high amino acid identity ...19921385386
vector construction, transformation, and gene amplification in clostridium acetobutylicum atcc 824.in order to alter the primary metabolism of c. acetobutylicum, we have constructed e. coli- or b. subtilis-c. acetobutylicum shuttle vectors that could be used to deliver homologous fermentative genes into c. acetobutylicum atcc 824. the plasmid copy number and plasmid stability in c. acetobutylicum for several of these plasmids were determined. we have also developed a protocol for the electrotransformation of c. acetobutylicum atcc 824. difficulty in the transformation of c. acetobutylicum atc ...19921416617
stable inheritance of shuttle vectors based on plasmid pim13 in a mutant strain of clostridium acetobutylicum.new shuttle vectors for clostridium acetobutylicum were constructed, using as replicons the gram-positive plasmid pim13, and derivatives of the gram-negative plasmid pbr322, including puc19. these vectors transformed c. acetobutylicum at a high frequency (up to 10(6) transformants per microgram dna) by peg-mediated protoplast transformation. a mutant host strain, ni-4082, was isolated on the basis of its ability to maintain plasmid pim13 stably in the absence of selection pressure. the shuttle v ...19921512567
replacement of the aliphatic chains of clostridium acetobutylicum by exogenous fatty acids: regulation of phospholipid and glycolipid composition.the membrane lipid aliphatic chains of clostridium acetobutylicum atcc 4259 have been extensively modified by growth in biotin-free medium containing vitamin-free casein hydrolysate supplemented with either elaidic acid, oleic acid, or mixtures of palmitic and oleic acids. growth with elaidic acid resulted in polar lipids containing 88.6% 18:1 acyl chains and 94.5% 18:1 ether-linked chains. growth with oleic acid resulted in comparable levels of enrichment of the lipids with 18:1 chains and c19 ...19921548233
molecular characterization of the dnak gene region of clostridium acetobutylicum, including grpe, dnaj, and a new heat shock gene.the dnak gene region of clostridium acetobutylicum was cloned in escherichia coli by using the pbluescript sk+ and puc18 vectors. by using the e. coli dnak gene as a probe and by in vivo chromosome walking, three positive clones harboring the recombinant plasmids pkg1, pkg2, and pkg3 containing 1.2-kbp hindiii, 3.55-kbp ecorv, and 1.2-kbp psti fragments of the chromosome of c. acetobutylicum, respectively, were isolated. the cloned fragments partially overlapped, and together they spanned 4,083 ...19921577695
purification and characterization of an extracellular muramidase of clostridium acetobutylicum atcc 824 that acts on non-n-acetylated peptidoglycan.an extracellular enzyme showing lytic activity on non-n-acetylated peptidoglycan has been isolated from clostridium acetobutylicum atcc 824. the lytic enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with a recovery of 24%. the enzyme was monomeric and had an estimated molecular weight of 41,000 and an isoelectric point of 3.8. it has been characterized as a muramidase whose 23-amino-acid n terminus displayed 39% homology with the n,o-diacetyl muramidase of ...19921599233
a general method for cloning reca genes of gram-positive bacteria by polymerase chain reaction.an internal fragment of the reca gene from eight gram-positive organisms has been amplified by using degenerate primers in a polymerase chain reaction. the internal 348- or 360-bp reca dna segments from bacillus subtilis, clostridium acetobutylicum, lactobacillus bulgaricus, lactobacillus helveticus, leuconostoc mesanteroides, listeria monocytogenes, staphylococcus aureus, and streptococcus salivarus subsp. thermophilus were amplified, cloned, and sequenced. the g + c contents of the dna from th ...19921629178
autolysis of clostridium acetobutylicum atcc 824.the optimum conditions for autolysis of clostridium acetobutylicum atcc 824 were determined. autolysis was optimal at ph 6.3 and 55 degrees c in 0.1 m-sodium acetate/phosphate buffer. the ability of cells to autolyse decreased sharply at the end of the exponential phase of growth. lysis was stimulated by monovalent cations and compounds that complex divalent cations, and inhibited by divalent cations. the autolysin of c. acetobutylicum, which was mainly cytoplasmic, was purified to homogeneity a ...19921645127
possible function of trna(thr)acg in regulation of solvent formation in clostridium acetobutylicum.the mutation of clostridium acetobutylicum mutant aa2, defective in the formation of acetone and butanol, was shown to be caused by a single insertion of tn916 close to the structural gene thra, encoding the trna(thr)acg. the dna region containing the thra gene was cloned and sequenced. start and end points of the transcript were determined by primer extension and s1-mapping analysis. the results obtained were identical to predictions derived from the dna sequence by various rna-analysing comput ...19921335943
cloning and characterization of a gene from bacillus stearothermophilus var. non-diastaticus encoding a glycerol dehydrogenase.a 4.1-kb ecori fragment which includes the gene (glda) encoding a glycerol dehydrogenase (g1dh; ec 1.1.1.6; glycerol:nad oxidoreductase) from bacillus stearothermophilus var. non-diastaticus has been cloned by virtue of its ability to restore glycerol utilisation to escherichia coli glycerol kinase (glpk) and glycerol-3-phosphate dehydrogenase (glpd) mutants. sequencing suggests that the glda gene is likely to be monocistronic and encodes a protein of 39450 da. the deduced amino acid composition ...19921339360
cloning, sequencing, and molecular analysis of the groesl operon of clostridium acetobutylicum.the groesl operon of clostridium acetobutylicum was cloned in escherichia coli by using a gene probe of e. coli groesl. sequencing of a positively reacting 2.2-kbp hindiii fragment contained in the recombinant plasmid pfn1 and a 2.5-kbp xbai fragment present in pfn4 revealed that both fragments partially overlapped and together spanned 3,493 bp of the clostridial chromosome. two complete open reading frames (288 and 1632 bp) were found and identified as the groes- and groel-homologous genes of c ...19921349602
reconstruction and expression of the autolytic gene from clostridium acetobutylicum atcc 824 in escherichia coli.the complete lyc gene encoding the autolytic lysozyme of clostridium acetobutylicum atcc 824 was reconstructed from two overlapping dna fragments and cloned into a suitable plasmid enabling escherichia coli to produce this lytic enzyme under the control of the lac promoter. a polypeptide with an apparent m(r) of 35,000, corresponding to that predicted from the nucleotide sequence, was observed by maxicell analysis of whole-cell extracts of e. coli harboring the clostridial gene. the enzyme yield ...19921355455
organization and nucleotide sequence of the glutamine synthetase (glna) gene from lactobacillus delbrueckii subsp. bulgaricus.a 3.3-kb bamhi fragment of lactobacillus delbrueckii subsp. bulgaricus dna was cloned and sequenced. it complements an escherichia coli glna deletion strain and hybridizes strongly to a dna containing the bacillus subtilis glna gene. dna sequence analysis of the l. delbrueckii subsp. bulgaricus dna showed it to contain the glna gene encoding class i glutamine synthetase, as judged by extensive homology with other prokaryotic glna genes. the sequence suggests that the enzyme encoded in this gene ...19921359838
differential expression of a clostridium acetobutylicum antisense rna: implications for regulation of glutamine synthetase.the clostridium acetobutylicum glutamine synthetase (gs) dna region is characterized by a downstream promoter, p3, oriented toward the glna gene, which controls the transcription of an rna complementary to the start of the glna mrna. expression of the predicted 43-base antisense rna was demonstrated in c. acetobutylicum and escherichia coli cells containing the cloned glna dna. antisense rna transcription from p3 was not regulated by nitrogen in e. coli cells, but the expression of antisense rna ...19921360004
physiological events in clostridium acetobutylicum during the shift from acidogenesis to solventogenesis in continuous culture and presentation of a model for shift induction.the ph of continuous cultures of clostridium acetobutylicum growing at ph 5.6 was allowed to decrease to 4.3 after acid production and thereby to shift the cultures from acetate and butyrate to acetone and butanol formation. several parameters were determined during the shift. an increase in the intracellular acid concentration to 440 mm was recorded. an excess of undissociated butyric acid but not of acetic acid just before the shift to solventogenesis was followed by a decline in acid producti ...199216348821
isolation of a degeneration-resistant mutant of clostridium acetobutylicum ncimb 8052.unless periodically grown from germinated spores, clostridium acetobutylicum tends to degenerate (that is, to spontaneously lose the capacity both to produce solvents and to develop into spores). to obtain mutants that are deficient in degeneration, c. acetobutylicum ncimb 8052 was mated with enterococcus faecalis bm4110 harboring transposon tn1545. we developed a degeneration resistance assay based on a secondary effect of degeneration, the production of toxic levels of acetic and butyric acids ...199316349119
role of the c-terminal domain of the lysozyme of clostridium acetobutylicum atcc 824 in a chimeric pneumococcal-clostridial cell wall lytic enzyme.an active chimeric cell wall lytic enzyme has been constructed by domain substitution between the major autolysins of clostridium acetobutylicum atcc 824 and streptococcus pneumoniae. the chimeric enzyme, built up by the fusion of the n-terminal domain of the pneumococcal lyta amidase and the c-terminal domain of the clostridial lyc lysozyme, exhibited an amidase activity capable of hydrolyzing choline-containing clostridial cell walls with an efficiency 250-times higher than when tested on pneu ...19937903254
cloning and sequence analysis of the dnak gene region of lactococcus lactis subsp. lactis.a 5.4 kb hindiii fragment of lactococcus lactis subsp. lactis was identified using a homologous dnak probe generated by pcr and cloned in escherichia coli. upstream sequences were generated by inverse pcr. the two cloned fragments partially overlapped, and sequencing of 5915 bp revealed the presence of four open reading frames in the order orf1-grpe-dnak-orf4. orf1 encodes a 39 kda protein of unknown function which shows considerable sequence homology with the orf39 and orfa proteins of bacillus ...19938126443
cloning, sequencing, and molecular analysis of the sol operon of clostridium acetobutylicum, a chromosomal locus involved in solventogenesis.a dna region of clostridium acetobutylicum contiguous with the adc operon has been cloned and sequenced. structural genes encoding the acetoacetyl coenzyme a:acetate/butyrate:coenzyme a transferase (ctfb and ctfa) and an alcohol/aldehyde dehydrogenase (adhe) could be identified. these three genes together with a small open reading frame (orf) of unknown function (upstream of adhe) formed an operon (sol operon), as shown by mrna analyses. the complete sol operon was transcriptionally induced or d ...19938226639
sequence and arrangement of two genes of the butyrate-synthesis pathway of clostridium acetobutylicum atcc 824.the genes encoding both clostridium acetobutylicum atcc 824 butyrate synthesis pathway enzymes, phosphotransbutyrylase (ptb) and butyrate kinase (buk), were sequenced. the genes are immediately adjacent on the chromosome, with ptb preceding buk. a single transcription start point (tsp) was identified 57 bp upstream from the ptb start codon by primer extension analysis. the ptb and buk genes appear to form an operon. a putative rho-independent terminator structure was identified 26 bp downstream ...19938244020
the xync gene from fibrobacter succinogenes s85 codes for a xylanase with two similar catalytic domains.the xync gene of fibrobacter succinogenes s85 codes for a 66.4-kda xylanase which consists of three distinct domains separated by two flexible regions rich in serine residues. domains a and b of xync code for catalytic domains with 56.5% identity and 9.6% similarity with each other, and both domains share homology with xylanases of ruminococcus flavefaciens, neocallimastix patriciarum, clostridium acetobutylicum, bacillus pumilus, bacillus subtilis, and bacillus circulans. more than 88% of the x ...19938244936
transposon mutagenesis of clostridium acetobutylicum p262: isolation and characterization of solvent deficient and metronidazole resistant mutants.an efficient transposon mutagenesis system using conjugative transposons tn916 and tn925::tn917 was established for clostridium acetobutylicum p262, an industrial strain which has proved difficult to manipulate genetically. transposon insertions occurred at several different locations to produce a variety of mutants. an oligosporogenous mutant deficient in acetone and butanol production, and two sporulation-deficient and metronidazole resistant mutants were characterized with respect to differen ...19938288111
purification and some properties of an alkaline xylanase from alkaliphilic bacillus sp. strain 41m-1.an alkaliphilic bacillus sp. strain, 41m-1, isolated from soil produced multiple xylanases extracellularly. one of these xylanases was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. the moleculr mass of this enzyme (xylanase j) was 36 kda, and the isoelectric point was ph 5.3. xylanase j was most active at ph 9.0. the optimum temperature for the activity at ph 9.0 was around 50 degrees c. the enzyme was stable up to 55 degrees c at ph 9.0 for 30 min. ...19938292206
a reporter gene vector to investigate the regulation of glutamine synthetase in bacteroides fragilis bf1.the clostridium acetobutylicum egla gene, encoding a beta-1,4-endoglucanase (eg), was shown to be a useful reporter gene for the study of gene expression in bacteroides fragilis. the egla reporter gene has the advantages that it can be easily identified in both escherichia coli and b. fragilis on agar media containing carboxymethylcellulose, and eg production can be rapidly quantified in liquid medium. since the b. fragilis glutamine synthetase (gs) is inactivated in permeabilized cells and cell ...19937680708
cloning, structure, and expression of acid and solvent pathway genes of clostridium acetobutylicum. 19937691287
nucleotide sequence of the clostridium stercorarium xyna gene encoding xylanase a: identification of catalytic and cellulose binding domains.the nucleotides of the xyna gene of clostridium stercorarium were sequenced. the structural gene consists of an open reading frame of 1533 bp encoding 511 amino acids with an m(r) of 56,519. the signal peptide cleavage site was identified by comparison with the n-terminal amino acid sequence of the enzyme produced by a recombinant escherichia coli. xylanase a consists of a catalytic domain belonging to family g at the n-terminus and two direct repeats of about 90 amino acids with a short spacing ...19937763496
cloning, nucleotide sequence and structural analysis of the clostridium acetobutylicum dnaj gene.the complete dnaj gene of clostridium acetobutylicum was isolated by chromosome walking using the previously cloned 5' end of the gene as a probe. nucleotide sequencing of a positively reacting 2.2-kb hincii fragment, contained in the recombinant plasmid pkg4, revealed that the reading frame of the dnaj gene of c. acetobutylicum consists of 1125 bp, encoding a protein of 374 amino acids with a calculated m(r) of 40376 and an isoelectric point of 9.54. the deduced amino acid sequence showed high ...19937507453
interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal-clostridial cell wall lytic enzyme.bacterial autolysins are endogenous enzymes that specifically cleave covalent bonds in the cell wall. these enzymes show both substrate and bond specificities. the former is related to their interaction with the insoluble substrate whereas the latter determine their site of action. the bond specificity allows their classification as muramidases (lysozymes), glucosaminidases, amidases, and endopeptidases. to demonstrate that the autolysin (lyc muramidase) of clostridium acetobutylicum atcc824 pre ...19937934908
in vivo methylation in escherichia coli by the bacillus subtilis phage phi 3t i methyltransferase to protect plasmids from restriction upon transformation of clostridium acetobutylicum atcc 824.the restriction endonuclease cac824i has been shown to be a major barrier to electrotransformation of clostridium acetobutylicum atcc 824 (l. d. mermelstein, n. e. welker, g. n. bennett, and e. t. papoutsakis, bio/technology 10:190-195, 1992). methylation by the phi 3t i methyltransferase encoded by bacillus subtilis phage phi 3t was shown to protect plasmid dna from restriction by cac824i. expression in escherichia coli of the phi 3ti gene (which encodes the phi 3t i methyltransferase) from pan ...19938386500
cloning and sequence analysis of the genes encoding phosphotransbutyrylase and butyrate kinase from clostridium acetobutylicum ncimb 8052.an 8.1-kb fragment of chromosomal dna from clostridium acetobutylicum ncimb 8052 (formerly ncib 8052) has been cloned into plasmid pat153 and shown to allow the growth of escherichia coli lj32 (f+ atoc2c atod32 fadr) on butyrate as the sole source of carbon and energy. deletion analysis delineated a 3.9-kb subfragment capable of complementation. the nucleotide sequence of this fragment was determined and it was shown to encode three complete, and two incomplete open reading frames (orfs). based ...19938396545
sequence and arrangement of genes encoding enzymes of the acetone-production pathway of clostridium acetobutylicum atcc824.the nucleotide sequence of three open reading frames in the acetone-production locus of clostridium acetobutylicum atcc824 has been established. the three gene products, corresponding to acetoacetate decarboxylase (ec 4.1.1.4) and both subunits of the acetoacetyl-coa:acetate/butyrate:coa transferase (ec 2.8.3.9) are transcribed in two convergently arranged operons. the intervening dna region separating the two transcripts is characterized by an inverted repeat which appears capable of forming a ...19938423010
cloning, nucleotide sequence, and regulatory analysis of the lactococcus lactis dnaj gene.the dnaj gene of lactococcus lactis was isolated from a genomic library of l. lactis nizo r5 and cloned into puc19. nucleotide sequencing revealed an open reading frame of 1,137 bp in length, encoding a protein of 379 amino acids. the deduced amino acid sequence showed homology to the dnaj proteins of escherichia coli, mycobacterium tuberculosis, bacillus subtilis, and clostridium acetobutylicum. the level of the dnaj monocistronic mrna increased approximately threefold after heat shock. the tra ...19938449872
sequence and molecular characterization of a dna region encoding a small heat shock protein of clostridium acetobutylicum.a dna region of clostridium acetobutylicum containing a gene (hsp 18) with significant homology to a family of small eukaryotic heat shock proteins was cloned and sequenced. it is the second reported sequence of a low-molecular-weight heat shock protein from gram-positive bacteria and is induced not only by heat shock but also at the onset of solventogenesis, as determined by northern (rna) blot analysis, thus confirming the results of an earlier study performed at the protein level (a. pich, f. ...19938501044
construction and characterization of a phage-plasmid hybrid (phagemid), pcak1, containing the replicative form of viruslike particle cak1 isolated from clostridium acetobutylicum ncib 6444.a bacteriophage-plasmid hybrid (phagemid) designated pcak1 was constructed by ligating 5-kbp escherichia coli plasmid pak102 (apremr) and the 6.6-kbp haeiii-linearized replicative form of the cak1 viruslike particle from clostridium acetobutylicum ncib 6444. phagemid pcak1 (11.6 kbp) replicated via the cole1 replication origin derived from pak102 in e. coli. single-stranded dna (ssdna) molecules complexed with protein in a manner which protected ssdna from nucleases were recovered from the super ...19938509336
determination of plasmid copy number and stability in clostridium acetobutylicum atcc 824.the copy number and stability of several plasmid vectors in clostridium acetobutylicum atcc 824 were determined. the protocols were modified from the traditional ones to overcome the problems associated with unusual behavior of c. acetobutylicum cells on solid medium. the plasmid copy numbers of psyl2, pfnk1, pfnk3, and pfnk5 in strain atcc 824 were 14, 8, 6, and 6, respectively. psyl2 and pfnk1 were segregationally stable, since the fractions of plasmid-carrying cells after 60 generations of gr ...19938514119
purification and characterization of the extracellular alpha-amylase from streptococcus bovis jb1.the extracellular alpha-amylase (1,4-alpha-d-glucanglucanohydrolase; ec 3.2.1.1) from maltose-grown streptococcus bovis jb1 was purified to apparent homogeneity by ion-exchange chromatography (mono q). the enzyme had an isoelectric point of 4.50 and an apparent molecular mass of 77,000 da, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. the enzyme was rich in acidic and hydrophobic amino acids. the 15-amino-acid nh2-terminal sequence was 40% homologous with the bacillu ...19938517735
metabolic engineering of clostridium acetobutylicum atcc 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon.the ability to genetically alter the product-formation capabilities of clostridium acetobutylicum is necessary for continued progress toward industrial production of the solvents butanol and acetone by fermentation. batch fermentations at ph 4.5, 5.5, or 6.5 were conducted using c. acetobutylicum atcc 824 (pfnk6). plasmid pfnk6 contains a synthetic operon (the "ace operon") in which the three homologous acetone-formation genas (adc, ctfa, and ctfb) are transcribed from the adc promoter. the corr ...199318613233
pervaporative butanol fermentation by clostridium acetobutylicum b18.extractive acetone-butanol-ethanol (abe) fermentation was carried out successfully using pervaporation and a low-acid-producing clostridium acetobutylicum b18. a pervaporation module with 0.17 m(2) of surface area was made of silicone membrane of 240 mum thickness. pervaporation experiments using make-up solutions showed that butanol and acetone fluxes increased linearly with their concentrations in the aqueous phase. fickian diffusion coefficients were constants for fixed air flow rates, and in ...199418615445
regulation of clostridium acetobutylicum metabolism as revealed by mixed-substrate steady-state continuous cultures: role of nadh/nad ratio and atp pool.glycerol-glucose-fed (molar ratio of 2) chemostat cultures of clostridium acetobutylicum were glucose limited but glycerol sufficient and had a high intracellular nadh/nad ratio (i. vasconcelos, l. girbal, and p. soucaille, j. bacteriol. 176:1443-1450, 1994). we report here that the glyceraldehyde-3-phosphate dehydrogenase, one of the key enzymes of the glycolytic pathway, is inhibited by high nadh/nad ratios. partial substitution of glucose by pyruvate while maintaining glycerol concentration a ...19947961393
sporulation and primary sigma factor homologous genes in clostridium acetobutylicum.using a pcr-based approach, we have cloned various sigma factor homologous genes from clostridium acetobutylicum dsm 792. the nucleotide sequence of the dnae-siga operon has been determined and predicts two genes encoding 69- and 43-kda proteins. the deduced dnae amino acid sequence has approximately 30% amino acid identity with protein sequences of other primases. the putative siga gene product shows high homology to primary sigma factors of various bacteria, most significantly to bacillus subt ...19947961408
purification and molecular characterization of the nad(+)-dependent acetaldehyde/alcohol dehydrogenase from entamoeba histolytica.a bifunctional 95 kda polypeptide (ehadh2) harbouring acetaldehyde dehydrogenase and alcohol dehydrogenase activities was purified to homogeneity from trophozoite extracts of the protozoan parasite entamoeba histolytica. kinetic studies revealed that the enzyme utilizes nad+ rather than nadp+ as cofactor. km values for acetyl-coa, acetaldehyde and ethanol were found to be 0.015, 0.15 and 80 mm respectively in the presence of 0.2 mm nad+. the primary structure of ehadh2 as deduced from respective ...19947980441
genes encoding homologues of three consecutive enzymes in the butyrate/butanol-producing pathway of clostridium acetobutylicum are clustered on the clostridium difficile chromosome.screening of a clostridium difficile lambda embl3 gene library with antisera raised against c. difficile culture supernatant identified several clones expressing a 31-kda protein. a 1.8-kb hindiii fragment subcloned from one of the clones was sufficient for expression of the 31-kda polypeptide. southern blot analysis showed a region homologous to this fragment to be present in all of 13 different c. difficile strains tested. sequence analysis of the 1.8-kb fragment revealed three adjacent open r ...19948001771
genetic and metabolic engineering of clostridium acetobutylicum atcc 824. 19948010697
studies of recombinant clostridium acetobutylicum with increased dosages of butyrate formation genes. 19948010698
molecular cloning of two new heat shock genes related to the hsp70 genes in staphylococcus aureus.we have identified two new heat shock protein genes, orf37 and orf35, in staphylococcus aureus, located upstream and downstream of grpe(hsp20), dnak(hsp70), and dnaj(hsp40) homologous genes in the order orf37-hsp20-hsp70-hsp40-orf35. the transcripts of both orf37 and orf35 were increased by thermal upshift of the culture from 37 to 46 degrees c. the heat shock promoters were located upstream of orf37 and upstream of hsp40. the deduced peptide of orf37 showed similarity with those of orfa in clos ...19948045913
expression of plasmid-encoded aad in clostridium acetobutylicum m5 restores vigorous butanol production.mutant m5 of clostridium acetobutylicum atcc 824, which produces neither butanol nor acetone and is deficient in butyraldehyde dehydrogenase (bydh), acetoacetate decarboxylase, and acetoacetyl-coenzyme a:acetate/butyrate:coenzyme a-transferase activities, was transformed with plasmid pcaad, which carries the gene aad (r. v. nair, g. n. bennett, and e. t. papoutsakis, j. bacteriol, 176:871-885, 1994). in batch fermentation studies, aad expression restored butanol formation (84 mm) in mutant m5 wi ...19948083176
regulation of carbon and electron flow in clostridium acetobutylicum grown in chemostat culture at neutral ph on mixtures of glucose and glycerol.the metabolism of clostridium acetobutylicum was manipulated, at neutral ph and in 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, and the intracellular enzymatic pattern indicated the absence of butyraldehyde dehydrogenase activity and very low levels of coenzyme a-transferase, butanol, and ethanol dehydrogenase activities. in contrast, cultures grown on mixtures of gluc ...19948113186
heterologous expression of endo-beta-1,4-d-glucanase from clostridium cellulovorans in clostridium acetobutylicum atcc 824 following transformation of the engb gene.heterologous expression of the clostridium cellulovorans engb gene by clostridium acetobutylicum bkw-1 was detected as zones of hydrolysis on carboxymethyl cellulose (cmc) trypticase glucose yeast plates stained with congo red. the extracellular cellulase preparation from c. acetobutylicum bkw-1 has a specific activity towards cmc which is more than fourfold that present in c. acetobutylicum atcc 824. western blot (immunoblot) analysis using the c. cellulovorans anti-engb primary antibody demons ...19948117087
characterization of spo0a homologues in diverse bacillus and clostridium species identifies a probable dna-binding domain.spo0a is a phosphorylation-activated transcription factor of bacillus subtilis. it is a member of the response regulator superfamily of bacterial signal transduction proteins and controls many of the changes in gene expression that occur during the transition into stationary phase and during the initiation of sporulation. to identify the domains of spo0a most critical for determining its structural and functional features, presumptive homologues of the spo0a gene were characterized in a collecti ...19947885226
cyclic amp in ruminal and other anaerobic bacteria.an examination of camp levels in predominant species of ruminal bacteria and other anaerobic bacteria was conducted. cellular camp concentrations of glucose-grown cultures of butyrivibrio fibrisolvens 49, prevotella ruminicola d31d, selenomonas ruminantium hd4 and d, megasphaera elsdenii b159, streptococcus bovis jb1, bacteroides thetaiotaomicron 5482, and clostridium acetobutylicum atcc 824 were determined at various times during growth by a competitive binding radioimmunoassay procedure. the r ...19947851742
identification of a grpe heat-shock gene homolog in the archaeon methanosarcina mazei.a grpe heat-shock gene was found by sequencing in the genome of the methanogenic archaeon methanosarcina mazei s-6. it is the first example of grpe from the phylogenetic domain archaea. since the other seven sequenced homologs are from the domain bacteria, it may be concluded that grpe appeared early in evolution, before the two domains separated. the archaeal grpe is located in the dnak locus, 431 base-pairs upstream of dnak, which is followed downstream by the dnaj gene. the organization of th ...19947517454
analysis of tn916-induced mutants of clostridium acetobutylicum altered in solventogenesis and sporulation.the conjugative transposon tn916 was used for mutagenesis of clostridium acetobutylicum atcc 824. tetracycline-resistant mutants were screened for loss of granulose synthesis and five classes of granulose mutants, that contained single transposon insertions, were identified on the basis of altered solvent production. class 1 mutants did not make acetone or butanol, lacked activity of enzymes induced during solventogenesis, and did not sporulate, indicating that they are regulatory mutants. the c ...19947765050
cloning and sequencing of a chromosomal fragment from clostridium acetobutylicum strain abkn8 conferring chemical-damaging agents and uv resistance to e. coli reca strains.a 3.3-kb dna fragment of clostridium acetobutylicum conferred methyl methane sulfonate (mms), mitomycin c (mc), and uv resistance to reca strains of e. coli when cloned on the puc19 plasmid. analysis of the nucleotide sequence of the total insert and results of in vitro transcription-translation experiments showed that the insert directed the synthesis of three polypeptides referred to as orfa, orfb, and orfc of 23.6, 15.3, and 21 kda, respectively. none of the polypeptides presented a relations ...19947765497
molecular cloning and nucleotide sequence of the beta-galactosidase gene from enterobacter cloacae gao.the gene encoding a beta-galactosidase from enterobacter cloacae gao was cloned and expressed in escherichia coli. the nucleotide sequence of the insert of a positive clone had an open reading frame of 3084 bp that encoded a polypeptide of 1028 amino acid residues with a calculated molecular mass of 116,677 daltons. the amino acid sequence of beta-galactosidase deduced from the nucleotide sequence, especially the sequence around the putative active site and of the fourteen regions, showed signif ...19947765512
molecular characterization of an aldehyde/alcohol dehydrogenase gene from clostridium acetobutylicum atcc 824.a gene (aad) coding for an aldehyde/alcohol dehydrogenase (aad) was identified immediately upstream of the previously cloned ctfa (j. w. cary, d. j. petersen, e. t. papoutsakis, and g. n. bennett, appl. environ. microbiol. 56:1576-1583, 1990) of clostridium acetobutylicum atcc 824 and sequenced. the 2,619-bp aad codes for a 96,517-da protein. primer extension analysis identified two transcriptional start sites 83 and 243 bp upstream of the aad start codon. the n-terminal section of aad shows hom ...19948300540
salmonella typhimurium loci involved in survival within macrophages.a set of tn10 mutants of salmonella typhimurium which have a diminished capacity to survive in murine macrophages and decreased virulence in mice has been described previously. in this study, we characterized 30 of these mutants and determined map locations of tn10 insertions for 23 of these strains. in addition, short fragments of transposon-flanking dna were cloned, and the nucleotide sequence was determined for 23 mutants. seven mutants carried transposon insertions in known genes, representi ...19948168923
molecular characterization of microbial alcohol dehydrogenases.there is an astonishing array of microbial alcohol oxidoreductases. they display a wide variety of substrate specificities and they fulfill several vital but quite different physiological functions. some of these enzymes are involved in the production of alcoholic beverages and of industrial solvents, others are important in the production of vinegar, and still others participate in the degradation of naturally occurring and xenobiotic aromatic compounds as well as in the growth of bacteria and ...19948185833
binding site-shaped repeated sequences of bacterial wall peptidoglycan hydrolases.the non-catalytic c-terminal regions of the n-acetylmuramidase (lysozyme) of clostridium acetobutylicum and n-acetylmuramoyl(d-lactyl)-l-alanine amidases cwla of bacillus subtilis, orfl3 and cwll of bacillus licheniformis were previously reported to have similarities with the amino acid sequence of the non-catalytic n-terminal module of the streptomyces albus g zn dd-peptidase. this peptidase is a bipartite protein of known three-dimensional structure. its non-catalytic n-terminal module possess ...19947908269
transmembrane ph of clostridium acetobutylicum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased.evidence is reported here that alkalinization of clostridium acetobutylicum cytoplasm involves hydrogenase activity. a decrease of in vivo hydrogenase activity is accompanied by intracellular accumulation of protons leading to a negative (interior acidic) ph gradient. however, the organism is able to maintain a constant proton motive force by interconverting chemical and electrical potentials.19947928980
intracellular concentrations of coenzyme a and its derivatives from clostridium acetobutylicum atcc 824 and their roles in enzyme regulation.intracellular levels of coenzyme a (coa) and its derivatives involved in the metabolic pathways for clostridium acetobutylicum atcc 824 were analyzed by using reverse-phase high-performance liquid chromatography (hplc). during the shift from the acidogenic to the solventogenic or stationary growth phase, the concentration of butyryl-coa increased rapidly and the concentrations of free coa and acetyl-coa decreased. these changes were accompanied by a rapid increase of the solvent pathway enzyme a ...199416349164
physical map of the clostridium beijerinckii (formerly clostridium acetobutylicum) ncimb 8052 chromosome.a combined physical and genetic map of the single, circular, 6.7-mbp chromosome of the ncimb 8052 strain of clostridium beijerinckii (formerly clostridium acetobutylicum) has been constructed by using a combination of cloned dna fragments as hybridization probes and a bank of strains harboring insertions of the conjugative transposon tn1545. the positions of 81 restriction endonuclease cleavage sites and 32 genes have been determined. eight genes concerned with solventogenic fermentation are fou ...19957814334
a clostridium acetobutylicum regulator gene (rega) affecting amylase production in bacillus subtilis.plasmid pmet7c containing a 6.05 kb dna insert from clostridium acetobutylicum p262 made escherichia coli f19 cells sensitive to metronidazole. the nucleotide sequence of the c. acetobutylicum dna controlling metronidazole sensitivity in e. coli f19 revealed an orf of 972 bp which encoded a protein of 324 amino acids with a calculated mr of 35,000. the amino acid sequence encoded by the orf contained a helix-turn-helix dna-binding domain and was homologous to the catabolite control protein, ccpa ...19957539689
taxonomy and phylogeny of industrial solvent-producing clostridia.we performed a systematic study of 55 solvent-producing clostridial strains, the majority of which are currently classified as clostridium acetobutylicum strains, by using a combination of biotyping and dna fingerprint analysis. the biotyping procedures used included rifampin susceptibility testing, bacteriocin typing, and bacteriophage typing. the 55 strains examined exhibited a good correlation between their biotypes and dna fingerprints, which allowed us to divide them into nine groups. the d ...19957547288
solventogenic enzymes of clostridium acetobutylicum: catalytic properties, genetic organization, and transcriptional regulation.the enzymes acetoacetate decarboxylase and coenzyme a transferase catalyse acetone production from acetoacetyl-coa in clostridium acetobutylicum. the adc gene encoding the former enzyme is organized in a monocistronic operon, while the ctf genes form a common transcription unit with the gene (adhe) encoding a probable polyfunctional aldehyde/alcohol dehydrogenase. this genetic arrangement could reflect physiological requirements at the onset of solventogenesis. in addition to adhe, two butanol d ...19957576767
alcohol dehydrogenase: multiplicity and relatedness in the solvent-producing clostridia.alcohol dehydrogenase (adh) is a key enzyme for the production of butanol, ethanol, and isopropanol by the solvent-producing clostridia. initial studies of adh in extracts of several strains of clostridium acetobutylicum and c. beijerinckii gave conflicting molecular properties. a more coherent picture has emerged because of the following results: (i) identification of adhs with different coenzyme specificities in these species; (ii) discovery of structurally conserved adhs (type 3) in three sol ...19957576768
molecular genetics and the initiation of solventogenesis in clostridium beijerinckii (formerly clostridium acetobutylicum) ncimb 8052.a physical map of the clostridium beijerinckii (formerly clostridium acetobutylicum) ncimb 8052 chromosome has been constructed, encompassing about 90 rare restriction sites. the 14 rrn operons together with about 40 genes have been assigned positions on the map. genetic analysis and gene transfer have been developed in this organism to enable in vivo analysis of the roles of cloned genes using marker replacement technology. experiments using the available genetic tools have shown that spo0a pla ...19957576769
regulation of nitrogen metabolism, starch utilisation and the beta-hbd-adh1 gene cluster in clostridium acetobutylicum.the successful genetic manipulation of clostridium acetobutylicum for the increased production of solvents will depend on an understanding of gene structure and regulation in the bacterium. the glutamine synthetase (glna) gene is regulated by antisense rna, transcribed from a downstream promoter, in the opposite direction to the glna gene. an open reading frame (orf) was detected downstream of the glna gene, which has sequence homology to response regulators with anti-termination activity and ma ...19957576770
expression of heat shock genes in clostridium acetobutylicum.characterization of the heat shock response in clostridium acetobutylicum has indicated that at least 15 proteins are induced by a temperature upshift from 30 to 42 degrees c. these so-called heat shock proteins include dnak and groel, two highly conserved molecular chaperones. several genes encoding heat shock proteins of c. acetobutylicum have been cloned and analysed. the dnak operon includes the genes orfa (a heat shock gene with an unknown function), grpe, dnak, and dnaj; and the groe opero ...19957576772
tracking the evolution of the bacterial choline-binding domain: molecular characterization of the clostridium acetobutylicum ncib 8052 cspa gene.the major secreted protein of clostridium acetobutylicum ncib 8052, a choline-containing strain, is cspa (clostridial secreted protein). it appears to be a 115,000-m(r) glycoprotein that specifically recognizes the choline residues of the cell wall. polyclonal antibodies raised against cspa detected the presence of the protein in the cell envelope and in the culture medium. the soluble cspa protein has been purified, and an oligonucleotide probe, prepared from the determined n-terminal sequence, ...19957860591
characterization of an acetyl-coa c-acetyltransferase (thiolase) gene from clostridium acetobutylicum atcc 824.thiolase (thl) is an important enzyme at the junction in the pathway leading to the production of either acids (acetate or butyrate) or solvents (acetone, butanol or ethanol) during the growth of clostridium acetobutylicum atcc 824. cloning and expression of the thl-encoding gene (thl) has been described [petersen and bennett, appl. environ. microbiol. 57 (1991) 2735-2741], as has the purification and properties of the enzyme [wiesenborn et al., appl. environ. microbiol. 54 (1988) 2717-2722]. he ...19957867955
structure and transcription of genes within the beta-hbd-adh1 region of clostridium acetobutylicum p262.the 1.2-kb dna fragment upstream of the linked beta-hbd (3-hydroxybutyryl-coa dehydrogenase) and adh1 (nadph-dependent alcohol dehydrogenase) genes from clostridium acetobutylicum p262 was sequenced. the upstream region contained an open reading frame (orfb) which was found to have 44% amino acid identity to the fixb gene products of rhizobium and azorhizobium. the beta-hbd and orfb genes were expressed during the acidogenic and solventogenic phases. the beta-hbd gene was transcribed on a single ...19957875566
sequence and arrangement of genes encoding sigma factors in clostridium acetobutylicum atcc 824.the nucleotide sequence of a 2.7-kb region of clostridium acetobutylicum atcc 824 dna containing three open reading frames was determined. they encoded homologs of three proteins of bacillus subtilis, and the gene arrangement in both organisms was identical. the first gene, orfa, was 801-bp long; the 31-kda (266 aa) product it encoded exhibited homology with the putative sigma e-processing enzyme. the second gene, sige, was 708-bp long encoding a 27-kda (235 aa) product; the third gene, sigg, wa ...19957883192
characterization and expression of the hydrogenase-encoding gene from clostridium acetobutylicum p262.the hydrogenase enzyme of clostridium acetobutylicum plays a pivotal role in controlling electron flow, and hence carbon flow, during the complex biphasic fermentation of carbohydrates to the neutral solvents acetone and butanol. we report here the cloning and molecular characterization of the hydrogenase-encoding gene (hyda) from c. acetobutylicum p262. this gene was isolated by colony hybridization, using the clostridium pasteurianum hydrogenase-1 gene as a probe. the dna sequence encoding the ...19957894709
enhanced acetone-butanol fermentation using repeated fed-batch operation coupled with cell recycle by membrane and simultaneous removal of inhibitory products by adsorption.a novel acetone-butanol production process was developed which integrates a repeated fed-batch fermentation with continuous product removal and cell recycle. the inhibitory product concentrations of the fermentation by clostridium acetobutylicum were reduced by the simultaneous extraction process using polyvinylpyridine (pvp) as an adsorbent. because of the reduced inhibition effect, a higher specific cell growth rate and thus a higher product formation rate was achieved. the cell recycle using ...199518623420
modulation of carbon and electron flow in clostridium acetobutylicum by iron limitation and methyl viologen addition.the metabolic flexibility of clostridium acetobutylicum during growth on glucose with methyl viologen addition (1 mm) and/or iron limitation was examined in batch cultures at ph 5.5. the physiological effects of iron limitation and methyl viologen addition are additive, suggesting that they have different and complementary sites of action.199516534918
modulation of metabolism of clostridium acetobutylicum grown in chemostat culture in a three-electrode potentiostatic system with methyl viologen as electron carrier.the metabolism of clostridium acetobutylicum was manipulated in chemostat culture at ph 5 and 6.5 in a three-electrode potentiostatic system with methyl viologen (mv) as the electron carrier. when a constant potential was applied at ph 5, the broth redox potential continuously decreased and, simultaneously, a high increase in the reduced mv concentration (mv(+.)) and the specific rate of butanol production was observed while butyric acid was taken up. a linear relationship was reported between t ...199618624366
molecular analysis of the anaerobic succinate degradation pathway in clostridium kluyveri.a region of genomic dna from clostridium kluyveri was cloned in escherichia coli by a screening strategy which was based on heterologous expression of the clostridial 4-hydroxybutyrate dehydrogenase gene. the gene region (6,575 bp) contained several open reading frames which encoded the coenzyme a (coa)- and nadp+-dependent succinate-semialdehyde dehydrogenase (sucd), the 4-hydroxybutyrate dehydrogenase (4hbd), and a succinyl-coa;coa transferase (cat1), as analyzed by heterologous expression in ...19968550525
mechanism of the reaction catalyzed by acetoacetate decarboxylase. importance of lysine 116 in determining the pka of active-site lysine 115.acetoacetate decarboxylase from clostridium acetobutylicum (aad) catalyzes the decarboxylation of acetoacetate via a schiff base intermediate [hamilton, g. a., & westheimer, f. h. (1959) j. am. chem. soc. 81, 6332; fridovich, i., & westheimer f. h. (1962) j. am. chem. soc. 84, 3208]. the pka of the active-site lysine (lys 115) is 6.0, 4.5 pka units less than the pka of lysine in solution [kokesh, f. c., & westheimer, f. h. (1971) j. am. chem. soc. 93, 7270; frey, p. a., kokesh, f. c., & westheim ...19968555196
molecular characterization and transcriptional analysis of the putative hydrogenase gene of clostridium acetobutylicum atcc 824.a 2.8-kbp dna region of clostridium acetobutylicum atcc 824 containing the putative hydrogenase gene (hyda) was cloned and sequenced. the 1,745-bp hyda encodes a 64,415-da protein and presents strong identity with the [fe] hydrogenase genes of desulfovibrio and clostridium species. the level of the putative hyda mrna was high in cells from an acidogenic or an alcohologenic phosphate-limited continuous culture, while it was comparatively very low in cells from a solventogenic phosphate-limited co ...19968626337
recombination-induced variants of clostridium acetobutylicum atcc 824 with increased solvent production.three sporulation-specific genes (orfa, sige, sigg) from clostridium acetobutylicum atcc 824 are arranged in a cluster, encoding the putative sigma e-processing enzyme, sigma e, and sigma sigma g respectively. when they were transformed into clostridium acetobutylicum while on a plasmid functional in this organism, transformants did not survive. three kinds of recombinations were then attempted with nonreplicative plasmids: duplication of orfa and sige, replacement of all of the three genes, and ...19968640107
cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme a (coa) dehydrogenase, crotonase, and butyryl-coa dehydrogenase from clostridium acetobutylicum atcc 824.the enzymes beta-hydroxybutyryl-coenzyme a (coa) dehydrogenase (bhbd), crotonase, and butyryl-coa dehydrogenase (bcd) from clostridium acetobutylicum are responsible for the formation of butyryl-coa from acetoacetyl-coa. these enzymes are essential to both acid formation and solvent formation by clostridia. clustered genes encoding bhbd, crotonase, bcd, and putative electron transfer flavoprotein alpha and beta subunits have been cloned and sequenced. the nucleotide sequence of the crt gene indi ...19968655474
inactivation of an aldehyde/alcohol dehydrogenase gene from clostridium acetobutylicum atcc 824.a nonreplicative plasmid containing an internal aad gene fragment has been integrated into the chromosome of clostridium acetobutylicum atcc 824. transformation was accomplished by electroporation with relatively high concentrations of methylated plasmid dna. southern hybridization experiments revealed that integration occurred by single crossover homologous recombination inactivating the aad gene. integrants were relatively stable after 25 generations. inactivation of the aad gene drastically r ...19968669898
cloning, sequencing, and expression of genes encoding phosphotransacetylase and acetate kinase from clostridium acetobutylicum atcc 824.the enzymes phosphotransacetylase (pta) and acetate kinase (ak) catalyze the conversion of acetyl coenzyme a to acetate in the fermentation of clostridium acetobutylicum. the acetate-producing step is an important element in the acidogenic fermentation stage and generates atp for clostridial cell growth. the genes pta and ack, encoding pta and ak, respectively, were cloned and sequenced. enzyme activity assays were performed on cell extracts from escherichia coli and c. acetobutylicum harboring ...19968702268
cloning and expression of a putative alcohol dehydrogenase gene of entamoeba histolytica and its application to immunological examination.to clone and express the genes encoding major antigens of entamoeba histolytica, we constructed a lambda gt11 cdna library for e. histolytica hm1:imss and screened it with pooled sera from patients with amoebiasis. a 1,223-bp cdna was cloned (clone 1223), and its nucleotide sequence was determined. the amino acid sequence predicted to be encoded by the open reading frame of clone 1223 consisted of 396 residues and showed 32.5 and 32.3% homology to the nadh-dependent butanol dehydrogenases i and ...19968705667
genetic manipulation of acid formation pathways by gene inactivation in clostridium acetobutylicum atcc 824.integrational plasmid technology has been used to disrupt metabolic pathways leading to acetate and butyrate formation in clostridium acetobutylicum atcc 824. non-replicative plasmid constructs, containing either clostridial phosphotransacetylase (pta) or butyrate kinase (buk) gene fragments, were integrated into homologous regions on the chromosome. integration was assumed to occur by a campbell-like mechanism, inactivating either pta or buk. inactivation of the pta gene reduced phosphotransace ...19968760920
proposed topology of the glucitol permeases of escherichia coli and clostridium acetobutylicum. 19968875915
transcriptional organization and regulation of the dnak and groe operons of chlamydia trachomatis.the transcriptional organization and regulation of the dnak and groe heat shock operons of chlamydia trachomatis were studied and found to resemble those of the cognate operons of bacillus subtilis and clostridium acetobutylicum. the gene order is conserved (hrca-grpe-dnak), but no dnaj homolog could be identified in this region. the dnak operon was transcribed as a low-abundance polycistronic mrna whose levels did not increase upon exposure to heat shock. in contrast, a more abundant 2.3-kb mrn ...19968955323
the effect of novobiocin on solvent production by clostridium acetobutylicum.cells of clostridium acetobutylicum treated with novoblocin, a dna gyrase inhibitor, produced higher butyrate levels and lower solvent levels with acetone being the most affected. seven enzyme activities involved in acid and solvent production were analyzed. among them, only coa transferase, required for acetone formation and acid uptake, experienced a significant decrease in activity. as in escherichia coli and bacillus subtilis, dna from c. acetobutylicum became less negatively supercoiled in ...19968987493
characterization of four outer membrane proteins that play a role in utilization of starch by bacteroides thetaiotaomicron.results of earlier work had suggested that utilization of polysaccharides by bacteroides spp. did not proceed via breakdown by extracellular polysaccharide-degrading enzymes. rather, it appeared that the polysaccharide was first bound to a putative outer membrane receptor complex and then translocated into the periplasm, where the degradative enzymes were located. in a recent article, we reported the cloning and sequencing of susc, a gene from bacteroides thetaiotaomicron that encoded a 115-kda ...19979006015
nad-independent lactate and butyryl-coa dehydrogenases of clostridium acetobutylicum p262.clostridium acetobutylicum p262 cells that were growing on lactate and acetate had an nad-independent lactate dehydrogenase(ildh) activity of 200 nmol mg protein-1 min-1. ammonium sulfate precipitation and deae cellulose caused a 35-fold purification. gel filtration indicated that the ildh had a molecular weight of approximately 55 kda, but two bands were always observed. phenyl sepharose could not separate the two proteins, and hydroxyapatite caused a complete loss of activity. the semi-purifie ...19979009069
sequence and transcriptional analysis of groes and groel genes from the thermophilic bacterium clostridium thermocellum.the groesl operon from clostridium thermocellum (ct) has been isolated and sequenced, revealing two orfs of 285 and 1626 nt, separated by 48 nt. the first orf encoded a 94-aa 10.6-kda groes homologue; the second encoded a 541-aa polypeptide of 57.6 kda, that exhibited 61% and 77% sequence identity with groel from escherichia coli (ec) and clostridium acetobutylicum (ca), respectively. a putative tsp, preceded by -10 and -35 consensus promoters, was identified upstream of groes. this was followed ...19979047357
cultures of "clostridium acetobutylicum" from various collections comprise clostridium acetobutylicum, clostridium beijerinckii, and two other distinct types based on dna-dna reassociation.the best-known acetone-butanol (solvent)-producing bacterium is the weizmann organism, clostridium acetobutylicum, which was used for starch-based industrial fermentation. in the past two decades, cultures of "c. acetobutylicum" from various culture collections have included organisms that were isolated for sugar (molasses)-based industrial solvent production. recent biochemical and genetic studies have revealed significant differences among some of these "c. acetobutylicum" strains. we used dna ...19979103631
evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite entamoeba histolytica.entamoeba histolytica is an amitochondriate protozoan parasite with numerous bacterium-like fermentation enzymes including the pyruvate:ferredoxin oxidoreductase (por), ferredoxin (fd), and alcohol dehydrogenase e (adhe). the goal of this study was to determine whether the genes encoding these cytosolic e. histolytica fermentation enzymes might derive from a bacterium by horizontal transfer, as has previously been suggested for e. histolytica genes encoding heat shock protein 60, nicotinamide nu ...19979171424
a small heat shock protein from leuconostoc oenos induced by multiple stresses and during stationary growth phase.in leuconostoc oenos, a malolactic bacterium, the synthesis of a stress protein called lo18 with an apparent molecular mass of 18 kda was greatly induced after heat (42 degrees c), acid (ph 3) or ethanolic (12% (v/v)) shocks. moreover, the lo18 protein synthesis was induced in stationary growth phase and was detected for a long time (30 h) during this growth phase. significant identity was found between the n-terminal parts of the lo18 protein and the hsp18 from clostridium acetobutylicum sugges ...19979172446
the pneumococcal cell wall degrading enzymes: a modular design to create new lysins?autolysins are enzymes that degrade different bonds in the peptidoglycan and, eventually, cause the lysis and death of the cell. streptococcus pneumoniae contains a powerful autolytic enzyme that has been characterized as an n-acetylmuramoyl-l-alanine amidase. we have cloned the lyta gene coding for this amidase and studied in depth the genetics and expression of this gene, which represented the first molecular analysis of a bacterial autolysin. two observations have been fundamental in revealin ...19979185148
the kdp system of clostridium acetobutylicum: cloning, sequencing, and transcriptional regulation in response to potassium concentration.the complete sequence of the kdp gene region of clostridium acetobutylicum has been determined. this part of the chromosome comprises two small open reading frames (orfz and orfy), putatively encoding hydrophobic peptides, and the genes kdpa, kdpb, kdpc, and kdpx, followed by an operon encoding a pair of sensor-effector regulatory proteins (kdpd and kdpe). except for orfz, orfy, and kdpx, all genes showed significant homology to the kdp genes of escherichia coli, encoding a high-affinity potassi ...19979226259
analysis of the bacillus subtilis genome: cloning and nucleotide sequence of a 62 kb region between 275 degrees (rrnb) and 284 degrees (pai).in the framework of the international project aimed at the sequencing of the bacillus subtilis genome, five dna fragments in the region between rrnb (275 degrees) and pai (284 degrees) were cloned by inverse and combinatorial long-range pcr and their nucleotide sequences were determined and analysed. together these sequences constituted a contig of 62229 bp. on the basis of the position of not1 and stil restriction sites, the orientation and order of known genetic markers was determined to be pa ...19979274030
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