Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis

In Bacillus subtilis, the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose. Inactivation of pta had only a weak inhibitory effect on growth. In contrast to pta and ackA in Escherichia coli, the corresponding B. subtilis genes are not cotranscribed. Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA. The effects of the predicted cis-acting catabolite response element (CRE) located upstream from the promoter and of the trans-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated. As for ackA, glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant. Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.     

Modulation of flagellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR.

During the search for unknown factors involved in motility, we have found that expression of the flagellar master operon flhDC is affected by mutations of the pta and ackA genes, encoding phosphotransacetylase and acetate kinase, respectively (S. Shin, J. Sheen, and C. Park, Korean J. Microbiol. 31:504-511, 1993). Here we describe results showing that this effect is modulated by externally added acetate, except when both pta and ackA are mutated, suggesting the role of acetyl phosphate, an intermediate of acetate metabolism, as a regulatory effector. Furthermore, the following evidence indicates that the phosphorylation of OmpR, a trans factor for osmoregulation, regulates flagellar expression. First, in a strain lacking ompR, the expression of flhDC is no longer responsive to a change in the level of acetyl phosphate. Second, an increase in medium osmolarity does not decrease flhDC expression in an ompR mutant. It is known that such an increase normally enhances OmpR phosphorylation. Third, OmpR protein binds to the DNA fragment containing the flhDC promoter, and its affinity is increased with phosphorylation by acetyl phosphate. DNase I footprinting revealed the regions of the flhDC promoter protected by OmpR in the presence or absence of phosphorylation. Therefore, we propose that the phosphorylated OmpR, generated by either osmolarity change or the internal level of acetyl phosphate, negatively regulates the expression of flagella.     

Instability of ackA (acetate kinase) mutations and their effects on acetyl phosphate and ATP amounts in Streptococcus pneumoniae D39

Acetyl phosphate (AcP) is a small-molecule metabolite that can act as a phosphoryl group donor for response regulators of two-component systems (TCSs). The serious human respiratory pathogen Streptococcus pneumoniae (pneumococcus) synthesizes AcP by the conventional pathway involving phosphotransacetylase and acetate kinase, encoded by pta and ackA, respectively. In addition, pneumococcus synthesizes copious amounts of AcP and hydrogen peroxide (H(2)O(2)) by pyruvate oxidase, which is encoded by spxB. To assess possible roles of AcP in pneumococcal TCS regulation and metabolism, we constructed strains with combinations of spxB, pta, and ackA mutations and determined their effects on ATP, AcP, and H(2)O(2) production. Unexpectedly, ΔackA mutants were unstable and readily accumulated primary suppressor mutations in spxB or its positive regulator, spxR, thereby reducing H(2)O(2) and AcP levels, and secondary capsule mutations in cps2E or cps2C. ΔackA ΔspxB mutants contained half the cellular amount of ATP as a ΔspxB or spxB(+) strain. Acetate addition and anaerobic growth experiments suggested decreased ATP, rather than increased AcP, as a reason that ΔackA mutants accumulated spxB or spxR suppressors, although experimental manipulation of the AcP amount was limited. This finding and other considerations suggest that coping with endogenously produced H(2)O(2) may require energy. Starting with a ΔspxB mutant, we constructed Δpta, ΔackA, and Δpta ΔackA mutants. Epistasis and microarray experiment results were consistent with a role for the SpxB-Pta-AckA pathway in expression of the regulons controlled by the WalRK(Spn), CiaRH(Spn), and LiaSR(Spn) TCSs involved in sensing cell wall status. However, AcP likely does not play a physiological role in TCS sensing in S. pneumoniae.     

Cloning, characterization, and functional expression of acs, the gene which encodes acetyl coenzyme A synthetase in Escherichia coli.

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.     

阻断嗜乙酰乙酸棒杆菌乙酸合成途径对其在缺氧条件下产琥珀酸的影响

为探究和阻断嗜乙酰乙酸棒杆菌乙酸代谢途径,提高缺氧条件下琥珀酸的产率,减少副产物乙酸的合成,以C. acetoacidophilum ΔldhA为出发菌株,利用同源重组的方法分别敲除磷酸乙酰转移酶、乙酸激酶、CoA转移酶和丙酮酸脱氢酶复合体的相关基因pta,ackA,ctfA与aceE,研究突变菌产琥珀酸过程中相关参数的变化。结果表明：敲除pta与ackA基因后,对乙酸浓度,糖耗速率和糖酸转化率影响不大;pta,ackA与ctfA基因的同时失活使得乙酸的浓度和摩尔转化率分别降低81.4%和77.2%,葡萄糖消耗速率下降28.3%,琥珀酸对葡萄糖摩尔转化率提高25.3%;而单独敲除aceE基因后,乙酸几乎不产生,葡萄糖消耗速率下降35.6%,琥珀酸对葡萄糖摩尔转化率提高34.7%。因此,缺氧条件下,嗜乙酰乙酸棒杆菌的乙酸合成几乎全部走乙酰CoA途径,pta,ackA与ctfA是由乙酰CoA合成乙酸途径中最主要的基因;敲除基因aceE, 可以完全阻断乙酸生成,有效提高琥珀酸产率。     

Long-term anaerobic survival of the opportunistic pathogen Pseudomonas aeruginosa via pyruvate fermentation

Denitrification and arginine fermentation are central metabolic processes performed by the opportunistic pathogen Pseudomonas aeruginosa during biofilm formation and infection of lungs of patients with cystic fibrosis. Genome-wide searches for additional components of the anaerobic metabolism identified potential genes for pyruvate-metabolizing NADH-dependent lactate dehydrogenase (ldhA), phosphotransacetylase (pta), and acetate kinase (ackA). While pyruvate fermentation alone does not sustain significant anaerobic growth of P. aeruginosa, it provides the bacterium with the metabolic capacity for long-term survival of up to 18 days. Detected conversion of pyruvate to lactate and acetate is dependent on the presence of intact ldhA and ackA-pta loci, respectively. DNA microarray studies in combination with reporter gene fusion analysis and enzyme activity measurements demonstrated the anr- and ihfA-dependent anaerobic induction of the ackA-pta promoter. Potential Anr and integration host factor binding sites were localized. Pyruvate-dependent anaerobic long-term survival was found to be significantly reduced in anr and ihfA mutants. No obvious ldhA regulation by oxygen tension was observed. Pyruvate fermentation is pH dependent. Nitrate respiration abolished pyruvate fermentation, while arginine fermentation occurs independently of pyruvate utilization.     

Down-regulation of acetate pathway through antisense strategy in Escherichia coli: improved foreign protein production

A problem with the use of Escherichia coli to produce foreign proteins is that although endogenously produced acetate is physiologically indispensable, it inhibits protein expression. Here we firstly employed an antisense RNA strategy as an elaborate metabolic engineering tool to partially block biosynthesis of two major acetate pathway enzymes, phosphotransacetylase (PTA) and acetate kinase (ACK). Three recombinant plasmids containing antisense genes targeting either or both of pta and ackA were constructed, and their effects on the acetate pathway and foreign protein productivity compared to control plasmid without any antisense genes were determined in E. coli BL21. Green fluorescent protein (GFP) was employed as a model foreign protein, and timing of antisense expression was controlled by using the intrinsic ackA promoter. We found that the antisense method partially reduced mRNA levels of target enzyme genes and, over time, lowered the concentration of acetate in culture media in all antisense-regulated strains. Notably, total production of GFP was enhanced 1.6- to 2.1-fold in antisense-regulated strains, even though the degree of acetate reduction was not significantly large. It was revealed that the acetate pathway has more critical roles in cellular physiology than expected in the previous reports. When the scale of culture was increased, enhancement of protein production became larger, demonstrating that this antisense strategy can be successfully applied to practical large-scale protein production processes.     

Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila.

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.     

The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli.

Mutants of Escherichia coli K12 have been isolated that grow on media containing pyruvate of proline as sole carbon sources despite the presence of 10 or 50 mM-sodium fluoroacetate. Such mutants lack either acetate kinase [ATP: acetate phosphotransferase; EC 2.7.2.1] or phosphotransacetylase [acetyl-CoA: orthophosphate acetyltransferase; EC 2.3.1.8] activity. Unlike wild-type E. coli, phosphotransacetylase mutants do not excrete acetate when growing aerobically or anaerobically on glucose; their anaerobic growth on this sugar is slow. The genes that specify acetate kinase (ack) and phosphotransacetylase (pta) activities are cotransducible with each other and with purF and are thus located at about min 50 on the E. coli linkage map. Although Pta- and Ack- mutants are greatly impaired in their growth on acetate, they incorporate [2-14C]acetate added to cultures growing on glycerol, but not on glucose. An inducible acetyl-CoA synthetase [acetate: CoA ligase (AMP-forming); EC 6.2.1.1] effects this uptake of acetate.     

Isolation and Characterization of acetate kinase and phosphotransacetylase mutants of Escherichia coli and Salmonella typhimurium.

Mutations in the ack (acetate kinase) and pta (phosphotransacetylase) genes in Salmonella typhimurium were characterized and determined to be analogous to those of previously described Escherichia coli mutants. We established that in both bacterial species these genes were cotransducible with the neighboring histidine transport operon and were distally located relative to purF. pta mutants were sensitive to the dye alizarin yellow and were unable to grow on medium containing inositol as a carbon source. We selected mutants of both species with deletions covering both the ack and the pta genes; some deletions extended into the histidine transport operon.     

Evaluation of Genetic Manipulation Strategies on d-Lactate Production by Escherichia coli

In order to rationally manipulate the cellular metabolism of Escherichia coli for D: -lactate production, single-gene and multiple-gene deletions with mutations in acetate kinase (ackA), phosphotransacetylase (pta), phosphoenolpyruvate synthase (pps), pyruvate formate lyase (pflB), FAD-binding D-lactate dehydrogenase (dld), pyruvate oxidase (poxB), alcohol dehydrogenase (adhE), and fumarate reductase (frdA) were tested for their effects in two-phase fermentations (aerobic growth and oxygen-limited production). Lactate yield and productivity could be improved by single-gene deletions of ackA, pta, pflB, dld, poxB, and frdA in the wild type E. coli strain but were unfavorably affected by deletions of pps and adhE. However, fermentation experiments with multiple-gene mutant strains showed that deletion of pps in addition to ackA-pta deletions had no effect on lactate production, whereas the additional deletion of adhE in E. coli B0013-050 (ackA-pta pps pflB dld poxB) increased lactate yield. Deletion of all eight genes in E. coli B0013 to produce B0013-070 (ackA-pta pps pflB dld poxB adhE frdA) increased lactate yield and productivity by twofold and reduced yields of acetate, succinate, formate, and ethanol by 95, 89, 100, and 93%, respectively. When tested in a bioreactor, E. coli B0013-070 produced 125 g/l D-lactate with an increased oxygen-limited lactate productivity of 0.61 g/g h (2.1-fold greater than E. coli B0013). These kinetic properties of D-lactate production are among the highest reported and the results have revealed which genetic manipulations improved D-lactate production by E. coli.     

Eliminating side products and increasing succinate yields in engineered strains of Escherichia coli C

Derivatives of Escherichia coli C were previously described for succinate production by combining the deletion of genes that disrupt fermentation pathways for alternative products (ldhA::FRT, adhE::FRT, ackA::FRT, focA-pflB::FRT, mgsA, poxB) with growth-based selection for increased ATP production. The resulting strain, KJ073, produced 1.2 mol of succinate per mol glucose in mineral salts medium with acetate, malate, and pyruvate as significant co-products. KJ073 has been further improved by removing residual recombinase sites (FRT sites) from the chromosomal regions of gene deletion to create a strain devoid of foreign DNA, strain KJ091(DeltaldhA DeltaadhE DeltaackA DeltafocA-pflB DeltamgsA DeltapoxB). KJ091 was further engineered for improvements in succinate production. Deletion of the threonine decarboxylase (tdcD; acetate kinase homologue) and 2-ketobutyrate formate-lyase (tdcE; pyruvate formate-lyase homologue) reduced the acetate level by 50% and increased succinate yield (1.3 mol mol(-1) glucose) by almost 10% as compared to KJ091 and KJ073. Deletion of two genes involved in oxaloacetate metabolism, aspartate aminotransferase (aspC) and the NAD(+)-linked malic enzyme (sfcA) (KJ122) significantly increased succinate yield (1.5 mol mol(-1) glucose), succinate titer (700 mM), and average volumetric productivity (0.9 g L(-1) h(-1)). Residual pyruvate and acetate were substantially reduced by further deletion of pta encoding phosphotransacetylase to produce KJ134 (DeltaldhA DeltaadhE DeltafocA-pflB DeltamgsA DeltapoxB DeltatdcDE DeltacitF DeltaaspC DeltasfcA Deltapta-ackA). Strains KJ122 and KJ134 produced near theoretical yields of succinate during simple, anaerobic, batch fermentations using mineral salts medium. Both may be useful as biocatalysts for the commercial production of succinate.     

A defect in the acetyl coenzyme A<-->acetate pathway poisons recombinational repair-deficient mutants of Escherichia coli

Recombinational repair-dependent mutants identify ways to avoid chromosomal lesions. Starting with a recBC(Ts) strain of Escherichia coli, we looked for mutants unable to grow at 42 degrees C in conditions that inactivate the RecBCD(Ts) enzyme. We isolated insertions in ackA and pta, which comprise a two-gene operon responsible for the acetate<-->acetyl coenzyme A interconversion. Using precise deletions of either ackA or pta, we showed that either mutation makes E. coli cells dependent on RecA or RecBCD enzymes at high temperature, suggesting dependence on recombinational repair rather than on the RecBCD-catalyzed linear DNA degradation. Complete inhibition of growth of pta/ackA rec mutants was observed only in the presence of nearby growing cells, indicating cross-inhibition. pta rec mutants were sensitive to products of the mixed-acid fermentation of pyruvate, yet none of these substances inhibited growth of the double mutants in low-millimolar concentrations. pta, but not ackA, mutants also depend on late recombinational repair functions RuvABC or RecG. pta/ackA recF mutants are viable, suggesting, together with the inviability of pta/ackA recBC mutants, that chromosomal lesions due to the pta/ackA defect are of the double-strand-break type. We have isolated three insertional suppressors that allow slow growth of pta recBC(Ts) cells under nonpermissive conditions; all three are in or near genes with unknown functions. Although they do not form colonies, ackA rec and pta rec mutants are not killed under the nonpermissive conditions, exemplifying a case of synthetic inhibition rather than synthetic lethality.