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For the efficient production of target metabolites from carbohydrates, syngas, or H 2 -CO 2 by genetically engineered Moorella thermoacetica , the control of acetate production (a main metabolite of M. thermoacetica ) is desired. Although propanediol utilization protein (PduL) was predicted to be a phosphotransacetylase (PTA) involved in acetate production in M. thermoacetica , this has not been confirmed. Our findings described herein directly demonstrate that two putative PduL proteins, encoded by Moth_0864 ( pduL1 ) and Moth_1181 ( pduL2 ), are involved in acetate formation as PTAs. To disrupt these genes, we replaced each gene with a lactate dehydrogenase gene from Thermoanaerobacter pseudethanolicus ATCC 33223 ( T-ldh ). The acetate production from fructose as the sole carbon source by the pduL1 deletion mutant was not deficient, whereas the disruption of pduL2 significantly decreased the acetate yield to approximately one-third that of the wild-type strain. The double-deletion (both pduL genes) mutant did not produce acetate but produced only lactate as the end product from fructose. These results suggest that both pduL genes are associated with acetate formation via acetyl-coenzyme A (acetyl-CoA) and that their disruption enables a shift in the homoacetic pathway to the genetically synthesized homolactic pathway via pyruvate. IMPORTANCE This is the first report, to our knowledge, on the experimental identification of PTA genes in M. thermoacetica and the shift of the native homoacetic pathway to the genetically synthesized homolactic pathway by their disruption on a sugar platform., (Copyright © 2017 American Society for Microbiology.)

In the dental caries pathogen Streptococcus mutans , phosphotransacetylase (Pta) and acetate kinase (Ack) convert pyruvate into acetate with the concomitant generation of ATP. The genes for this pathway are tightly regulated by multiple environmental and intracellular inputs, but the basis for differential expression of the genes for Pta and Ack in S. mutans had not been investigated. Here, we show that inactivation in S. mutans of ccpA or codY reduced the activity of the ackA promoter, whereas a ccpA mutant displayed elevated pta promoter activity. The interactions of CcpA with the promoter regions of both genes were observed using electrophoretic mobility shift and DNase protection assays. CodY bound to the ackA promoter region but only in the presence of branched-chain amino acids (BCAAs). DNase footprinting revealed that the upstream region of both genes contains two catabolite-responsive elements ( cre1 and cre2 ) that can be bound by CcpA. Notably, the cre2 site of ackA overlaps with a CodY-binding site. The CcpA- and CodY-binding sites in the promoter region of both genes were further defined by site-directed mutagenesis. Some differences between the reported consensus CodY binding site and the region protected by S. mutans CodY were noted. Transcription of the pta and ackA genes in the ccpA mutant strain was markedly different at low pH relative to transcription at neutral pH. Thus, CcpA and CodY are direct regulators of transcription of ackA and pta in S. mutans that optimize acetate metabolism in response to carbohydrate, amino acid availability, and environmental pH. IMPORTANCE The human dental caries pathogen Streptococcus mutans is remarkably adept at coping with extended periods of carbohydrate limitation during fasting periods. The phosphotransacetylase-acetate kinase (Pta-Ack) pathway in S. mutans modulates carbohydrate flux and fine-tunes the ability of the organisms to cope with stressors that are commonly encountered in the oral cavity. Here, we show that CcpA controls transcription of the pta and ackA genes via direct interaction with the promoter regions of both genes and that branched-chain amino acids (BCAAs), particularly isoleucine, enhance the ability of CodY to bind to the promoter region of the ackA gene. A working model is proposed to explain how regulation of pta and ackA genes by these allosterically controlled regulatory proteins facilitates proper carbon flow and energy production, which are essential functions during infection and pathogenesis as carbohydrate and amino acid availability continually fluctuate., (Copyright © 2017 American Society for Microbiology.)

We previously reported the metabolic analysis of butanol and acetone production from exogenous acetate by (13)C tracer experiments (Gao et al., RSC Adv., 5, 8486-8495, 2015). To clarify the influence of acetate on acetone-butanol-ethanol (ABE) production, we first performed an enzyme assay in Clostridium saccharoperbutylacetonicum N1-4. Acetate addition was found to drastically increase the activities of key enzymes involved in the acetate uptake (phosphate acetyltransferase and CoA transferase), acetone formation (acetoacetate decarboxylase), and butanol formation (butanol dehydrogenase) pathways. Subsequently, supplementation of acetate during acidogenesis and early solventogenesis resulted in a significant increase in ABE production. To establish an efficient ABE production system using acetate as a co-substrate, several shot strategies were investigated in batch culture. Batch cultures with two substrate shots without pH control produced 14.20 g/L butanol and 23.27 g/L ABE with a maximum specific butanol production rate of 0.26 g/(g h). Furthermore, pH-controlled (at pH 5.5) batch cultures with two substrate shots resulted in not only improved acetate consumption but also a further increase in ABE production. Finally, we obtained 15.13 g/L butanol and 24.37 g/L ABE at the high specific butanol production rate of 0.34 g/(g h) using pH-stat co-feeding method. Thus, in this study, we established a high ABE production system using glucose and acetate as co-substrates in a pH-stat co-feeding system with C. saccharoperbutylacetonicum N1-4., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Although many Archaea have AMP-Acs (acetyl-coenzyme A synthetase) and ADP-Acs, the extant methanogenic genus Methanosarcina is the only identified Archaeal genus that can utilize acetate via acetate kinase (Ack) and phosphotransacetylase (Pta). Despite the importance of ack as the potential urkinase in the ASKHA phosphotransferase superfamily, an origin hypothesis does not exist for the acetate kinase in Bacteria, Archaea, or Eukarya. Here we demonstrate that Archaeal AMP-Acs and ADP-Acs contain paralogous ATPase motifs previously identified in Ack, which demonstrate a novel relation between these proteins in Archaea. The identification of ATPase motif conservation and resulting structural features in AMP- and ADP-acetyl-CoA synthetase proteins in this study expand the ASKHA superfamily to include acetyl-CoA synthetase. Additional phylogenetic analysis showed that Pta and MaeB sequences had a common ancestor, and that the Pta lineage within the halophilc archaea was an ancestral lineage. These results suggested that divergence of a duplicated maeB within an ancient halophilic, archaeal lineage formed a putative pta ancestor. These results provide a potential scenario for the establishment of the Ack/Pta pathway and provide novel insight into the evolution of acetate metabolism for all three domains of life.

In the dental caries pathogen Streptococcus mutans, phosphotransacetylase (Pta) catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate (AcP), which can be converted to acetate by acetate kinase (Ack), with the concomitant generation of ATP. A ΔackA mutant displayed enhanced accumulation of AcP under aerobic conditions, whereas little or no AcP was observed in the Δpta or Δpta ΔackA mutant. The Δpta and Δpta ΔackA mutants also had diminished ATP pools compared to the size of the ATP pool for the parental or ΔackA strain. Surprisingly, when exposed to oxidative stress, the Δpta ΔackA strain appeared to regain the capacity to produce AcP, with a concurrent increase in the size of the ATP pool compared to that for the parental strain. The ΔackA and Δpta ΔackA mutants exhibited enhanced (p)ppGpp accumulation, whereas the strain lacking Pta produced less (p)ppGpp than the wild-type strain. The ΔackA and Δpta ΔackA mutants displayed global changes in gene expression, as assessed by microarrays. All strains lacking Pta, which had defects in AcP production under aerobic conditions, were impaired in their abilities to form biofilms when glucose was the growth carbohydrate. Collectively, these data demonstrate the complex regulation of the Pta-Ack pathway and critical roles for these enzymes in processes that appear to be essential for the persistence and pathogenesis of S. mutans., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes., (© 2014 American Society of Plant Biologists. All rights reserved.)

Acetogen strain Clostridium sp. MT1121 produced 300 mM acetate (p<0.005) and 321 mM ethanol (p<0.005) from synthesis gas (syngas) blend 60 % CO and 40 % H(2). Clostridium sp. MT1121 was metabolically engineered to eliminate production of either acetate or acetaldehyde during syngas fermentation. We used Cre-lox66/lox71-based gene removal system to eliminate either phosphotransacetylase (pta), or acetaldehyde dehydrogenase (aldh). The resulted biocatalyst with eliminated pta increased ethanol yield to 610 mM (p<0.005). Inactivation of pta rendered only 502 mM of ethanol (p<0.005). The acetogen biocatalyst with eliminated aldh produced 450 mM acetate (p<0.005). The role of cell energy pool preservation for re-directed carbon flux is discussed. This is the first report on time- and cost-efficient gene elimination in acetogens using lox66/lox71 gene elimination system.

Microbial biosynthesis of fatty acid like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Wild type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and do not accumulate free fatty acids as intermediates in lipid biosynthesis. However, free fatty acids can be produced by breaking the fatty acid elongation through the overexpression of an acyl-ACP thioesterase. Since acetyl-CoA might be an important factor for fatty acid synthesis (acetate formation pathways are the main competitive pathways in consuming acetyl-CoA or pyruvate, a precursor of acetyl-CoA), and the long chain fatty acid CoA-ligase (FadD) plays a pivotal role in the transport and activation of exogenous fatty acids prior to their subsequent degradation, we examined the composition and the secretion of the free fatty acids in four different strains including the wild type MG1655, a mutant strain with inactivation of the fatty acid beta-oxidation pathway (fadD mutant (ML103)), and mutant strains with inactivation of the two major acetate production pathways (an ack-pta (acetate kinase/phosphotransacetylase), poxB (pyruvate oxidase) double mutant (ML112)) and a fadD, ack-pta, poxB triple mutant (ML115). The engineered E. coli cells expressing acyl-ACP thioesterase with glucose yield is higher than 40% of theoretical yield. Compared to MG1655(pXZ18) and ML103(pXZ18), acetate forming pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar quantity of total free fatty acids, which indicated that acetyl-CoA availability does not appear to be limiting factor for fatty acid production in these strains. However, these strains did show significant differences in the composition of free fatty acids. Different from MG1655(pXZ18) and ML103(pXZ18), acetate formation pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar level of C14, C16:1 and C16 free fatty acids, and the free fatty acid compositions of both strains did not change significantly with time. In addition, the strains bearing the fadD mutation showed significant differences in the quantities of free fatty acids found in the broth. Finally, we examined two potential screening methods for selecting and isolating high free fatty acids producing cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Individual deletions of acs and aceA genes in E. coli B (BL21) showed little difference in the metabolite accumulation patterns but deletion of the ackA gene alone or together with pta showed acetic acid gradually accumulated to 3.1 and 1.7 g/l, respectively, with a minimal extended lag in bacterial growth and a higher pyruvate formation. Single poxB deletion in E. coli B (BL21) or additional poxB deletion in the ackA-pta mutants did not change the acetate accumulation pattern. When the acetate production genes (ackA-pta-poxB) were deleted in E. coli B (BL21) acetate still accumulated. This may be an indication that perhaps acetate is not only a by-product of carbon metabolism; it is possible that acetate plays also a role in other cellular metabolite pathways. It is likely that there are alternative acetate production pathways.

Background: The biotechnology industry has extensively exploited Escherichia coli for producing recombinant proteins, biofuels etc. However, high growth rate aerobic E. coli cultivations are accompanied by acetate excretion i.e. overflow metabolism which is harmful as it inhibits growth, diverts valuable carbon from biomass formation and is detrimental for target product synthesis. Although overflow metabolism has been studied for decades, its regulation mechanisms still remain unclear., Results: In the current work, growth rate dependent acetate overflow metabolism of E. coli was continuously monitored using advanced continuous cultivation methods (A-stat and D-stat). The first step in acetate overflow switch (at μ = 0.27 ± 0.02 h(-1)) is the repression of acetyl-CoA synthetase (Acs) activity triggered by carbon catabolite repression resulting in decreased assimilation of acetate produced by phosphotransacetylase (Pta), and disruption of the PTA-ACS node. This was indicated by acetate synthesis pathways PTA-ACKA and POXB component expression down-regulation before the overflow switch at μ = 0.27 ± 0.02 h(-1) with concurrent 5-fold stronger repression of acetate-consuming Acs. This in turn suggests insufficient Acs activity for consuming all the acetate produced by Pta, leading to disruption of the acetate cycling process in PTA-ACS node where constant acetyl phosphate or acetate regeneration is essential for E. coli chemotaxis, proteolysis, pathogenesis etc. regulation. In addition, two-substrate A-stat and D-stat experiments showed that acetate consumption capability of E. coli decreased drastically, just as Acs expression, before the start of overflow metabolism. The second step in overflow switch is the sharp decline in cAMP production at μ = 0.45 h(-1) leading to total Acs inhibition and fast accumulation of acetate., Conclusion: This study is an example of how a systems biology approach allowed to propose a new regulation mechanism for overflow metabolism in E. coli shown by proteomic, transcriptomic and metabolomic levels coupled to two-phase acetate accumulation: acetate overflow metabolism in E. coli is triggered by Acs down-regulation resulting in decreased assimilation of acetic acid produced by Pta, and disruption of the PTA-ACS node.

Background: Acetate metabolism in Escherichia coli plays an important role in the control of the central metabolism and in bioprocess performance. The main problems related to the use of E. coli as cellular factory are i) the deficient utilization of carbon source due to the excretion of acetate during aerobic growth, ii) the inhibition of cellular growth and protein production by acetate and iii) the need for cofactor recycling (namely redox coenzymes and free CoASH) to sustain balanced growth and cellular homeostasis., Results: This work analyzes the effect of mutations in the acetate excretion/assimilation pathways, acetyl-CoA synthethase (acs) and phosphotransacetylase (pta), in E. coli BW25113 grown on glucose or acetate minimal media. Biomass and metabolite production, redox (NADH/NAD+) and energy (ATP) state, enzyme activities and gene expression profiles related to the central metabolism were analyzed. The knock-out of pta led to a more altered phenotype than that of acs. Deletion of pta reduced the ability to grow on acetate as carbon source and strongly affected the expression of several genes related to central metabolic pathways., Conclusion: Results showed that pta limits biomass yield in aerobic glucose cultures, due to acetate production (overflow metabolism) and its inefficient use during glucose starvation. Deletion of pta severely impaired growth on acetate minimal medium and under anaerobiosis due to decreased acetyl-coenzyme A synthethase, glyoxylate shunt and gluconeogenic activities, leading to lower growth rate. When acetate is used as carbon source, the joint expression of pta and acs is crucial for growth and substrate assimilation, while pta deletion severely impaired anaerobic growth. Finally, at an adaptive level, pta deficiency makes the strain more sensitive to environmental changes and de-regulates the central metabolism.

In vivo accumulation of D-serine by Escherichia coli CFT073 leads to elevated expression of PAP fimbriae and hemolysin by an unknown mechanism. Loss of D-serine catabolism by CFT073 leads to a competitive advantage during murine urinary tract infection (UTI), but loss of both D- and L-serine catabolism results in attenuation. Serine is the first amino acid to be consumed in closed tryptone broth cultures and precedes the production of acetyl phosphate, a high-energy molecule involved in intracellular signaling, and the eventual secretion of acetate. We propose that the colonization defect associated with the loss of serine catabolism is due to perturbations of acetate metabolism. CFT073 grows more rapidly on acetogenic substrates than does E. coli K-12 isolate MG1655. As shown by transcription microarray results, D-serine is catabolized into acetate via the phosphotransacetylase (pta) and acetate kinase (ackA) genes while downregulating expression of acetyl coenzyme A synthase (acs). CFT073 acs, which is unable to reclaim secreted acetate, colonized mouse bladders and kidneys in the murine model of UTI indistinguishably from the wild type. Both pta and ackA are involved in the maintenance of intracellular acetyl phosphate. CFT073 pta and ackA mutants were screened to investigate the role of acetyl phosphate in UTI pathogenesis. Both single mutants are at a competitive disadvantage relative to the wild type in the kidneys but normally colonize the bladder. CFT073 ackA pta was attenuated in both the bladder and the kidneys. Thus, we demonstrate that CFT073 is adapted to acetate metabolism as a result of requiring a proper cycling of the acetyl phosphate pathway for colonization of the upper urinary tract.

A tightly regulated gene expression system composed of a single-copy target gene under the control of a lac promoter derivative and lacI gene in a multicopy plasmid is proposed, and its ability to control the flux of a metabolic pathway is demonstrated. A model system to control the flux of acetyl-CoA to acetyl phosphate was constructed by integrating pta, a gene encoding phosphotransacetylase, under a tac promoter into the chromosome of E. coli with a pta-negative background and transforming a multicopy plasmid containing the lac iota(Q) gene into the strain. The production rate of acetate was shown to be tightly controlled when varying the concentration of the inducer (IPTG) in the model system.

Corynebacterium glutamicum R efficiently produces valuable chemicals from glucose under oxygen-deprived conditions. In an effort to reduce acetate as a byproduct, acetate productivity of several mutant-disrupted genes encoding possible key enzymes for acetate formation was determined. Disruption of the aceE gene that encodes the E1 enzyme of the pyruvate dehydrogenase complex resulted in almost complete elimination of acetate formation under oxygen-deprived conditions, implying that acetate synthesis under these conditions was essentially via acetyl-coenzyme A (CoA). Simultaneous disruption of pta, encoding phosphotransacetylase, and ack, encoding acetate kinase, resulted in no measurable change in acetate productivity. A mutant strain with disruptions in pta, ack and as-yet uncharacterized gene (cgR2472) exhibited 65% reduced acetate productivity compared to the parental strain, although a single disruption of cgR2472 exhibited no effect on acetate productivity. The gene cgR2472 was shown to encode a CoA-transferase (CTF) that catalyzes the formation of acetate from acetyl-CoA. These results indicate that PTA-ACK as well as CTF is involved in acetate production in C. glutamicum. This study provided basic information to reduce acetate production under oxygen-deprived conditions.

This report shows that Salmonella enterica catabolizes ethanolamine to acetyl-CoA (Ac-CoA), which enters the glyoxylate bypass and tricarboxylic acid cycle for the generation of energy and central metabolites. During growth on ethanolamine, S. enterica excreted acetate, whose recapture depended on Ac-CoA synthetase (Acs) and the housekeeping phosphotransacetylase (Pta) enzyme activities. The Pta enzyme did not play a role in acetate excretion during growth of S. enterica on ethanolamine. It is proposed that during growth on ethanolamine, acetate excretion is necessary to maintain a pool of free CoA. Acetate excretion requires the eut operon-encoded phosphotransacetylase (EutD) and acetate kinase (Ack) enzymes. EutD function was not required for growth on ethanolamine, and an eutD strain showed only a slight reduction in growth rate. The existence of an as-yet-unidentified system that releases acetate was revealed during growth of a strain lacking Acs, the housekeeping phosphotransacetylase (Pta), and EutD. The functions of pyruvate oxidase (PoxB), Ack and STM3118 protein [a homologue of the Saccharomyces cerevisiae Ac-CoA hydrolase (Ach1p) enzyme] were not involved in the release of acetate by the acs pta eutD strain.

Although the bacterium E. coli is chosen as the host in many bioprocesses, the accumulation of a common byproduct, acetate, is often problematic. Acetate, when present at high levels, will inhibit both cell growth and recombinant protein productivity. In addition, products derived from the central aerobic metabolic pathway often compete with the acetate-producing pathways poxB and ackA-pta for glucose as the substrate. As such, a significant portion of the glucose may be excreted as acetate, wasting substrate that otherwise could have been used for the desired product. We have created mutant E. coli strains with a deletion of either the poxB or the ackA-pta pathway. These two strains, along with the wild-type strain, have been studied in batch reactors over a 12 h time period, at pH 7.0 and 6.0. The wild-type strain has also been studied using glucose as the carbon source. Data were collected to correlate cellular growth, extracellular metabolite production, enzyme activity, and gene expression. Results show that the ackA-pta pathway dominates in exponential phase, and the poxB pathway dominates in stationary phase. The ackA-pta pathway is repressed in acidic environments, whereas the poxB pathway is activated.

During growth of Corynebacterium glutamicum on acetate as its carbon and energy source, the expression of the pta-ack operon is induced, coding for the acetate-activating enzymes, which are phosphotransacetylase (PTA) and acetate kinase (AK). By transposon rescue, we identified the two genes amrG1 and amrG2 found in the deregulated transposon mutant C. glutamicum G25. The amrG1 gene (NCBI-accession: AF532964) has a size of 732 bp, encoding a polypeptide of 243 amino acids and apparently is partially responsible for the regulation of acetate metabolism in C. glutamicum. We constructed an in-frame deletion mutant and an over-expressing strain of amrG1 in the C. glutamicum ATCC13032 wildtype. The strains were then analyzed with respect to their enzyme activities of PTA and AK during growth on glucose, acetate and glucose or acetate alone as carbon sources. Compared to the parental strain, the amrG1 deletion mutant showed higher specific AK and PTA activities during growth on glucose but showed the same high specific activities of AK and PTA on medium containing acetate plus glucose and on medium containing acetate. In contrast to the gene deletion, overexpression of the amrG1 gene in C. glutamicum 13032 had the adverse regulatory effect. These results indicate that the amrG1 gene encodes a repressor or co-repressor of the pta-ack operon.

In order to study the physiological role of acetate metabolism in Escherichia coli, the growth characteristics of an E. coli W3100 pta mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated. The pta mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate, D-lactate, and L-glutamate instead of acetate. In an analysis of the sequential consumption of amino acids by the pta mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain. The pta mutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate. The defective growth and starvation survival of the pta mutant were restored by the introduction of poly-beta-hydroxybutyrate (PHB) synthesis genes (phbCAB) from Alcaligenes eutrophus, indicating that the growth defect of the pta mutant was due to a perturbation of acetyl coenzyme A (CoA) flux. By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. These results suggest that E. coli excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the pta mutant.

In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes (aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum.

The rate of acid production and end-products from sorbitol were measured under anaerobic conditions in washed-cell suspensions of oral strains of Lactobacillus casei subsp. casei and Lactobacillus casei subsp. rhamnosus. The enzymatic activities were assayed in cell extracts of these strains. The cells fermented sorbitol to lactate, formate, ethanol and acetate under anaerobic conditions. Exposure of the cells to air (oxygen) led to inactivation of pyruvate formate-lyase and inhibition of anaerobic sorbitol fermentation. In the presence of acetate, air-exposed cells fermented sorbitol with a concomitant consumption of acetate and production of ethanol and lactate. Acetate also enhanced acid production from sorbitol in cells kept under anaerobic conditions and resulted in formation of lactate and ethanol. Cell extracts of all the strains had NADH-coupled acetate-reducing activity, which consisted of sequential reactions of acetate kinase, phosphotransacetylase, acylating aldehyde dehydrogenase and alcohol dehydrogenase. These findings indicate that oral lactobacilli can utilize acetate as an electron acceptor for maintaining their intracellular redox balance during anaerobic sorbitol fermentation in the absence of pyruvate formate-lyase activity.

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.

A five-component enzyme system which catalyzes synthesis of acetylphosphate from methyltetrahydrofolate (CH3THF) plus pyruvate has been purified from the homoacetate-fermenting bacterium, Clostridium thermoaceticum. One of the components was identified as the low potential electron carrier, ferredoxin, and the other 4 protein components have been designated F1, F2, F3, and F4. F1, F2, and F4 have been purified to homogeneity and, as estimated by gel filtration, have native molecular weights of 88,100, 58,900, and 255,000, respectively, while the subunit molecular weights obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis are 20,000, 25,500, and 120,000, respectively. F3 contains 3 to 4 protein bands and has not been characterized with respect to molecular weights. Acetylphosphate synthesis by the purified system is optimal at pH 6.0 and 65 degrees C and requires ATP, CoA, and, to a lesser extent, thiamin pyrophosphate and Fe2+. S-Adenosylmethionine is not required. The F1 component has been identified as phosphotransacetylase and in its absence, the product is acetyl-CoA. Some properties of the phosphotransacetylase are presented. A scheme is given indicating present views of the functions of the individual components.

Phosphotransacetylase (EC 2.3.1.8) was purified 83-fold to a specific activity of 2.5 mmol of acetyl-CoA synthesized per min/mg of protein from Methanosarcina thermophila cultivated on acetate. This rate was 10-fold greater than the rate of acetyl phosphate synthesis. The native enzyme (Mr 42,000-52,000) was a monomer and was not integral to the membrane. Activity was optimum at pH 7.0, and 35-45 degrees C. The enzyme was stable to air and to temperatures up to 70 degrees C, but was inactivated at higher temperatures. Phosphate and sulfate partially protected against heat inactivation. Potassium or ammonium ion concentrations above 10 mM were required for maximum activity of the purified enzyme; the intracellular potassium concentration of M. thermophila approximated 175 mM. Sodium, phosphate, sulfate, and arsenate ions were inhibitory to enzyme activity. Western blots of cell extracts showed that phosphotransacetylase was synthesized in higher quantity in acetate-grown cells than in methanol-grown cells.

The carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila was further studied by EPR spectroscopy. The as purified enzyme exhibited no paramagnetic species at 113 K; however, enzyme reduced with CO exhibited a complex EPR spectrum comprised of two paramagnetic species with g values of g1 = 2.089, g2 = 2.078, and g3 = 2.030 (signal I) and g1 = 2.057, g2 = 2.049, and g3 = 2.027 (signal II). Isotopic substitution with 61Ni, 57Fe, or 13CO resulted in broadening of the EPR spectra indicating a Ni-Fe-C spin-coupled complex. Pure signal II was obtained following treatment of the CO-reduced enzyme with acetyl-CoA but not by addition of acetyl phosphate or CoASH. Acetate-grown cells were highly enriched in acetate kinase (EC 2.7.2.1) and CoASH-dependent phosphotransacetylase (EC 2.3.1.8) activities. These results suggest acetyl-CoA is a physiological substrate for the carbon monoxide dehydrogenase complex synthesized in acetate-grown cells of M. thermophila.

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.