1. |
Fontaine L,
Meynial-Salles I,
Girbal L,
Yang X,
Croux C,
Soucaille P,
( 2002 ) 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. PMID : 11790753 : DOI : 10.1128/jb.184.3.821-830.2002 PMC : PMC139506 Abstract >>
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 mutant of C. acetobutylicum, a mutant cured of the pSOL1 megaplasmid, restored butanol production and provided elevated activities of NADH-dependent butyraldehyde and butanol dehydrogenases. The recombinant AdhE2 protein expressed in E. coli as a Strep-tag fusion protein and purified to homogeneity also demonstrated NADH-dependent butyraldehyde and butanol dehydrogenase activities. This is the second AADH identified in C. acetobutylicum ATCC 824, and to our knowledge this is the first example of a bacterium with two AADHs. It is noteworthy that the two corresponding genes, adhE and adhE2, are carried by the pSOL1 megaplasmid of C. acetobutylicum ATCC 824.
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2. |
Tangney M,
Winters GT,
Mitchell WJ,
( 2001 ) Characterization of a maltose transport system in Clostridium acetobutylicum ATCC 824. PMID : 11781805 : DOI : 10.1038/sj/jim/7000125 Abstract >>
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 entirely within the membrane in this organism. A putative gene system for the maltose PTS was identified (from the C. acetobutylicum ATCC 824 genome sequence), encoding an enzyme II(Mal) and a maltose 6-phosphate hydrolase.
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3. |
Sabathé F,
Croux C,
Cornillot E,
Soucaille P,
( 2002 ) amyP, a reporter gene to study strain degeneration in Clostridium acetobutylicum ATCC 824. PMID : 12023083 : DOI : 10.1111/j.1574-6968.2002.tb11165.x Abstract >>
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 not in acidogenic chemostat cultures. These results are in agreement with the extracellular alpha-amylase activities indicating that the expression of amyP is regulated at the transcriptional level. amyP is located on the pSOL1 megaplasmid that carries all the genes involved in the final steps of solvent formation. Degeneration of C. acetobutylicum has been associated to the loss of pSOL1. We demonstrate here that amyP can be used as a reporter system to quantitatively follow this phenomenon.
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4. |
Behrens S,
Mitchell W,
Bahl H,
( 2001 ) Molecular analysis of the mannitol operon of Clostridium acetobutylicum encoding a phosphotransferase system and a putative PTS-modulated regulator. PMID : 11160802 : DOI : 10.1099/00221287-147-1-75 Abstract >>
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 (IICB) component. The mannitol (mtl) operon of C. acetobutylicum DSM 792 comprises four genes in the order mtlARFD. Sequence analysis of deduced protein products indicated that the mtlA and mtlF genes respectively encode the IICB and IIA components of the mannitol PTS, which is a member of the fructose-mannitol (Fru) family. The mtlD gene product is a mannitol-1-phosphate dehydrogenase, while mtlR encodes a putative transcriptional regulator. MtlR contains two PTS regulatory domains (PRDs), which have been found in a number of DNA-binding transcriptional regulators and in transcriptional antiterminators of the Escherichia coli BglG family. Also, near the C-terminus is a well-conserved signature motif characteristic of members of the IIA(Fru)/IIA(Mtl)/IIA(Ntr) PTS protein family. These regions are probably the sites of PTS-dependent phosphorylation to regulate the activity of the protein. A helix-turn-helix DNA-binding motif was not found in MtlR. Transcriptional analysis of the mtl genes by Northern blotting indicated that the genes were transcribed as a polycistronic operon, expression of which was induced by mannitol and repressed by glucose. Primer extension experiments identified a transcriptional start point 42 bp upstream of the mtlA start codon. Two catabolite-responsive elements (CREs), one of which overlapped the putative -35 region of the promoter, were located within the 100 bp upstream of the start codon. These sequences may be involved in regulation of expression of the operon.
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5. |
Tangney M,
( 2000 ) Analysis of a catabolic operon for sucrose transport and metabolism in Clostridium acetobutylicum ATCC 824. PMID : 10937490 : Abstract >>
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 hydrolase respectively. While the pathway for sucrose metabolism is conserved between C. acetobutylicum and C. beijerinckii, the operons show considerable differences in organization and regulatory elements. The C. acetobutylicum scr operon contains the elements of an antiterminator-mediated regulation mechanism, typical of the BgIG family of regulators. The scrT gene, located upstream of scrA encodes an antiterminator that is preceded by a transcription terminator, which is overlapped by a classical ribonucleic antiterminator (RAT) sequence. We also propose the existence of a new variant RAT-like sequence which overlaps a terminator between scrT and the downstream structural genes.
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6. |
Winzer K,
Lorenz K,
Zickner B,
Dürre P,
( 2000 ) Differential regulation of two thiolase genes from Clostridium acetobutylicum DSM 792. PMID : 11075929 : Abstract >>
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 transcriptional start site was identified 102 bp upstream of the thlA start codon. This site was preceded by a region that exhibits high similarity to the sigma70 consensus promoter sequences of Gram-positive and -negative bacteria. Regulation of thlA and thlB was studied at the transcriptional level to elucidate the specific function of each gene. Non-radioactive primer extension analysis using fluorescein-labelled oligonucleotides and Northern blot analysis revealed high levels of thlA transcripts in acid- and solvent-producing cells. During an induced shift of a continuous culture from acid to solvent formation, the transcript level transiently decreased to a minimum, 3 to 7 h after induction. The thlA transcript length is about 1.4 kb, indicating a monocistronic organisation, whereas genetic organization and reverse transcription (RT)-PCR analysis indicated that thlB forms an operon with two other adjacent genes, thlR and thlC. Transcription and regulation of the thlB operon was studied using RTPCR and showed a very low expression in acid- and solvent-producing cells. Heterologously expressed clostridial ThlB showed high thiolase activity in Escherichia coli. The N-terminal part of ThlR possesses a potential helix-turn-helix motif and shows significant homology to regulatory proteins belonging to the TetR/AcrR family of transcriptional regulators. ThlR possibly acts as a transcriptional repressor of thlB operon expression. The data provide strong evidence that ThlA is involved in the metabolism of both acid and solvent formation, whereas the physiological function of ThlB has yet to be elucidated.
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7. |
Hujer S,
Externbrink T,
( 2000 ) Sequence analysis of the atp operon of Clostridium acetobutylicum DSM 792 encoding the F0F1 ATP synthase. PMID : 10902917 : Abstract >>
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 (without corresponding promoter sequence) upstream of atpE, indicating posttranscriptional processing for massive expression of this gene product.
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8. |
Iddar A,
Valverde F,
Assobhei O,
Serrano A,
Soukri A,
( 2005 ) Widespread occurrence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase among gram-positive bacteria. PMID : 16562377 : Abstract >>
The non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDHN, NADP+-specific, EC 1.2.1.9) is present in green eukaryotes and some Streptococcus strains. The present report describes the results of activity and immunoblot analyses, which were used to generate the first survey of bacterial GAPDHN distribution in a number of Bacillus, Streptococcus and Clostridium strains. Putative gapN genes were identified after PCR amplification of partial 700-bp sequences using degenerate primers constructed from highly conserved protein regions. Alignment of the amino acid sequences of these fragments with those of known sequences from other eukaryotic and prokaryotic GAPDHNs, demonstrated the presence of conserved residues involved in catalytic activity that are not conserved in aldehyde dehydrogenases, a protein family closely linked to GAPDHNs. The results confirm that the basic structural features of the members of the GAPDHN family have been conserved throughout evolution and that no identity exists with phosphorylating GAPDHs. Furthermore, phylogenetic trees generated from multiple sequence alignments suggested a close relationship between plant and bacterial GAPDHN families.
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9. |
Petersen DJ,
Bennett GN,
( 1991 ) Cloning of the Clostridium acetobutylicum ATCC 824 acetyl coenzyme A acetyltransferase (thiolase; EC 2.3.1.9) gene. PMID : 1685080 : PMC : PMC183649 Abstract >>
Thiolase (acetyl coenzyme A acetyltransferase; EC 2.3.1.9) from Clostridium acetobutylicum is a key enzyme in the production of acids and solvents in this organism. The purification and properties of the enzyme have already been described (D. P. Wiesenborn, F. B. Rudolph, and E.T. Papoutsakis, Appl. Environ. Microbiol. 54:2717-2722, 1988). The thl gene encoding the thiolase has been cloned by using primary antibodies raised to the purified enzyme. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was prepared and screened by immunoblots with the antithiolase antibodies. Phage DNA was purified from positive plaques, and restriction enzyme digests identified an approximately 4.8-kb AccI fragment common to all positive plaques. A corresponding fragment was also found in AccI digests of C. acetobutylicum chromosomal DNA. The fragment was purified and EcoRI linkers were attached before being subcloned into pUC19. Maxicell analysis showed the production of an approximately 42-kDa protein, whose size corresponded to the molecular size of the purified thiolase, from the clostridial insert. Enzyme activity assays and Western blot (immunoblot) analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell extracts of Escherichia coli harboring the cloned thl confirmed the presence of the thiolase encoded within the cloned DNA.
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10. |
Tangney M,
Galinier A,
Deutscher J,
Mitchell WJ,
( 2003 ) Analysis of the elements of catabolite repression in Clostridium acetobutylicum ATCC 824. PMID : 14593248 : DOI : 10.1159/000073403 Abstract >>
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 in an ATP-dependent manner in cell-free extracts of C. Acetobutylicum. Furthermore, purified His-tagged HPr from Bacillus Subtilis was also a substrate for the clostridial HPr kinase/phosphorylase. This phosphorylation reaction is a key step in the mechanism of carbon catabolite repression proposed to operate in B. Subtilis and other low-GC gram-positive bacteria. Putative genes encoding the HPr kinase/phosphorylase and the other element of this model, namely the catabolite control protein CcpA, were identified from the C. Acetobutylicum genome sequence, suggesting that a similar mechanism of carbon catabolite repression may operate in this industrially important organism.
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11. |
Jia K,
Zhu Y,
Zhang Y,
Li Y,
( 2011 ) Group II intron-anchored gene deletion in Clostridium. PMID : 21304965 : DOI : 10.1371/journal.pone.0016693 PMC : PMC3031624 Abstract >>
Clostridium plays an important role in commercial and medical use, for which targeted gene deletion is difficult. We proposed an intron-anchored gene deletion approach for Clostridium, which combines the advantage of the group II intron "ClosTron" system and homologous recombination. In this approach, an intron carrying a fragment homologous to upstream or downstream of the target site was first inserted into the genome by retrotransposition, followed by homologous recombination, resulting in gene deletion. A functional unknown operon CAC1493-1494 located in the chromosome, and an operon ctfAB located in the megaplasmid of C. acetobutylicum DSM1731 were successfully deleted by using this approach, without leaving antibiotic marker in the genome. We therefore propose this approach can be used for targeted gene deletion in Clostridium. This approach might also be applicable for gene deletion in other bacterial species if group II intron retrotransposition system is established.
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12. |
Hancock KR,
Rockman E,
Young CA,
Pearce L,
Maddox IS,
Scott DB,
( 1991 ) Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli. PMID : 1850729 : DOI : 10.1128/jb.173.10.3084-3095.1991 PMC : PMC207901 Abstract >>
A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the beta-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the beta-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium beta-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for beta-galactosidase expression in E. coli. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and Klebsiella pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of beta-galactosidase in E. coli.
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13. |
( 1996 ) Molecular characterization of a family of choline-binding proteins of Clostridium beijerinckii NCIB 8052. Evolution and gene redundancy in prokaryotic cell. PMID : 8973341 : DOI : 10.1016/s0378-1119(96)00390-3 Abstract >>
Three genes homologous to cspA, which encodes the major secretable protein of Clostridium beijerinckii NCIB 8052 have been cloned and sequenced. The Csp proteins showed the typical modular structure of cell-wall associated proteins and, that found in the choline-binding proteins of Streptococcus pneumoniae. The variable number of repeats that constitute the C-terminal choline-binding domain suggests that the csp genes have evolved by deletion-duplication events. Northern blot analysis indicated that under the culture conditions employed only two genes, cspA and cspC, are efficiently expressed and their products are detected in the culture medium. The csp genes are not contiguously located in the chromosome and appear to be expressed independently. Primer extension experiments located a transcription start site 29 bp upstream of the cspA initiation codon. The -10 and -35 promoter regions are closely related to the consensus sequence of Escherichia coli sigma 70 promoters. The cell wall binding capacity of the clostridial proteins, their abundance in the extracellular media, together with the existence of gene redundancy suggest that the Csp proteins should play an important role in the interaction of this microorganism with its surrounding environment.
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14. |
( 1997 ) The kdp system of Clostridium acetobutylicum: cloning, sequencing, and transcriptional regulation in response to potassium concentration. PMID : 9226259 : DOI : 10.1128/jb.179.14.4501-4512.1997 PMC : PMC179285 Abstract >>
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 potassium transport ATPase and its regulators. The complete genome sequence of Synechocystis sp. strain PCC 6803 and a recently published part of the Mycobacterium tuberculosis genome indicate the existence of a kdp system in these organisms as well, but all three systems comprise neither a second orf upstream of kdpA nor an additional kdpX gene. Expression of the clostridial kdp genes, including the unique kdpX gene, was found to be inducible by low potassium concentrations. A transcription start point could be mapped upstream of orfZ. A promoter upstream of kdpD was active only under noninducing conditions. Lowering the potassium content of the medium led to formation of a common transcript (orfZYkdpABCXDE), with a putative internal RNase E recognition site, which could be responsible for the instability of the common transcript. Except for the two small peptides, all gene products could be detected in in vitro transcription-translation experiments.
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15. |
( 1996 ) Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824. PMID : 8626337 : DOI : 10.1128/jb.178.9.2668-2675.1996 PMC : PMC177994 Abstract >>
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 continuous culture. These results were in agreement with the hydrogenase protein level, indicating that expression of hydA is regulated at the transcriptional level. Primer extension analysis identified a major transcriptional start site 90 bp upstream of the hydA start codon. The position of a putative rho-independent transcription terminator immediately downstream of the termination codon is in agreement with the size of the hydA transcript (1.9 kb) determined by Northern (RNA) blot experiments and confirms that the gene is transcribed as a monocistronic operon. Two truncated open reading frames (ORFs) were identified downstream and upstream of hydA and in opposite directions. The amino acid sequence deduced from ORF2 presents strong identity with ortho phosphoribosyl transferases involved in pyrimidine synthesis. The amino acid sequence deduced from ORF3 presents no significant similarity to any sequence in various available databases.
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16. |
( 1993 ) Sequence and arrangement of two genes of the butyrate-synthesis pathway of Clostridium acetobutylicum ATCC 824. PMID : 8244020 : DOI : 10.1016/0378-1119(93)90182-3 Abstract >>
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 from buk.
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17. |
( 1996 ) Cloning, sequencing, and expression of genes encoding phosphotransacetylase and acetate kinase from Clostridium acetobutylicum ATCC 824. PMID : 8702268 : PMC : PMC168061 Abstract >>
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 the subclone, and both AK and PTA activities were shown to be elevated. DNA sequence analysis showed that the pta and ack genes are adjacent in the clostridial chromosome, with pta upstream. The pta gene encodes a protein of 333 amino acid residues with a calculated molecular mass of 36.2 kDa, and ack encodes a polypeptide of 401 residues with a molecular mass of 44.3 kDa. Primer extension analysis identified a single transcriptional start site located 70 bp upstream of the start codon for the pta gene, suggesting an operon arrangement for these tandem genes. The results from overexpression of ack and pta in C. acetobutylicum showed that the final ratios of acetate to other major products were higher and that there was a greater proportion of two- versus four-carbon-derived products.
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18. |
( 1993 ) Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis. PMID : 8226639 : DOI : 10.1128/jb.175.21.6959-6969.1993 PMC : PMC206823 Abstract >>
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 derepressed before the onset of solventogenesis, thus confirming earlier results of Northern hybridizations with a ctfB gene probe (U. Gerischer and P. D?rre, J. Bacteriol. 174:426-433, 1992). Upstream of the sol operon, we identified two putative promoters that were located in regions with possible stem-loop structures formed by several inverted repeats. The distal promoter P1 showed only minor transcription initiation in solventogenic C. acetobutylicum cells but was recognized in Escherichia coli, presumably because of its high similarity to the sigma 70 consensus sequence. The adhE-proximal promoter P2 directed the major transcription start point in solventogenic C. acetobutylicum but was not recognized in E. coli. The clostridial AdhE showed high similarity to a novel family (type III) of alcohol dehydrogenases. Two other ORFs (ORF 5 and ORF 6) were found on the cloned DNA region that showed no significant similarity to sequences in various available data bases. mRNA studies revealed that ORF 5 formed a monocistronic operon and showed increased expression before onset of solventogenesis.
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19. |
Azeddoug H,
Reysset G,
( 1994 ) Cloning and sequencing of a chromosomal fragment from Clostridium acetobutylicum strain ABKn8 conferring chemical-damaging agents and UV resistance to E. coli recA strains. PMID : 7765497 : Abstract >>
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 relationship with the RecA protein of E. coli or products of genes involved in the SOS response. The deduced amino acid sequence of ORFb and ORFc are highly homologous to those deduced from two genes specifying resistance to tellurium salts present on plasmid pMER610 harbored by Alcaligenes sp.strains and to an AMP-binding protein (CABP1) found in Dictyostelium discoideum. The existence of these homologous proteins suggests that they may perform a similar key function in the three unrelated organisms.
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20. |
Sanchez-Beato AR,
Ronda C,
Garcia JL,
( 1995 ) Tracking the evolution of the bacterial choline-binding domain: molecular characterization of the Clostridium acetobutylicum NCIB 8052 cspA gene. PMID : 7860591 : DOI : 10.1128/jb.177.4.1098-1103.1995 PMC : PMC176709 Abstract >>
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, has been used to clone the cspA gene which encodes a protein with 590 amino acids and an M(r) of 63,740. According to the predicted amino acid sequence, CspA is synthesized with an N-terminal segment of 26 amino acids characteristic of prokaryotic signal peptides. Expression of the cspA gene in Escherichia coli led to the production of a major anti-CspA-labeled protein of 80,000 Da which was purified by affinity chromatography on DEAE-cellulose. A comparison of CspA with other proteins in the EMBL database revealed that the C-terminal half of CspA is homologous to the choline-binding domains of the major pneumococcal autolysin (LytA amidase), the pneumococcal antigen PspA, and other cell wall-lytic enzymes of pneumococcal phages. This region, which is constructed of four repeating motifs, also displays a high similarity with the glucan-binding domains of several streptococcal glycosyltransferases and the toxins of Clostridium difficile.
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21. |
( 1994 ) Sporulation and primary sigma factor homologous genes in Clostridium acetobutylicum. PMID : 7961408 : DOI : 10.1128/jb.176.21.6572-6582.1994 PMC : PMC197012 Abstract >>
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 subtilis and Staphylococcus aureus. Northern (RNA) blot analysis revealed that both genes from an operon, which is clearly expressed under conditions that allow for cell division. A promoter sequence with significant homology to the sigma H-dependent Bacillus promoters preceded the determined transcriptional start point, 182 bp upstream of the GUG start codon of dnaE. The homologous genes to Bacillus spp. sporulation sigma factors G, E, and K have been cloned and sequenced. Indirect evidence for the existence of sigma F was obtained by identification of a DNA sequence homologous to the respective Bacillus consensus promoter. Southern hybridization analysis indicated the presence of sigma D and sigma H homologous genes in C. acetobutylicum. A new gene group conserved within the eubacteria, but with yet unspecified functions, is described. The data presented here provide strong evidence that at least some of the complex regulation features of sporulation in B. subtilis are conserved in C. acetobutylicum and possibly Clostridium spp.
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22. |
Dürre P,
Fischer RJ,
Kuhn A,
Lorenz K,
Schreiber W,
Stürzenhofecker B,
Ullmann S,
Winzer K,
Sauer U,
( 1995 ) Solventogenic enzymes of Clostridium acetobutylicum: catalytic properties, genetic organization, and transcriptional regulation. PMID : 7576767 : DOI : 10.1111/j.1574-6976.1995.tb00209.x Abstract >>
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 dehydrogenase isozymes and a thiolase are involved in butanol synthesis. RNA analyses showed a sequential order of induction for the different butanol dehydrogenase genes, indicating an in vivo function of BdhI in low level butanol formation. The physiological roles of AdhE and BdhII most likely involve high level butanol formation, with AdhE being responsible for the onset of solventogenesis and BdhII ensuring continued butanol production. Addition of methyl viologen results in artificially induced butanol synthesis which seems to be mediated by a still unknown set of enzymes. Although the signal that triggers the shift to solventogenesis has not yet been elucidated, recent investigations suggest a possible function of DNA supercoiling as a transcriptional sensor of the respective environmental stimuli.
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