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The heterofermentative lactic acid bacterium Oenococcus oeni requires pantothenic acid for growth. In the presence of sufficient pantothenic acid, glucose was converted by heterolactic fermentation stoichiometrically to lactate, ethanol and CO2. Under pantothenic acid limitation, substantial amounts of erythritol, acetate and glycerol were produced by growing and resting bacteria. Production of erythritol and glycerol was required to compensate for the decreasing ethanol production and to enable the synthesis of acetate. In ribose fermentation, there were no shifts in the fermentation pattern in response to pantothenate supply. In the presence of pantothenate, growing O. oeni contained at least 10.2 microM HSCoA, whereas the HSCoA content was tenfold lower after growth in pantothenate-depleted media. HSCoA and acetyl-CoA are cosubstrates of phosphotransacetylase and acetaldehyde dehydrogenase from the ethanol pathway. Both enzymes were found with activities commensurate with their function in ethanol production during heterolactic fermentation. From the kinetic data of the enzymes and the HSCoA and acetyl-CoA contents, it can be calculated that, under pantothenate limitation, phosphotransacetylase, and in particular acetaldehyde dehydrogenase activities become limiting due to low levels of the cosubstrates. Thus HSCoA deficiency represents the major limiting factor in heterolactic fermentation of glucose under pantothenate deficiency and the reason for the shift to erythritol, acetate, and glycerol fermentation.

The phosphotransacetylase (PTA) (EC 2.3.1.8) catalyzes a key branch point reaction in the carbohydrate pathway of Lactobacillus sanfranciscensis. In this report, we describe the cloning of the pta gene. The DNA sequence analysis revealed a 987 bp open reading frame encoding a protein with a molecular mass of 35.5 kD. These are the first studies on a PTA of an organism representative for the heterofermentative lactic acid bacteria. Unlike in most other bacteria analysed so far, in L. sanfranciscensis the pta gene is not adjacent located to the gene encoding acetate-kinase. The PTA was heterologously expressed as a biotinylated fusion protein in E. coli and purified to homogeneity. Rate dependence on all substrates followed Michaelis-Menten kinetics. The apparent Km values for acetylphosphate and CoA (forward reaction) were 1.3 and 0.1 mM, respectively. The apparent Vmax was 194 U/mg. The enzyme also catalyzed in vitro the reverse reaction with apparent Km values for acetylCoA and phosphate of 0.6 and 6.7 mM, respectively (Vmax of 38 U/mg). The PTA showed a wide range of temperature for optimal activity (49 degrees C to 58 degrees C). It was inactivated after 15 min at 60 degrees C. Its activity was not affected by addition of MgCl2 (10 mM) or KCl (100 mM).

Active butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) were purified in three steps: ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on Matrex Red A from recombinant Escherichia coli K2006 (pJC7). They were then successfully exploited for in vitro synthesis of 3-hydroxybutyryl-CoA (3HBCoA), 4-hydroxybutyryl-CoA (4HBCoA), 4-hydroxyvaleryl-CoA (4HVCoA) and poly(hydroxyalkanoic acid) (PHA). In addition, the ability of the PHA synthase of Chromatium vinosum, PhaEC(Cv), to use these CoA thioesters was evaluated. Combination of Buk and Ptb with PhaEC(Cv) established a new system for in vitro synthesis of poly(3-hydroxybutyric acid) [poly(3HB)]. In this system, 3-hydroxybutyric acid was converted to 3HBCoA by Buk and Ptb at the expense of ATP. Formation of 3HBCoA was further driven by the polymerization of 3HBCoA molecules to poly(3HB) by PHA synthase, and the released CoA was recycled by Ptb. This system therefore also ensured the regeneration of CoA. With ATP as the energy supply, which was hydrolyzed to ADP and phosphate, 2.6 mg poly(3HB) was obtained from a 1-ml reaction mixture containing 7.6 mg 3-hydroxybutyrate at the beginning. Studies showed that Ptb and PHA synthase were the rate-limiting steps in this system, and initial CoA concentrations ranging from 1 to 7 mM did not inhibit poly(3HB) synthesis. Synthesis of various polyesters of 3HB and 4HB with this system was also tested, and copolyesters containing 4HB of 1-46 mol % were obtained.

A new pathway to synthesize poly(hydroxyalkanoic acids) (PHA) was constructed by simultaneously expressing butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) genes of Clostridium acetobutylicum and the two PHA synthase genes (phaE and phaC) of Thiocapsa pfennigii in Escherichia coli. The four genes were cloned into the BamHI and EcoRI sites of pBR322, and the resulting hybrid plasmid, pBPP1, conferred activities of all three enzymes to E. coli JM109. Cells of this recombinant strain accumulated PHAs when hydroxyfatty acids were provided as carbon sources. Homopolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), or 4-hydroxyvalerate (4HV) were obtained from each of the corresponding hydroxyfatty acids. Various copolyesters of those hydroxyfatty acids were also obtained when two of these hydroxyfatty acids were fed at equal amounts: cells fed with 3HB and 4HB accumulated a copolyester consisting of 88 mol% 3HB and 12 mol% 4HB and contributing to 68.7% of the cell dry weight. Cells fed with 3HB and 4HV accumulated a copolyester consisting of 94 mol% 3HB and 6 mol% 4HV and contributing to 64.0% of the cell dry weight. Cells fed with 3HB, 4HB, and 4HV accumulated a terpolyester consisting of 85 mol% 3HB, 13 mol% 4HB, and 2 mol% 4HV and contributing to 68.4% of the cell dry weight.

In the cultivation of gene engineered strain of Escherichia coli on glucose medium, excretion and accumulation of acetic acid inhibit not only cell growth but also the the expression of heterologous protein. It is obvious that the desirable host strain maintaining acetate at a low level is one of the approaches to increase the production of recombinant protein. The present article deals with the selection of mutants of E. coli DP19, DP8, which grow on the medium containing pyruvate as the sole carbon source in the presence of 50 mmol/L fluoroacetic acid. It is shown that mutant DP19 is defective in its phosphotransacetylase(PTA) activity and accumulates less acetate in the medium, while DP8 is defective in acetate kinase (ACK) and accumulates similar level of acetate comparing with its parent. Using pta- mutant E. coli DP19 as host, the expression of GL-7ACA acylase gene on the recombinant plasmid pMR24 is improved, and the yield of enzyme activity in flask fermentation is about twice as much as its parent.

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 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.

We investigated metabolic engineering of fermentation pathways in Escherichia coli for production of optically pure D- or L-lactate. Several pta mutant strains were examined, and a pta mutant of E. coli RR1 which was deficient in the phosphotransacetylase of the Pta-AckA pathway was found to metabolize glucose to D-lactate and to produce a small amount of succinate by-product under anaerobic conditions. An additional mutation in ppc made the mutant produce D-lactate like a homofermentative lactic acid bacterium. When the pta ppc double mutant was grown to higher biomass concentrations under aerobic conditions before it shifted to the anaerobic phase of D-lactate production, more than 62.2 g of D-lactate per liter was produced in 60 h, and the volumetric productivity was 1.04 g/liter/h. To examine whether the blocked acetate flux could be reoriented to a nonindigenous L-lactate pathway, an L-lactate dehydrogenase gene from Lactobacillus casei was introduced into a pta ldhA strain which lacked phosphotransacetylase and D-lactate dehydrogenase. This recombinant strain was able to metabolize glucose to L-lactate as the major fermentation product, and up to 45 g of L-lactate per liter was produced in 67 h. These results demonstrate that the central fermentation metabolism of E. coli can be reoriented to the production of D-lactate, an indigenous fermentation product, or to the production of L-lactate, a nonindigenous fermentation product.

Phosphate acetyltransferase (PTA) and acetate kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (alpha4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from Clostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi -->/<-- acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5. The apparent Km values for acetyl-CoA, Pi, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 microM, respectively; the apparent Vmax values (at 55 degrees C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation). In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%). The enzyme had a temperature optimum at 90 degrees C and was not inactivated by heat upon incubation at 80 degrees C for more than 2 h. AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (alpha2) structure. The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon Methanosarcina thermophila. The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP -->/<-- acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent Vmax values (at 50 degrees C) were 2,600 U/mg (acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90 degrees C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4 (1 M), which was most effective, the enzyme did not lose activity upon incubation at 100 degrees C for 3 h. The temperature optimum at 90 degrees C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.

We examined the effectiveness of antisense RNA (as RNA) strategies for metabolic engineering of Clostridium acetobutylicum. Strain ATCC 824(pRD4) was developed to produce a 102-nucleotide asRNA with 87% complementarity to the butyrate kinase (BK) gene. Strain ATCC 824(pRD4) exhibited 85 to 90% lower BK and acetate kinase specific activities than the control strain. Strain ATCC 824(pRD4) also exhibited 45 to 50% lower phosphotransbutyrylase (PTB) and phosphotransacetylase specific activities than the control strain. This strain exhibited earlier induction of solventogenesis, which resulted in 50 and 35% higher final concentrations of acetone and butanol, respectively, than the concentrations in the control. Strain ATCC 824(pRD1) was developed to putatively produce a 698-nucleotide asRNA with 96% complementarity to the PTB gene. Strain ATCC 824(pRD1) exhibited 70 and 80% lower PTB and BK activities, respectively, than the control exhibited. It also exhibited 300% higher levels of a lactate dehydrogenase activity than the control exhibited. The growth yields of ATCC 824(pRD1) were 28% less than the growth yields of the control. While the levels of acids were not affected in ATCC 824(pRD1) fermentations, the acetone and butanol concentrations were 96 and 75% lower, respectively, than the concentrations in the control fermentations. The lower level of solvent production by ATCC 824(pRD1) was compensated for by approximately 100-fold higher levels of lactate production. The lack of any significant impact on butyrate formation fluxes by the lower PTB and BK levels suggests that butyrate formation fluxes are not controlled by the levels of the butyrate formation enzymes.

The Corynebacterium glutamicum ack and pta genes encoding the acetate-activating enzymes acetate kinase and phosphotransacetylase were isolated, subcloned on a plasmid and re-introduced into Corynebacterium glutamicum. Relative to the wild-type, the recombinant strains showed about tenfold higher specific activities of both enzymes. Sequence analysis of a 3657 bp DNA fragment revealed that the ack and pta genes are contiguous in the corynebacterial chromosome, with pta upstream and the last nucleotide of the pta stop codon (TAA) overlapping the first of the ack start codon (ATG). The predicted gene product of pta consists of 329 amino acids (Mr 35242), that of ack consists of 397 amino acids (Mr 43098) and the amino acid sequences of the two polypeptides show up to 60 % (phosphotransacetylase) and 53% (acetate kinase) identity in comparison with respective enzymes from other organisms. Northern (RNA) blot hybridizations using pta- and ack-specific probes and transcriptional cat fusion experiments revealed that the two genes are transcribed as a 2.5 kb bicistronic mRNA and that the expression of this operon is induced when Corynebacterium glutamicum grows on acetate instead of glucose as a carbon source. Directed inactivation of the chromosomal pta and ack genes led to the absence of detectable phosphotransacetylase and acetate kinase activity in the respective mutants and to their inability to grow on acetate. These data indicate that no isoenzymes of acetate kinase and phosphotransacetylase are present in Corynebacterium glutamicum and that a functional acetate kinase/phosphotransacetylase pathway is essential for growth of this organism on acetate.

Carbon sources that can be converted to acetate were added to the growth medium of Escherichia coli wild-type cells. Cells responded with an increased cell division rate. The addition of acetate also caused a decreased synthesis of flagella. Mutants in phosphotransacetylase, which are incapable of synthesizing acetyl phosphate, and mutants in the osmoregulator OmpR divided at a lower rate than did wild-type cells. The mutants did not increase their cell division rate upon the addition of serine, as observed for wild-type cells. These data are consistent with the idea that the previously described effect of serine upon the cell division rate is mediated by acetyl phosphate and phosphorylation of OmpR.

Phosphotransacetylase catalyzes the following reaction: CoASH + CH3CO2PO3(2-) <==> CH3COSCoA + HPO4(2-) (where CoA is coenzyme A). Based on biochemical characterization of the enzyme from the obligate anaerobe Clostridium kluyveri, a ternary mechanism was proposed in which an unspecified cysteine abstracts a proton from CoASH forming a nucleophilic thiolate anion which attacks acetyl phosphate (J. Henkin and R. H. Abeles, Biochemistry 15:3472-3479, 1976). Heterologous production in Escherichia coli of the phosphotransacetylase from Methanosarcina thermophila, an obligately anaerobic methanoarchaeon, allowed site-specific replacements to identify essential residues. All four cysteines present in the sequence were individually replaced with alanine, and the kinetic constants of the altered enzymes were determined. The results indicated that only C159 is essential for activity; however, replacement with serine resulted in a fully active enzyme. Activity of the unaltered phosphotransacetylase was sensitive to N-ethylmaleimide. Inhibition kinetics of altered enzymes indicated that this sensitivity resulted from modification of C312, which is at the active site but itself is nonessential for catalysis. Five arginines were individually replaced with glutamine. Kinetic analysis of the altered enzymes identified R310 as essential for activity. Of the four nonessential for activity, R87 and R133 appear to be involved in binding CoA.

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.

Poly-beta-hydroxybutyrate (PHB) synthesis in a cyanobacterium, Synechococcus sp. strain MA19, is controlled at the enzyme level and is dependent on the C/N balance in the culture medium. The control involves at least two enzymes. The first enzyme is PHB synthase. Little PHB synthase activity was detected in crude extracts from cells grown under nitrogen-sufficient conditions (MA19(+N)). The activity was detected exclusively in membrane fractions from nitrogen-deprived cells (MA19(-N)) under light but not dark conditions. The shift in the enzyme activity was insensitive to chloramphenicol, which suggests posttranslational activation. Acetyl phosphate activated PHB synthase in membrane fractions from MA19(+N). In vitro, the activation level of PHB synthase changed, depending on the concentration of acetyl phosphate. The second enzyme was phosphotransacetylase (EC 2.3.1.8), which catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate. The activity was detected in crude extracts from MA19(-N) but not in those from MA19(+N). The results suggested that intracellular acetyl phosphate concentration could be controlled, depending on C/N balance and intracellular acetyl-CoA concentration. Acetyl phosphate probably acts as a signal of C/N balance affecting PHB metabolism in MA19.

Biologically-produced CH4 derives from either the reduction of CO2 or the methyl group of acetate by two separate pathways present in anaerobic mierobes from the Archaea domain. Elucidation of the pathway for CO2 reduction to CH4, the first to be investigated, has yielded several novel enzymes and cofactors. Most of the CH4 produced in nature derives from the methyl group of acetate. Methanosarcina thermophila is a moderate thermophile which ferments acetate by reducing the methyl group to CH4 with electrons derived from oxidation of the carbonyl group to CO2. The pathway in M. thermophila is now understood on a biochemical and genetic level comparable to understanding of the CO2-reducing pathway. Enzymes have been purified and characterized. The genes encoding these enzymes have been cloned, sequenced, transcriptionally mapped, and their regulation defined on a molecular level. This review emphasizes recent developments concerning the enzymes which are unique to the acetate fermentation pathway in M. thermophila.

Clostridum acetobutylicum strain P262 fermented glucose, pyruvate, or lactate, and the butyrate production was substrate-dependent. Differences in butyrate yield could not be explained by changes in butyrate kinase activities, but the butyrate production was inversely related to acetate kinase activity. The acetate kinase had a pH optimum of 8.0, a Km for acetate of 160 mM, and a kcat of 16, 800 min-1. The enyzme had a native molecular mass of 78 kDa; the size of 42 kDa on SDS-PAGE indicated that the acetate kinase of strain P262 was a homodimer.

The aerobic metabolism of lactate by oral Actinomyces was studied. Six of 7 strains of Actinomyces naeslundii increased their growth in the presence of lactate under aerobic conditions. Washed cells grown on lactate aerobically degraded lactate and pyruvate to acetate with a concomitant consumption of oxygen. In the presence of catalase, the molar ratios of oxygen consumed to acetate produced were 1 for lactate degradation and 0.5 for pyruvate degradation. The enzymatic activities found in cell extracts revealed that lactate could be converted to pyruvate by NAD-independent lactate dehydrogenase (iLDH) and further to acetyl CoA by pyruvate dehydrogenase (PDH). The acetyl CoA formed could be metabolized into acetate by phosphotransacetylase (PTA) and acetate kinase (AK) with the formation of ATP. These results indicate that A. naeslundii metabolizes lactate into acetate by the sequential enzymatic reactions iLDH, PDH, PTA and AK and that hydrogens produced by iLDH and PDH are transferred to oxygen. The activity of lactate degradation and oxygen consumption may modify the environmental conditions of dental plaque.

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.

In Escherichia coli carrying the poly(3-hydroxyalkanoate) (PHA) biosynthesis pathway on a plasmid (pha+), the function of the ackA (acetate kinase) and pta (phosphotransacetylase) genes is necessary for efficient incorporation of 3-hydroxyvalerate (3-HV) into the copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)). Recombinant pha+ E. coli fadR atoC(Con) strains possessing mutations in ackA, pta, or both ackA and pta exhibited substantially reduced levels of 3-HV formation. Conversely, the same strains carrying the ackA gene on a multicopy plasmid exhibited an increase in 3-HV formation concomitant with a large increase in acetate kinase activity. However, if the strain possessing the multicopy ackA+ plasmid was mutant at the pta locus, it lost the ability to incorporate significant amounts of 3-HV into P(3HB-co-3HV). In addition to the ackA pta pathway, there is an inducible activity that can also mediate the incorporation of 3-HV into P(3HB-co-3HV). This pathway is repressed by glucose and is not normally operative in P(3HB-co-3HV) production in recombinant pha+ E. coli strains that are grown using glucose as the major carbon source. It appears likely that this activity is due to an inducible acetyl-CoA synthetase that converts propionate to propionyl-CoA.

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.

We investigated the relationship between Escherichia coli flagellar expression and the regulation of acetyl phosphate synthesis and degradation. Using cells either wild type for acetyl phosphate metabolism or defective for phosphotransacetylase or acetate kinase, or both, we measured flagellar expression and the intracellular concentration of acetyl phosphate relative to growth phase and temperature. Under the conditions tested, we found that elevated levels of acetyl phosphate corresponded to inhibition of flagellar synthesis. To extend these observations, we measured the intracellular concentration of acetyl-CoA, the level of expression from the pta and ackA promoters, and the activities of phosphotransacetylase and acetate kinase derived from cell lysates. Relative to increasing culture density, acetyl-CoA levels and expression from both the pta and ackA promoters decreased. Relative to increasing temperature, expression from the ackA promoter decreased and phosphotransacetylase activity increased. In contrast, temperature had little or no effect on either acetate kinase activity or expression from the pta promoter. We propose that cells regulate intracellular acetyl phosphate concentrations relative to growth phase and temperature by modulating the availability of acetyl-CoA, the expression of ackA, and the activity of phosphotransacetylase.

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.

Two controls of the phosphate (PHO) regulon require sensor proteins that are protein kinases that phosphorylate the regulator, PhoB, which in turn activates transcription only when phosphorylated. Pi control requires the Pi sensor PhoR; the other control is Pi independent and requires the sensor CreC (formerly called PhoM). Here we describe an additional control of the PHO regulon which is Pi independent and requires neither PhoR nor CreC. This control is regulated by a two-step pathway in carbon metabolism in which acetyl coenzyme A, Pi, and ADP are converted into acetate, coenzyme A, and ATP via the enzymes phosphotransacetylase (Pta) and acetate kinase (AckA). It responds to the synthesis of acetyl phosphate, an intermediate in the Pta-AckA pathway. Since the synthesis of acetyl phosphate via this pathway leads to the incorporation of Pi into ATP, the primary phosphoryl donor in metabolism, we propose that a regulatory coupling(s) may exist between the PHO regulon, which encodes genes for Pi uptake, and genes for enzymes in central metabolism for incorporation of Pi into ATP. Regulatory interactions of this sort may be important in global control. Further, it provides a functional basis for the concept of cross-regulation in the PHO regulon. This is also the first evidence that acetyl phosphate may have a role as an effector of gene regulation.

The mechanism behind the hypolipidaemic effect of the drug Acipimox was studied in rat. The triacylglycerol (TG) lowering effect of the drug was accompanied by a marked reduction in free fatty acid (FFA) levels in the plasma. Also, the flux of TG from the liver to the plasma was substantially reduced. The liver TG content was not significantly changed. No significant effect of Acipimox treatment on the activities of glycerol 3-phosphate acyltransferase, phosphatidate phosphohydrolase or diacylglycerol acyltransferase in rat liver was observed. These findings strongly support the contention that Acipimox exerts its TG-lowering effect mainly by reducing FFA influx to the liver and thereby reducing the de novo biosynthesis of TG in the liver.

The endogenous respiration of the rumen ciliate Dasytricha ruminantium maintained under an O2 tension of 2kPa (approximately 0.02 atm) was partially inhibited by KCN (40% inhibition) and NaN3 (58% inhibition). The organisms lack cytochromes, and sensitivity of respiration to KCN, NaN3, chloroquine and quercetin suggest that the operation of flavoprotein-iron-sulphur-mediated electron transport. As in Tritrichomonas foetus, hydrogenosomal respiration can be stimulated by the addition of CoA in the presence of 0.025% Triton X-100; stimulation by ADP was not detected. Stimulation of pyruvate-supported O2 uptake by Pi suggests that acetate is produced via acetyl phosphate.

Clostridial phosphotransacetylase catalyzes acyl group transfer between coenzyme A (CoA) and inorganic phosphate and also the arsenolysis of acetyl-coenzyme A (AcCoA) to yield acetate and CoA-SH. The enzyme mobility on sodium dodecyl sulfate electrophoresis corresponds to a molecular weight of 70 000. Kinetics of both forward and reverse reactions are of the ternary type as previously reported and product inhibition data are consistent with a random binding scheme. One essential sulfhydryl group per 70 000 daltons was inactivated in a pseudo-first-order process by either N-ethylmaleimide or 5,5'-dithiobis (nitrobenzoic acid). Reduction of the rate of this inactivation by 50% in the presence of AcCoA or acetyl phosphate concentrations near their kinetic K values demonstrates binding of these acyl donors in simple enzyme-substrate complexes. Moreover, pulse-chase experiments show these binary complexes to be functional and also show that they do not dissociate rapidly compared with their rates of catalytic turnover. Incubation of the enzyme with 14C-labeled acyl donors failed to produce labeled protein after passage through Sephadex. This was true despite efforts to mimic "substrate synergism" with desulfo-CoA or to compensate for unfavorable equilibria by means of CoA traps. Very slow isotope exchange reactions of 32Pi into acetyl phosphate and [3H]CoA into AcCoA were at first observed. As in the cases of several other enzymes recently reexamined, these were shown on careful inspection to be artifacts of contamination by second substrates. Attempts to detect exchange reactions between acetyl phosphate and Pi, even in the presence of the CoA analogue, desulfo-CoA, were also unsuccessful. Therefore, no evidence for an acyl-enzyme could be detected. Furthermore, our data allow us to develop arguments which, we believe, indicate that an acyl-enzyme intermediate is extremely improbable in the reaction catalyzed by phosphotransacetylase.

Spores of C. tyrobutyricum do not contain 3-phosphoglyceric acid (PGA) but a polysaccharide which could replace PGA as an energy source during germination. The absence of PGA, which is an inhibitor of phosphotransacetylase, confirms the role of the acetyl-CoA synthesizing system in the germination initiated by acetate. Spore extracts of C. tyrobutyricum, as extracts of vegetative cells, were found to contain a ferredoxin and exhibited a NADH-ferredoxin oxydase activity which required the presence of an acetyl-CoA regenerating system, suggesting that this enzyme is also involved in germination. From this results, an hypothesis on the role of initiators (acetate and NH4+) in the mechanism of initiation of spore germination in C. tyrobutyricum is proposed. Acetate would have an effect on the utilisation of the endogenous polysaccharide and on glucose catabolism, and therefore, would be an effector for the production of the energy required particularly to transport cations into the spore.

In Escherichia coli, acetyl phosphate can be formed from acetyl-CoA via the phosphotransacetylase (phosphate acetyltransferase; acetyl-CoA:orthophosphate acetyltransferase, EC 2.3.1.8) reaction and from acetate (plus ATP) via the acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) reaction. By restricting acetyl phosphate formation to the phosphotransacetylase reaction alone, through the use of metabolic inhibitors, we were able to show that, with pyruvate as a source of energy, mutants defective in phosphotransacetylase are unable to transport glutamine, histidine, and methionine. However, with the same energy source, mutants defective in acetate kinase are normal in the transport of these amino acids. The inability of the phosphotransacetylase mutants to transport is due to their presumed inability to form acetyl phosphate, because pyruvate is found to be metabolized to acetyl-CoA in these mutants. Thus acetyl phosphate has been implicated in active transport. Evidence is also presented that neither the protonmotive force nor the ecf gene product is required for the shock-sensitive transport systems.

The synthesis of a new coenzyme A analogue, N6-[N-(6-aminohexyl)carbamoylmethyl]-CoA, suitable for immobilisation through its terminal amino group to support matrices, is described. The synthetic route starts with bis(CoA) and involves the following steps: alkylation with iodoacetic acid and rearrangement yielding bis(N6-carboxymethyl-CoA), elongation of the carboxymethyl terminal with 1,6-diaminohexane using carbodiimide to yield bis(N6-[N-(6-aminohexyl)-carbamoylmethyl]-CoA) and finally the splitting of this bis[CoA analogue) through reduction with dithiothreitol to give the final product in approximately 10% overall yield. This CoA analogue showed 'coenzymic activity' with the enzymes acetyl-CoA synthetase, phosphotransacetylase and succinic thiokinase. Covalent binding of the CoA analogue to Sepharose 4B was normally carried out using its S-(5-thio-2-nitrobenzoic acid) derivative as this allows a convenient way for determining the amount of ligand coupled, based on the amount of 5-thio-2-nitrobenzoic acid liberated from the gel after reduction with dithiothreitol. After covalent binding of the CoA analogue to water-soluble activated dextran 70, the analogue was recycled while present in an ultrafiltration cell using the enzymes phosphotransacetylase and citrate synthase. The reaction was followed by measuring the citrate formed on addition of acetylphosphate and oxaloacetate. In affinity chromatographic studies it was shown that the CoA-Sepharose preparation could bind the CoA-dependent enzymes citrate synthase and succinic thiokinase and these could be biospecifically eluted using soluble CoA.

Group N streptococci formed acetaldehyde and ethanol from glucose. As the enzymes aldehyde dehydrogenase, phosphotransacetylase and acetate kinase were present this would enable these organisms to reduce acetyl-CoA to acetaldehyde and convert acetyl-CoA to acetyl phosphate and acetate. A pentose phosphate pathway which converted ribose-5-phosphate to glyceraldehyde-3-phosphate was also present. Acetaldehyde could not be formed via the hexose monophosphate shunt or by direct decarboxylation of pyruvate, as the enzymes phosphoketolase and alpha-carboxylase were absent. Phosphoketolase activity was induced in Streptococcus lactis subsp. diacetylactis after growth on D-xylose. Group N streptococci also contained an NAD-dependent alcohol dehydrogenase which reduced acetaldehyde to ethanol while both NAD- and NADP-dependent alcohol dehydrogenase activities were found in Leuconostoc cremoris.

Catabolism of L-threonine in the anaerobic bacteria Cl. sticklandii has been studied. Degradation of this amino acid was shown to occur with participation of the following enzymes: NAD-dependent L-threonine-3-dehydrogenase (EC 1.1.1.103), glycine acetyltransferase (EC 2.3.1.29), phosphotransacetylase (EC 2.3.1.8) and acetate kinase (EC 2.7.2.1). The presence of the first two enzymes in Clostridia has been shown for the first time. A scheme of threonine oxidation down to acetic acid and glycine by Cl. sticklandii was proposed. The oxidation of one threonine molecule is coupled with phosphorylation of one ADP molecule and reduction of one NAD+ molecule.

Cell-free extracts of Treponema pallidum catalyzed the decarboxylation of pyruvate. This activity was suppressed at low O2 tensions and appeared to be coenzyme A independent. Pyruvate decarboxylation was inorganic phosphate dependent, and evidence suggested that acetyl phosphate was a product. Oxygen was consumed, and data indicated that H2O2 was produced. These results indicated that the overall oxidation of pyruvate was: pyruvate + O2 + inorganic phosphate leads to CO2 + acetyl phosphate + H2O2. Phosphotransacetylase and acetate kinase activities were also observed in the cell-free extracts and could catalyze formation of acetyl coenzyme A and adenosine 5'-triphosphate, respectively, from acetyl phosphate.

Phosphotransbutyrylase (phosphate butyryltransferase [EC 2.3.1.19]) from Clostridium acetobutylicum ATCC 824 was purified approximately 200-fold to homogeneity with a yield of 13%. Steps used in the purification procedure were fractional precipitation with (NH4)2SO4, Phenyl Sepharose CL-4B chromatography, DEAE-Sephacel chromatography, high-pressure liquid chromatography with an anion-exchange column, and high-pressure liquid chromatography with a hydrophobic-interaction column. Gel filtration and denaturing gel electrophoresis data were consistent with a native enzyme having eight 31,000-molecular-weight subunits. Within the physiological range of pH 5.5 to 7, the enzyme was very sensitive to pH change in the butyryl phosphate-forming direction and showed virtually no activity below pH 6. This finding indicates that a change in internal pH may be one important factor in the regulation of the enzyme. The enzyme was less sensitive to pH change in the reverse direction. The enzyme could use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA) but had the highest relative activity with butyryl-CoA, isovaleryl-CoA, and valeryl-CoA. The Km values at 30 degrees C and pH 8.0 for butyryl-CoA, phosphate, butyryl phosphate, and CoASH (reduced form of CoA) were 0.11, 14, 0.26, and 0.077 mM, respectively. Results of product inhibition studies were consistent with a random Bi Bi binding mechanism in which phosphate binds at more than one site.

Aerobacter (Enterobacter) aerogenes wild type and three mutants deficient in the formation of acetoin and 2,3-butanediol were grown in a glucose minimal medium. Culture densities, pH, and diacetyl, acetoin, and 2,3-butanediol levels were recorded. The pH in wild-type cultures dropped from 7.0 to 5.8, remained constant while acetoin and 2,3-butanediol were formed, and increased to pH 6.5 after exhaustion of the carbon source. More 2,3-butanediol than acetoin was formed initially, but after glucose exhaustion reoxidation to acetoin occurred. The three mutants differed from the wild type in yielding acid cultures (pH below 4.5). The wild type and one of the mutants were grown exponentially under aerobic and anaerobic conditions with the pH fixed at 7.0, 5.8, and 5.0, respectively. Growth rates decreased with decreasing pH values. Aerobically, this effect was weak, and the two strains were affected to the same degree. Under anaerobic conditions, the growth rates were markedly inhibited at a low pH, and the mutant was slightly more affected than the wild type. Levels of alcohol dehydrogenase were low under all conditions, indicating that the enzyme plays no role during exponential growth. The levels of diacetyl (acetoin) reductase, lactate dehydrogenase, and phosphotransacetylase were independent of the pH during aerobic growth of the two strains. Under anaerobic conditions, the formation of diacetyl (acetoin) reductase was pH dependent, with much higher levels of the enzyme at pH 5.0 than at pH 7.0. Lactate dehydrogenase and phosphotransacetylase revealed the same pattern of pH-dependent formation in the mutant, but not in the wild type.

Six loci coding for arginine biosynthetic enzymes in Pseudomonas aeruginosa strain PAO were identified by enzyme assay: argA (N-acetylglutamate synthase), argB (N-acetylglutamate 5-phosphotransferase), argC (N-acetylglutamate 5-semialdehyde dehydrogenase), argF (anabolic ornithine carbamoyl-transferase), argG (argininosuccinate synthetase), and argH (argininosuccinase). One-step mutants which had a requirement for arginine and uracil were defective in carbamoylphosphate synthase, specified by a locus designated car. To map these mutations we used the sex factor FP2 in an improved interrupted mating technique as well as the generalized transducing phages F116L and G101. We confirmed earlier studies, and found no clustering of arg and car loci. However, argA, argH, and argB were mapped on a short chromosome segment (approx. 3 min long), and argF and argG were cotransducible, but not contiguous. N-Acetylglutamate synthase, the enzyme which replenishes the cycle of acetylated intermediates in ornithine synthesis of Pseudomonas, appears to be essential for arginine synthesis since argA mutants showed no growth on unsupplemented minimal medium.

The structure of the CoA affinity analog-oxidized CoA disulfide (o-CoAS2) (Collier, G. E., and Nishimura, J. S. (1978) J. Biol. Chem. 253, 4938-4939) has been deduced to be that of the thiosulfonate of CoA, i.e. coenzyme A disulfide-S,S-dioxide. This deduction is based on several considerations among which are: the cleavage of o-CoAS2 by dithiothreitol under anaerobic conditions to equimolar amounts of CoASH and CoASO2H; the alkali-catalyzed dismutation of 3 mol of o-CoAS2 to 4 mol of CoASO2H and 1 mol of CoA disulfide; and comparison of the 13C-NMR spectra of CoA disulfide and o-CoAS2. The results of studies with Clostridial phosphotransacetylase (EC 2.3.1.8) and pigeon muscle carnitine acetyltransferase (EC 2.3.1.7) were consistent with the action of o-CoAS2 as a CoA affinity analog on these enzymes. Inactivation was characterized by what appeared to be disulfide bonding between CoA and important sulfhydryl groups of the proteins.

A phosphotransacetylase was purified to apparent homogeneity from photolithoautotrophically grown Rhodopseudomonas palustris by liquid chromatography methods. A 400-fold increase in specific activity could be achieved. The enzyme was characterized by a relative molecular mass of 54,500, an isoelectric point of 6.3 and the absence of dissociable subunits. The enzyme appeared very labile at elevated temperatures or in diluted solutions. The stability could be increased distinctly in case sulfate or ammonium ions were added to the enzyme solution. The activity also was influenced by inorganic salts. Potassium and ammonium ions activated the enzymatic reaction. Sulfate ions revealed an inhibitory influence. A strong substrate inhibition was found with coenzyme A as substrate. The Arrhenius plot revealed a discontinuity at 15 degrees C which most likely corresponds to a conformational change of the enzyme protein.

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.

The 3'-pyrophosphate derivative of CoA was synthesized by using the excreted 5'-to-3' pyrophosphoryl-transferring enzyme from Streptomyces adephospholyticus and ATP as donor and dephospho-CoA as acceptor. Cofactor activity of this new coenzyme A derivative was tested with Clostridium kluyveri phosphotransacetylase and hog heart succinic thiokinase. With the phosphotransacetylase, 3'-pyrophospho-CoA was found to be twice as active as CoA whereas dephospho-CoA was inactive. However, succinic thiokinase utilized all three types of CoA equally well. Adenosine 5'-monophosphate 3'-pyrophosphate also was synthesized and used as an analog of adenosine 5'-monophosphate 3'-monophosphate in the dog liver's sulfotransferase-catalyzed sulfate transfer from p-nitrophenyl sulfate to phenol. In contrast to the pyrophospho derivative of coenzyme A, adenosine 5'-monophosphate 3'-pyrophosphate was inactive as a cofactor.

Syntrophomonas wolfei is an anaerobic fatty acid degrader that can only be grown in coculture with H2-using bacteria such as Methanospirillum hungatei. Cells of S. wolfei were selectively lysed by lysozyme treatment, and unlysed cells of M. hungatei were removed by centrifugation. The cell extract of S. wolfei obtained with this method had low levels of contamination by methanogenic cofactors. However, lysozyme treatment was not efficient in releasing S. wolfei protein; only about 15% of the L-3-hydroxyacyl-coenzyme A (CoA) dehydrogenase activity was found in the lysozyme supernatant. Cell extracts of S. wolfei obtained with this method had high specific activities of acyl-CoA dehydrogenase, enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. These activities were not detected in cell extracts of M. hungatei grown alone, confirming that these activities were present in S. wolfei. The acyl-CoA dehydrogenase activity was high when a C4 but not a C8 or C16 acyl-CoA derivative served as the substrate. S. Wolfei cell extracts had high CoA transferase specific activities and no detectable acyl-CoA synthetase activity, indicating that fatty acid activation occurred by transfer of CoA from acetyl-CoA. Phosphotransacetylase and acetate kinase activities were detected in cell extracts of S. wolfei, indicating that S. wolfei is able to perform substrate-level phosphorylation.

One-carbon metabolic transformations associated with cell carbon synthesis and methanogenesis were analyzed by long- and short-term (14)CH(3)OH or (14)CO(2) incorporation studies during growth and by cell suspensions. (14)CH(3)OH and (14)CO(2) were equivalently incorporated into the major cellular components (i.e., lipids, proteins, and nucleic acids) during growth on H(2)-CO(2)-methanol. (14)CH(3)OH was selectively incorporated into the C-3 of alanine with decreased amounts fixed in the C-1 and C-2 positions, whereas (14)CO(2) was selectively incorporated into the C(1) moiety with decreasing amounts assimilated into the C-2 and C-3 atoms. Notably, (14)CH(4) and [3-(14)C]alanine synthesized from (14)CH(3)OH during growth shared a common specific activity distinct from that of CO(2) or methanol. Cell suspensions synthesized acetate and alanine from (14)CO(2). The addition of iodopropane inhibited acetate synthesis but did not decrease the amount of (14)CH(3)OH or (14)CO(2) fixed into one-carbon carriers (i.e., methyl coenzyme M or carboxydihydromethanopterin). Carboxydihydromethanopterin was only labeled from (14)CH(3)OH in the absence of hydrogen. Cell extracts catalyzed the synthesis of acetate from (14)CO ( approximately 1 nmol/min per mg of protein) and an isotopic exchange between CO(2) or CO and the C-1 of pyruvate. Acetate synthesis from (14)CO was stimulated by methyl B(12) but not by methyl tetrahydrofolate or methyl coenzyme M. Methyl coenzyme M and coenzyme M were inhibitory to acetate synthesis. Cell extracts contained high levels of phosphotransacetylase (>6 mumol/min per mg of protein) and acetate kinase (>0.14 mumol/min per mg of protein). It was not possible to distinguish between acetate and acetyl coenzyme A as the immediate product of two-carbon synthesis with the methods employed.

Carbon from glycerol and palmitate, but not significantly from five other carbon sources tested, was incorporated into lipids by suspensions of non-growing Mycobacterium leprae organisms. However, of the five other substrates three-citrate, glucose and pyruvate-were taken up. Nongrowing Mycobacterium microti and Mycobacterium avium incorporated carbon into lipids from most simple carbon sources tested unless they were obtained from growth media including palmitate or from experimentally infected animals, when incorporation of carbon into lipids from carbon sources except palmitate occurred up to 20 times more slowly. Thus, utilization of simple carbon appeared to be repressible while utilization of the one fatty acid tested, palmitate, appeared constitutive. In M. leprae, carbon from glycerol was incorporated into the glycerol moiety of acylglycerols but not into the fatty acid moieties or into free fatty acids. M. microti and M. avium incorporated carbon from simple carbon sources into fatty acids, even (though very slowly) when these organisms were obtained from host tissue. Isocitrate lyase, malate synthase and acetate kinase were detected in M. leprae. However acetyl-CoA synthetase was not detectable and phosphoacetylase was deficient; thus, M. leprae may be incapable of making acetyl-CoA from acetate. Phosphotransacetylase was readily detected in both host-grown M. avium and M. microti.

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.

Levels of the five enzymes involved in the fermentation of 1,2-ethanediol and 1,2-propanediol in the strictly anaerobic bacterium, Clostridium glycolicum, were investigated. All enzymes with the exception of the first enzyme in the pathway, diol dehydratase, were found to be constitutive, stable to exposure to oxygen, and present in the cytosol. Diol dehydratase was found to be extremely oxygen sensitive and strongly associated with the cell membrane. Treatment with ionic and nonionic detergents, butanol, phospholipase A2, or osmotic shock procedures failed to solubilize any diol dehydratase activity. Limited proteolysis using subtilisin released small amounts of activity. Diol dehydratase was found to be specific for 1,2-ethanediol and 1,2-propanediol and required the addition of a reducing agent for maximal activity. The enzyme was strongly inhibited by low concentrations of EDTA, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, o-phenanthroline, hydroxylamine, hydroxyurea, and sulfhydryl reagents. Addition of adenosylcobalamin or high levels of intrinsic factor did not affect the reaction rate. Irradiation with light also did not inhibit the enzyme activity. These results suggest that the catalytic mechanism of diol dehydratase from C. glycolicum does not involve a cobamide coenzyme.