Cloning and sequence analysis of the genes encoding phoshotransbutyrylase and butyrate kinase from Clostridium acetobutylicum NCIMB 8052

Oultram, John D.; Burr, lan D.; Elmore, Michael J.; Minton, Nigel P.

doi:10.1016/0378-1119(93)90677-u

Cited by 27 publications

(26 citation statements)

References 19 publications

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“…3. Phylogenetic tree of part of the deduced protein sequence (amino acid positions 22 to 127 of strain L2-50) of the butyrate kinase gene (buk) from bacteria examined in this study (shown in boldface) and several clostridial sequences (13,34,35,47). Accession numbers for the sequences used are given in brackets.…”

Section: Discussionmentioning

confidence: 99%

Restricted Distribution of the Butyrate Kinase Pathway among Butyrate-Producing Bacteria from the Human Colon

Louis¹,

Duncan²,

McCrae³

et al. 2004

J Bacteriol

397

319

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The final steps in butyrate synthesis by anaerobic bacteria can occur via butyrate kinase and phosphotransbutyrylase or via butyryl-coenzyme A (CoA):acetate CoA-transferase. Degenerate PCR and enzymatic assays were used to assess the presence of butyrate kinase among 38 anaerobic butyrate-producing bacterial isolates from human feces that represent three different clostridial clusters (IV, XIVa, and XVI). Only four strains were found to possess detectable butyrate kinase activity. These were also the only strains to give PCR products (verifiable by sequencing) with degenerate primer pairs designed within the butyrate kinase gene or between the linked butyrate kinase/phosphotransbutyrylase genes. Further analysis of the butyrate kinase/phosphotransbutyrylase genes of one isolate, L2-50, revealed similar organization to that described previously from different groups of clostridia, along with differences in flanking sequences and phylogenetic relationships. Butyryl-CoA:acetate CoA-transferase activity was detected in all 38 strains examined, suggesting that it, rather than butyrate kinase, provides the dominant route for butyrate formation in the human colonic ecosystem that contains a constantly high concentration of acetate.The human colon harbors a diverse range of bacteria that form a complex anaerobic ecosystem. Apart from interacting with each other, for example by cross-feeding, colonic bacteria also interact with the human host and play a vital role in maintaining human health through mechanisms such as protecting against pathogenic bacteria, influencing the host's immune system, and supplying nutrients (23).The recent development of molecular techniques for studying phylogenetic diversity of the colonic microflora is leading to a better understanding of its composition (9, 21). One group of bacteria, which has received relatively little previous attention because of their fastidious growth requirements, are the strictly anaerobic gram-positive bacteria of low mol% GϩC-content clostridial groups. Several studies employing a variety of molecular approaches indicate that members of this group related to clostridial clusters IV and XIVa (14) might make up about 30 to 60% of the total bacterial population within the colon (22,25,38,46,50).Bacteria have been identified within these groups that carry out a fermentative metabolism leading to the buildup of butyrate; lactate and formate are often being produced, while acetate can be either produced or consumed (18). Butyrate is considered to exert health-promoting effects on the colon. It serves as a major energy source for the colonocytes and has also been claimed to be protective against colon cancer and inflammatory bowel diseases via effects on gene expression and cellular development (reviewed in references 36 and 51).The pathway for butyrate formation and its respective genes have been examined in the solventogenic bacterium Clostridium acetobutylicum. Two molecules of acetyl coenzyme A (acetyl-CoA) are condensed and subsequently reduced to butyryl-CoA in a ...

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Section: Discussionmentioning

confidence: 99%

Restricted Distribution of the Butyrate Kinase Pathway among Butyrate-Producing Bacteria from the Human Colon

Louis¹,

Duncan²,

McCrae³

et al. 2004

J Bacteriol

397

319

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“…The 750 bp fragment was digested with AluI and one of the resulting subfragments could be cloned in HincIIdigested pUC18. The nucleotide sequence of the 246-bp insert of the resulting recombinant plasmid pAK12 represented part of an open reading frame and its deduced amino acid sequence exhibited high homology (66-50 Yo identity and 83-68 '/O similarity) to the acetate kinases of the organisms mentioned above and ORFX from E. coli (Schweizer & Datta, 1991), but no significant homology to the butyrate kinases of C. acetobutylicum ATCC 824 (Walter et al, 1993) and a Clostridium beijerinckii strain (Oultram et al, 1993 ; formerly C. acetobutylicum NCIMB 8052 - Wilkinson et al, 1995). A recent report described cloning and sequencing of the acetate kinase gene of C. acetobutylicum ATCC 824 (Boynton et al, 1996), confirming that the 246 bp fragment was indeed part of the ack gene (nt 1786-2032).…”

Section: Regulationmentioning

confidence: 99%

“…A polycistronic organization has also been suggested for the respective genes of E. coli and M . thermophila (Latimer & Ferry 1993;Matsuyama et al, 1994;Singh-Wissmann & Ferry, 1995) as well as the butyrate kinase and phosphotransbutyrylase genes of C. acetobutylicurn ATCC 824 (Walter et al, 1993) and strain NCIMB 8052 (Oultram et al, 1993 ; now recognized as a C. beijevinckii strain -Wilkinson et al, 1995), whereas the acetate kinase gene of B. subtilis appears to be monocistronically organized (Grundy et al, 1993). In M .…”

Section: Regulationmentioning

confidence: 99%

Acetate kinase from Clostridium acetobutylicum: a highly specific enzyme that is actively transcribed during acidogenesis and solventogenesis

1997

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Acetate kinase (ATP: phosphotransferase, EC 2.7.2.1) has been purified 294-fold from acid-producing cells of CIostridium acetdbutylicum DSM 1731 to a specific activity of 1087 U mg' ' (ADP-forming direction). The dimeric enzyme consisted of subunits with a molecular mass of 43 kDa. The molecular mass of the native acetate kinase was in the range 87-94 kDa as judged by gel filtration and native gel electrophoresis. The enzyme showed high specificity for the substrates acetate and ATP, and maximal activity was obtained with Mnz+ as divalent cation. The presence of mercury compounds such as HgCI, and plhydroxymercuri benzoate resulted in an essential loss of activity. The apparent K,,, values for acetate, MgATP, acetyl phosphate, and Mg-ADP were 73,0.37,0.58 and 0.71 mM. An activity-staining procedure for detection of acetate kinase in crude cell extracts after separation on native polyacrylamide gels was developed. A DNA fragment encoding 246 bp of the acetate kinase gene of C. acetobutylicum DSM 792 was cloned by a PCR-based approach.Northern blot analysis revealed transcription of the gene under acid-and solvent-producing conditions with no significant differences at the level of transcription.

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“…Genes encoding enzymes from this pathway are widespread in genome-sequenced clostridia and related species (1)(2)(3)(4)(5)(6)(7)(8). In spite of the central importance of butyrate-forming genes in these organisms, only individual enzymes from a comparably small selection of organisms have been purified and carefully studied in the past (9)(10)(11)(12)(13)(14)(15). In particular, Clostridium acetobutylicum enzymes were of great interest due to the organism's capability of producing acetone and butanol (16)(17)(18).…”

mentioning

confidence: 99%

Effect of an Oxygen-Tolerant Bifurcating Butyryl Coenzyme A Dehydrogenase/Electron-Transferring Flavoprotein Complex from Clostridium difficile on Butyrate Production in Escherichia coli

et al. 2013

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The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro. While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD ؉ -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a cooxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases (Rhodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed. G enome sequencing of organisms provides information regarding the distribution of genes encoding biotechnologically important metabolic pathways. This is true for the clostridial butyrogenic pathway, which converts acetyl-conenzyme A (CoA)-the terminal oxidation product of glucose via glycolysis-to butyrate. Genes encoding enzymes from this pathway are widespread in genome-sequenced clostridia and related species (1-8). In spite of the central importance of butyrate-forming genes in these organisms, only individual enzymes from a comparably small selection of organisms have been purified and carefully studied in the past (9-15). In particular, Clostridium acetobutylicum enzymes were of great interest due to the organism's capability of producing acetone and butanol (16)(17)(18). Driven by the urgent need to replace oil-derived fossil fuels, genetic engineering of microbes for production of butan-1-ol, a promising alternative transportation fuel, is proceeding worldwide (16,(18)(19)(20)(21)(22)(23)(24).A long-known metabolic route such as the butyrate pathway (Fig. 1A) can suddenly gain fundamentally new meanings when hitherto-unknown properties of individual enzymes within this pathway become known. While for decades the role of butyrate formation was generally a...

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Cloning and sequence analysis of the genes encoding phoshotransbutyrylase and butyrate kinase from Clostridium acetobutylicum NCIMB 8052

Cited by 27 publications

References 19 publications

Restricted Distribution of the Butyrate Kinase Pathway among Butyrate-Producing Bacteria from the Human Colon

Restricted Distribution of the Butyrate Kinase Pathway among Butyrate-Producing Bacteria from the Human Colon

Acetate kinase from Clostridium acetobutylicum: a highly specific enzyme that is actively transcribed during acidogenesis and solventogenesis

Effect of an Oxygen-Tolerant Bifurcating Butyryl Coenzyme A Dehydrogenase/Electron-Transferring Flavoprotein Complex from Clostridium difficile on Butyrate Production in Escherichia coli

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