Cloning, characterization, and functional expression of acs, the gene which encodes acetyl coenzyme A synthetase in Escherichia coli.
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Acetyl coenzyme A synthetase (Acs) activates acetate to acetyl coenzyme A through an acetyladenylate intermediate; two other enzymes, acetate kinase (Ack) and phosphotransacetylase (Pta), activate acetate through an acetyl phosphate intermediate. We subcloned acs, the Escherichia coli open reading frame purported to encode Acs (F. R. Blattner, V. Burland, G. Plunkett III, H. J. Sofia, and D. L. Daniels, Nucleic Acids Res. 21:5408-5417, 1993). We constructed a mutant allele, delta acs::Km, with the central 0.72-kb BclI-BclI portion of acs deleted, and recombined it into the chromosome. Whereas wild-type cells grew well on acetate across a wide range of concentrations (2.5 to 50 mM), those deleted for acs grew poorly on low concentrations (< or = 10 mM), those deleted for ackA and pta (which encode Ack and Pta, respectively) grew poorly on high concentrations (> or = 25 mM), and those deleted for acs, ackA, and pta did not grow on acetate at any concentration tested. Expression of acs from a multicopy plasmid restored growth to cells deleted for all three genes. Relative to wild-type cells, those deleted for acs did not activate acetate as well, those deleted for ackA and pta displayed even less activity, and those deleted for all three genes did not activate acetate at any concentration tested. Induction of acs resulted in expression of a 72-kDa protein, as predicted by the reported sequence. This protein immunoreacted with antiserum raised against purified Acs isolated from an unrelated species, Methanothrix soehngenii. The purified E. coli Acs then was used to raise anti-E. coli Acs antiserum, which immunoreacted with a 72-kDa protein expressed by wild-type cells but not by those deleted for acs. When purified in the presence, but not in the absence, of coenzyme A, the E. coli enzyme activated acetate across a wide range of concentrations in a coenzyme A-dependent manner. On the basis of these and other observations, we conclude that this open reading frame encodes the acetate-activating enzyme, Acs. (+info)
Construction of Pta-Ack pathway deletion mutants of Escherichia coli and characteristic growth profiles of the mutants in a rich medium.
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Escherichia coli grown in a rich medium excreted acetate and reused the acetate. Using cloned genes and a plasmid with a temperature-sensitive replication origin, three kinds of Pta-Ack pathway deletion mutants were constructed. Acetate production and reuse by wild-type cells grown in the rich medium was confirmed to largely occur through the Pta-Ack pathway. The deletion mutants of the gene encoding phosphotransacetylase secreted pyruvate before the secretion of acetate into the medium. A deletion mutant of the gene encoding acetate kinase grew at a slow rate, but its secretion and use of acetate were rapid. These results indicated that a pathway(s), other than the Pta-Ack pathway, functions in the control of excess carbon flow in the mutants. (+info)
Identification and characterization of the ackA (acetate kinase A)-pta (phosphotransacetylase) operon and complementation analysis of acetate utilization by an ackA-pta deletion mutant of Escherichia coli.
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The pta gene encoding phosphotransacetylase was cloned on a high copy plasmid with or without the ackA gene encoding acetate kinase in Escherichia coli. The acetate kinase and phosphotransacetylase were overproduced in cells harboring the plasmid possessing both genes. Nucleotide sequencing of the pta gene revealed that it is able to produce a polypeptide comprising 714 amino acid residues, which starts at 70 base pairs downstream from the stop codon of the ackA gene. The 77-kDa protein band of overproduced phosphotransacetylase was observed on SDS-polyacrylamide gel electrophoresis, of which the amino terminal sequence corresponds to that of the deduced polypeptide without the amino terminal methionine. Two transcripts of pta of different sizes were found in the cells. A 3,700 nucleotide transcript, which covers the ackA and pta genes, seemed to be produced by the first promoter in the operon and a 2,300 nucleotide transcript, which covers just pta, seemed to be produced by the second promoter. In a synthetic medium containing acetate as the sole carbon source, the growth of an ackA-pta double mutant was greatly impaired. Complementation analyses revealed that both the acetate kinase and phosphotransacetylase were required for the rapid growth in the acetate medium. (+info)
Transcriptional regulation of the phosphotransacetylase-encoding and acetate kinase-encoding genes (pta and ack) from Methanosarcina thermophila.
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Phosphotransacetylase and acetate kinase catalyze the activation of acetate to acetyl coenzyme A in the first step of methanogenesis from acetate in Methanosarcina thermophila. The genes encoding these enzymes (pta and ack) have been cloned and sequenced. They are arranged on the chromosome with pta upstream of ack (M.T. Latimer, and J. G. Ferry, J. Bacteriol. 175:6822-6829, 1993). The activities of phosphotransacetylase and acetate kinase are at least 8- to 11-fold higher in acetate-grown cells than in cells grown on methanol, monomethylamine, dimethylamine, or trimethylamine. Northern blot (RNA) analyses demonstrated that pta and ack are transcribed as an approximately 2.4-kb polycistronic message and that the regulation of enzyme synthesis occurs at the mRNA level. Primer extension analyses revealed a transcriptional start site located 27 bp upstream from the translational start of the pta gene and 24 bp downstream from a consensus archaeal boxA promoter sequence. S1 nuclease protection assays detected transcripts with four different 3' ends, each of which mapped to the beginning of four consecutive direct repeats. Northern blot analysis using an ack-specific probe detected both the 2.4-kb polycistronic transcript and a smaller 1.4-kb transcript which is the estimated size of monocistronic ack mRNA. A primer extension product was detected with an ack-specific primer; the 5' end of the product was in the intergenic region between the pta and ack genes but did not follow a consensus archaeal boxA sequence. This result, as well as detection of an additional 1.4-kb mRNA species, suggests processing of the polycistronic 2.4-kb transcript. (+info)
Change in direction of flagellar rotation in Escherichia coli mediated by acetate kinase.
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Strains of Escherichia coli lacking all cytoplasmic chemotaxis proteins except CheY swim smoothly under most conditions. However, they tumble when exposed to acetate. Acetate coenzyme A synthetase (EC 6.2.1.1) was thought to be essential for this response. New evidence suggests that the tumbling is mediated instead by acetate kinase (EC 2.7.2.1), which might phosphorylate CheY via acetyl phosphate. In strains that were wild type for chemotaxis, neither acetate coenzyme A synthetase, acetate kinase, nor phosphotransacetylase (EC 2.3.1.8) (and thus acetyl phosphate) was required for responses to aspartate, serine, or sugars sensed by the phosphotransferase system. Thus, acetate-induced tumbling does not appear to play an essential role in chemotaxis in wild-type cells. (+info)
Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila.
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The genes for the acetate-activating enzymes, acetate kinase and phosphotransacetylase (ack and pta), from Methanosarcina thermophila TM-1 were cloned and sequenced. Both genes are present in only one copy per genome, with the pta gene adjacent to and upstream of the ack gene. Consensus archaeal promoter sequences are found upstream of the pta coding region. The pta and ack genes encode predicted polypeptides with molecular masses of 35,198 and 44,482 Da, respectively. A hydropathy plot of the deduced phosphotransacetylase sequence indicates that it is a hydrophobic polypeptides; however, no membrane-spanning domains are evident. Comparison of the amino acid sequences deduced from the M. thermophila and Escherichia coli ack genes indicate similar subunit molecular weights and 44% identity (60% similarity). The comparison also revealed the presence of several conserved arginine, cysteine, and glutamic acid residues. Arginine, cysteine, and glutamic acid residues have previously been implicated at or near the active site of the E. coli acetate kinase. The pta and ack genes were hyperexpressed in E. coli, and the overproduced enzymes were purified to homogeneity with specific activities higher than those of the enzymes previously purified from M. thermophila. The overproduced phosphotransacetylase and acetate kinase migrated at molecular masses of 37,000 and 42,000 Da, respectively. The activity of the acetate kinase is optimal at 65 degrees C and is protected from thermal inactivation by ATP. Diethylpyrocarbonate and phenylglyoxal inhibited acetate kinase activity in a manner consistent with the presence of histidine and arginine residues at or near the active site; however, the thiol-directed reagents 5,5'-dithiobis (2-nitrobenzoic acid) and N-ethylmaleimide were ineffective. (+info)
Regulation of the Bacillus subtilis acetate kinase gene by CcpA.
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The Bacillus subtilis gene encoding acetate kinase was identified on the basis of sequence similarity to the Escherichia coli ackA gene and to a second E. coli gene closely related to ackA. Insertional inactivation of this region of the B. subtilis chromosome resulted in the disappearance of acetate kinase enzyme activity in cell extracts. The ackA gene was mapped to a site close to the ccpA gene, at 263 degrees. The transcriptional start site for B. subtilis ackA was located 90 bp upstream from the start of the coding region, and expression was increased by growth in the presence of excess glucose. Growth of the AckA- mutant was inhibited by glucose, suggesting that acetate kinase is important for excretion of excess carbohydrate. The stimulation of ackA expression by glucose was blocked in a CcpA- mutant, indicating that CcpA, which is required for glucose repression of certain carbon source utilization genes, including amyE, may also be involved in activation of carbon excretion pathways. Two sequences resembling the amyO operator site were identified upstream of the ackA promoter; removal of this region resulted in loss of glucose activation of ackA expression. (+info)
Cloning, sequencing, and expression of genes encoding phosphotransacetylase and acetate kinase from Clostridium acetobutylicum ATCC 824.
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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. (+info)