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Genetic truncations in a gene encoding a putative glucose-phosphotransferase system (PTS) protein (manL, EIIAB(Man)) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. To understand the emergence of these mutants, we engineered deletion mutants of manL and showed that the ManL-deficient strain had improved bacterial viability in the stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H(2)O(2)) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small but significant percentage (∼10%) of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. To our surprise, significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e., excess carbohydrates and low pH, are those associated with caries development, we propose that glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H(2)O(2). IMPORTANCE A health-associated dental microbiome provides a potent defense against pathogens and diseases. Streptococcus sanguinis is an abundant member of a health-associated oral flora that antagonizes pathogens by producing hydrogen peroxide. There is a need for a better understanding of the mechanisms that allow bacteria to survive carbohydrate-rich and acidic environments associated with the development of dental caries. We report the isolation and characterization of spontaneous mutants of S. sanguinis with impairment in glucose transport. The resultant reprograming of the central metabolism in these mutants reduced the production of lactic acid and increased pyruvate accumulation; the latter enables these bacteria to better cope with hydrogen peroxide and low pH. The implications of these discoveries in the development of dental caries are discussed.

Pyruvate is a key metabolite in living cells and has been shown to play a crucial role in the virulence of several bacterial pathogens. The bioluminescent Vibrio campbellii, a severe infectious burden for marine aquaculture, excretes extraordinarily large amounts of pyruvate during growth and rapidly retrieves it by an as-yet-unknown mechanism. We have now identified the responsible pyruvate transporter, here named BtsU, and our results show that it is the only pyruvate transporter in V. campbellii. Expression of btsU is tightly regulated by the membrane-integrated LytS-type histidine kinase BtsS, a sensor for extracellular pyruvate, and the LytTR-type response regulator BtsR. Cells lacking either the pyruvate transporter or sensing system show no chemotactic response toward pyruvate, indicating that intracellular pyruvate is required to activate the chemotaxis system. Moreover, pyruvate sensing and uptake were found to be important for the resuscitation of V. campbellii from the viable but nonculturable state and the bacterium’s virulence against brine shrimp larvae. IMPORTANCE Bacterial infections are a serious threat to marine aquaculture, one of the fastest growing food sectors on earth. Therefore, it is extremely important to learn more about the pathogens responsible, one of which is Vibrio campbellii. This study sheds light on the importance of pyruvate sensing and uptake for V. campbellii, and reveals that the bacterium possesses only one pyruvate transporter, which is activated by a pyruvate-responsive histidine kinase/response regulator system. Without the ability to sense or take up pyruvate, the virulence of V. campbellii toward gnotobiotic brine shrimp larvae is strongly reduced.

The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. Following our previous studies on key enzymes of this pathway, we now focus on the characterization of enzymes involved in 3-phosphoglycerate conversion to pyruvate, in anaplerosis, and in acetyl coenzyme A (acetyl-CoA) formation from pyruvate. These enzymes include phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenolpyruvate carboxylase, and pyruvate-ferredoxin oxidoreductase. The essential function of these enzymes were shown by transcript analyses and growth experiments with respective deletion mutants. Furthermore, we show that H. volcanii—during aerobic growth on glucose—excreted significant amounts of acetate, which was consumed in the stationary phase (acetate switch). The enzyme catalyzing the conversion of acetyl-CoA to acetate as part of the acetate overflow mechanism, an ADP-forming acetyl-CoA synthetase (ACD), was characterized. The functional involvement of ACD in acetate formation and of AMP-forming acetyl-CoA synthetases (ACSs) in activation of excreted acetate was proven by using respective deletion mutants. Together, the data provide a comprehensive analysis of enzymes of the spED pathway and of anaplerosis and report the first genetic evidence of the functional involvement of enzymes of the acetate switch in archaea. IMPORTANCE In this work, we provide a comprehensive analysis of glucose degradation via the semiphosphorylative Entner-Doudoroff pathway in the haloarchaeal model organism Haloferax volcanii. The study includes transcriptional analyses, growth experiments with deletion mutants. and characterization of all enzymes involved in the conversion of 3-phosphoglycerate to acetyl coenzyme A (acetyl-CoA) and in anaplerosis. Phylogenetic analyses of several enzymes indicate various lateral gene transfer events from bacteria to haloarchaea. Furthermore, we analyzed the key players involved in the acetate switch, i.e., in the formation (overflow) and subsequent consumption of acetate during aerobic growth on glucose. Together, the data provide novel aspects of glucose degradation, anaplerosis, and acetate switch in H. volcanii and thus expand our understanding of the unusual sugar metabolism in archaea.

Pyruvate is an important intermediate of central carbon metabolism and connects a variety of metabolic pathways in Escherichia coli . Although the intracellular pyruvate concentration is dynamically altered and tightly balanced during cell growth, the pyruvate transport system remains unclear. Here, we identified a pyruvate transporter in E. coli using high-throughput transposon sequencing. The transposon mutant library (a total of 5 × 10 5 mutants) was serially grown with a toxic pyruvate analog (3-fluoropyruvate [3FP]) to enrich for transposon mutants lacking pyruvate transport function. A total of 52 candidates were selected on the basis of a stringent enrichment level of transposon insertion frequency in response to 3FP treatment. Subsequently, their pyruvate transporter function was examined by conventional functional assays, such as those measuring growth inhibition by the toxic pyruvate analog and pyruvate uptake activity. The pyruvate transporter system comprises CstA and YbdD, which are known as a peptide transporter and a conserved protein, respectively, whose functions are associated with carbon starvation conditions. In addition to the presence of more than one endogenous pyruvate importer, it has been suggested that the E. coli genome encodes constitutive and inducible pyruvate transporters. Our results demonstrated that CstA and YbdD comprise the constitutive pyruvate transporter system in E. coli , which is consistent with the tentative genomic locus previously suggested and the functional relationship with the extracellular pyruvate sensing system. The identification of this pyruvate transporter system provides valuable genetic information for understanding the complex process of pyruvate metabolism in E. coli . IMPORTANCE Pyruvate is an important metabolite as a central node in bacterial metabolism, and its intracellular levels are tightly regulated to maintain its functional roles in highly interconnected metabolic pathways. However, an understanding of the mechanism of how bacterial cells excrete and transport pyruvate remains elusive. Using high-throughput transposon sequencing followed by pyruvate uptake activity testing of the selected candidate genes, we found that a pyruvate transporter system comprising CstA and YbdD, currently annotated as a peptide transporter and a conserved protein, respectively, constitutively transports pyruvate. The identification of the physiological role of the pyruvate transporter system provides valuable genetic information for understanding the complex pyruvate metabolism in Escherichia coli .

The present study investigated the simultaneous oxidation of pyruvate and amino acids during H 2 -evolving growth of the hyperthermophilic archaeon Thermococcus kodakarensis . The comparison of mass balance between a cytosolic hydrogenase (HYH)-deficient strain (the Δ hyhBGSL strain) and the parent strain indicated that NADPH generated via H 2 uptake by HYH was consumed by reductive amination of 2-oxoglutarate catalyzed by glutamate dehydrogenase. Further examinations were done to elucidate functions of three enzymes potentially involved in pyruvate oxidation: pyruvate formate-lyase (PFL), pyruvate:ferredoxin oxidoreductase (POR), and 2-oxoisovalerate:ferredoxin oxidoreductase (VOR) under the HYH-deficient background in T. kodakarensis . No significant change was observed by deletion of pflDA , suggesting that PFL had no critical role in pyruvate oxidation. The growth properties and mass balances of Δ porDAB and Δ vorDAB strains indicated that POR and VOR specifically functioned in oxidation of pyruvate and branched-chain amino acids, respectively, and the lack of POR or VOR was compensated for by promoting the oxidation of another substrate driven by the remaining oxidoreductase. The H 2 yields from the consumed pyruvate and amino acids were increased from 31% by the parent strain to 67% and 82% by the deletion of hyhBGSL and double deletion of hyhBGSL and vorDAB , respectively. Significant discrepancies in the mass balances were observed in excess formation of acetate and NH 3 , suggesting the presence of unknown metabolisms in T. kodakarensis grown in the rich medium containing pyruvate.

The bacterial uptake of steroids and their metabolites remains poorly understood. We investigated two transporters associated with cholate catabolism in Rhodococcus jostii RHA1. Reverse transcriptase quantitative-PCR indicated that an ATP-binding cassette (ABC) transporter and a major facilitator superfamily (MFS) transporter were upregulated 16.7- and 174-fold, respectively, during the exponential phase of growth on cholate compared to growth on pyruvate. Gene knockout analysis established that these transporters are required for the reassimilation of distinct metabolites that accumulate during growth on cholate. The ABC transporter, encoded by camABCD , was essential for uptake of 1β(2′-propanoate)-3aα- H -4α(3″ (R) -hydroxy-3″-propanoate)-7aβ-methylhexahydro-5-indanone and a desaturated analog. The MFS transporter, encoded by camM , was essential for uptake of 3,7 (R) ,12 (S) -trihydroxy-9-oxo-9,10-seco-23,24-bisnorchola-1,3,5(10)-trien-22-oate. These metabolites differ from cholate metabolites reported to be excreted by proteobacteria in that they retain an isopropanoyl side chain at C-17. The uptake of these metabolites was necessary for maximal growth on cholate: a Δ camB mutant lacking the permease component of the ABC transporter and a Δ camM mutant lacking the MFS transporter grew to 74% and 77%, respectively, of the yield of the wild type. This study demonstrates for the first time the requirement for specific transporters for uptake of cholate metabolites and highlights the importance and complexity of transport processes associated with bacterial steroid catabolism.

The carbon monoxide-sensing transcriptional factor CooA has been studied only in hydrogenogenic organisms that can grow using CO as the sole source of energy. Homologs for the canonical CO oxidation system, including CooA, CO dehydrogenase (CODH), and a CO-dependent Coo hydrogenase, are present in the sulfate-reducing bacterium Desulfovibrio vulgaris , although it grows only poorly on CO. We show that D. vulgaris Hildenborough has an active CO dehydrogenase capable of consuming exogenous CO and that the expression of the CO dehydrogenase, but not that of a gene annotated as encoding a Coo hydrogenase, is dependent on both CO and CooA. Carbon monoxide did not act as a general metabolic inhibitor, since growth of a strain deleted for cooA was inhibited by CO on lactate-sulfate but not pyruvate-sulfate. While the deletion strain did not accumulate CO in excess, as would have been expected if CooA were important in the cycling of CO as a metabolic intermediate, global transcriptional analyses suggested that CooA and CODH are used during normal metabolism.

Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5′phosphate (PLP)-dependent enzyme that catalyzes the conversion of diaminopropionate (DAP) to pyruvate and ammonia and plays an important role in cell metabolism. We have investigated the role of the ygeX gene of Escherichia coli K-12 and its ortholog, STM1002 , in Salmonella enterica serovar Typhimurium LT2, presumed to encode DAPAL, in the growth kinetics of the bacteria. While Salmonella Typhimurium LT2 could grow on dl- DAP as a sole carbon source, the wild-type E. coli K-12 strain exhibited only marginal growth on dl-DAP , suggesting that DAPAL is functional in S. Typhimurium . The expression of ygeX in E. coli was low as detected by reverse transcriptase PCR (RT-PCR), consistent with the poor growth of E. coli on dl- DAP. Strains of S. Typhimurium and E. coli with STM1002 and ygeX , respectively, deleted showed loss of growth on dl-DAP , confirming that STM1002 ( ygeX ) is the locus encoding DAPAL. Interestingly, the presence of dl- DAP caused a growth inhibition of the wild-type E. coli strain as well as the knockout strains of S. Typhimurium and E. coli in minimal glucose/glycerol medium. Inhibition by dl- DAP was rescued by transforming the strains with plasmids containing the STM1002 ( ygeX ) gene encoding DAPAL or supplementing the medium with Casamino Acids. Growth restoration studies using media lacking specific amino acid supplements suggested that growth inhibition by dl- DAP in the absence of DAPAL is associated with auxotrophy related to the inhibition of the enzymes involved in the biosynthetic pathways of pyruvate and aspartate and the amino acids derived from them.

Genetic analysis of alanine synthesis in the model genetic organism Escherichia coli has implicated avtA , the still uncharacterized alaA and alaB genes, and probably other genes. We identified alaA as yfbQ . We then transferred mutations in several transaminase genes into a yfbQ mutant and isolated a mutant that required alanine for optimal growth. For cells grown with carbon sources other than pyruvate, the major alanine-synthesizing transaminases are AvtA, YfbQ (AlaA), and YfdZ (which we designate AlaC). Growth with pyruvate as the carbon source and multicopy suppression suggest that several other transaminases can contribute to alanine synthesis. Expression studies showed that alanine modestly repressed avtA and yfbQ but had no effect on yfdZ . The leucine-responsive regulatory protein (Lrp) mediated control by alanine. We purified YfbQ and YfdZ and showed that both are dimers with K m s for pyruvate within the intracellular range of pyruvate concentration.

In the diazotrophic filaments of heterocyst-forming cyanobacteria, an exchange of metabolites takes place between vegetative cells and heterocysts that results in a net transfer of reduced carbon to the heterocysts and of fixed nitrogen to the vegetative cells. Open reading frame alr2355 of the genome of Anabaena sp. strain PCC 7120 is the ald gene encoding alanine dehydrogenase. A strain carrying a green fluorescent protein (GFP) fusion to the N terminus of Ald (Ald-N-GFP) showed that the ald gene is expressed in differentiating and mature heterocysts. Inactivation of ald resulted in a lack of alanine dehydrogenase activity, a substantially decreased nitrogenase activity, and a 50% reduction in the rate of diazotrophic growth. Whereas production of alanine was not affected in the ald mutant, in vivo labeling with [ 14 C]alanine (in whole filaments and isolated heterocysts) or [ 14 C]pyruvate (in whole filaments) showed that alanine catabolism was hampered. Thus, alanine catabolism in the heterocysts is needed for normal diazotrophic growth. Our results extend the significance of a previous work that suggested that alanine is transported from vegetative cells into heterocysts in the diazotrophic Anabaena filament.

The human pathogenL. monocytogenesis a facultatively intracellular bacterium that survives and replicates in the cytosol of many mammalian cells. The listerial metabolism, especially under intracellular conditions, is still poorly understood. Recent studies analyzed the carbon metabolism ofL. monocytogenesby the13C isotopologue perturbation method in a defined minimal medium containing [U-13C6]glucose. It was shown that these bacteria produce oxaloacetate mainly by carboxylation of pyruvate due to an incomplete tricarboxylic acid cycle. Here, we report that apycAinsertion mutant defective in pyruvate carboxylase (PYC) still grows, albeit at a reduced rate, in brain heart infusion (BHI) medium but is unable to multiply in a defined minimal medium with glucose or glycerol as a carbon source. Aspartate and glutamate of thepycAmutant, in contrast to the wild-type strain, remain unlabeled when [U-13C6]glucose is added to BHI, indicating that the PYC-catalyzed carboxylation of pyruvate is the predominant reaction leading to oxaloacetate inL. monocytogenes. ThepycAmutant is also unable to replicate in mammalian cells and exhibits high virulence attenuation in the mouse sepsis model.

The metabolism of monocarboxylic acids is of central importance for bacteria in their natural habitat as well as during biotechnological production. Although biosynthesis and degradation are well understood, the transport of such compounds is still a matter of discussion. Here we present the identification and characterization of a new transport system in Corynebacterium glutamicum with high affinity for acetate and propionate and with lower affinity for pyruvate. Biochemical analysis of this monocarboxylic acid transporter (MctC) revealed for the first time a quantitative discrimination of passive diffusion and active transport of acetate by bacterial cells. MctC is a secondary transporter and belongs to the class of sodium solute symporters, but it is driven by the electrochemical proton potential. The mctC gene is preceded by and cotranscribed with cg0952, a locus encoding a small membrane protein, and the transcription of the cg0952- mctC operon is under the control of the transcriptional regulators RamA and RamB. Both of these proteins directly bind to the promoter region of the operon; RamA is essential for expression and RamB exerts a slightly negative control on expression of the cg0952- mctC operon. mctC expression is induced in the presence of pyruvate and beneficial under substrate-limiting conditions for C. glutamicum .

The YjgF/YER057c/UK114 family of proteins is conserved across the three domains of life, yet no biochemical function has been clearly defined for any member of this family. InSalmonella enterica, a deletion ofyjgFresults in a requirement for isoleucine when the mutant strain is grown in glucose-serine or pyruvate medium. Feedback inhibition of IlvA is required for the curative effect of isoleucine on glucose-serine medium. On pyruvate medium,yjgFmutants are unable to synthesize enough isoleucine for growth. From this study, we conclude that the isoleucine requirement of ayjgFmutant on pyruvate is a consequence of the decreased transaminase B (IlvE) activity that has previously been characterized in these mutants.

Mycothiol ([MSH] AcCys-GlcN-Ins, where Ac is acetyl) is the major thiol produced by Mycobacterium smegmatis and other actinomycetes. Mutants deficient in MshA (strain 49) or MshC (transposon mutant Tn 1 ) of MSH biosynthesis produce no MSH. However, when stationary phase cultures of these mutants were incubated in medium containing MSH, they actively transported it to generate cellular levels of MSH comparable to or greater than the normal content of the wild-type strain. When these MSH-loaded mutants were transferred to MSH-free preconditioned medium, the cellular MSH was catabolized to generate GlcN-Ins and AcCys. The latter was rapidly converted to Cys by a high deacetylase activity assayed in extracts. The Cys could be converted to pyruvate by a cysteine desulfhydrase or used to regenerate MSH in cells with active MshC. Using MSH labeled with [U- 14 C]cysteine or with [6- 3 H]GlcN, it was shown that these residues are catabolized to generate radiolabeled products that are ultimately lost from the cell, indicating extensive catabolism via the glycolytic and Krebs cycle pathways. These findings, coupled with the fact the myo -inositol can serve as a sole carbon source for growth of M. smegmatis , indicate that MSH functions not only as a protective cofactor but also as a reservoir of readily available biosynthetic precursors and energy-generating metabolites potentially important under stress conditions. The half-life of MSH was determined in stationary phase cells to be ∼50 h in strains with active MshC and 16 ± 3 h in the MshC-deficient mutant, suggesting that MSH biosynthesis may be a suitable target for drugs to treat dormant tuberculosis.

The opportunistic pathogen Pseudomonas aeruginosa produces colorful, redox-active antibiotics called phenazines. Excretion of pyocyanin, the best-studied natural phenazine, is responsible for the bluish tint of sputum and pus associated with P. aeruginosa infections in humans. Although the toxicity of pyocyanin for other bacteria, as well as its role in eukaryotic infection, has been studied extensively, the physiological relevance of pyocyanin metabolism for the producing organism is not well understood. Pyocyanin reduction by P. aeruginosa PA14 is readily observed in standing liquid cultures that have consumed all of the oxygen in the medium. We investigated the physiological consequences of pyocyanin reduction by assaying intracellular concentrations of NADH and NAD + in the wild-type strain and a mutant defective in phenazine production. We found that the mutant accumulated more NADH in stationary phase than the wild type. This increased accumulation correlated with a decrease in oxygen availability and was relieved by the addition of nitrate. Pyocyanin addition to a phenazine-null mutant also decreased intracellular NADH levels, suggesting that pyocyanin reduction facilitates redox balancing in the absence of other electron acceptors. Analysis of extracellular organic acids revealed that pyocyanin stimulated stationary-phase pyruvate excretion in P. aeruginosa PA14, indicating that pyocyanin may also influence the intracellular redox state by decreasing carbon flux through central metabolic pathways.

The pyruvate dehydrogenase (PDH) multienzyme complex plays a key role in the metabolic interconnection between glycolysis and the citric acid cycle. Transcription of the Escherichia coli genes for all three components of the PDH complex in the pdhR-aceEF-lpdA operon is repressed by the pyruvate-sensing PdhR, a GntR family transcription regulator, and derepressed by pyruvate. After a systematic search for the regulation targets of PdhR using genomic systematic evolution of ligands by exponential enrichment (SELEX), we have identified two novel targets, ndh , encoding NADH dehydrogenase II, and cyoABCDE , encoding the cytochrome bo -type oxidase, both together forming the pathway of respiratory electron transport downstream from the PDH cycle. PDH generates NADH, while Ndh and CyoABCDE together transport electrons from NADH to oxygen. Using gel shift and DNase I footprinting assays, the PdhR-binding site (PdhR box) was defined, which includes a palindromic consensus sequence, ATTGGTNNNACCAAT. The binding in vitro of PdhR to the PdhR box decreased in the presence of pyruvate. Promoter assays in vivo using a two-fluorescent-protein vector also indicated that the newly identified operons are repressed by PdhR and derepressed by the addition of pyruvate. Taken together, we propose that PdhR is a master regulator for controlling the formation of not only the PDH complex but also the respiratory electron transport system.

A mutant strain of Bradyrhizobium japonicum USDA110 lacking isocitrate dehydrogenase activity was created to determine whether this enzyme was required for symbiotic nitrogen fixation with soybean ( Glycine max cv. Williams 82). The isocitrate dehydrogenase mutant, strain 5051, was constructed by insertion of a streptomycin resistance gene cassette. The mutant was devoid of isocitrate dehydrogenase activity and of immunologically detectable protein, indicating there is only one copy in the genome. Strain 5051 grew well on a variety of carbon sources, including arabinose, pyruvate, succinate, and malate, but, unlike many microorganisms, was a glutamate auxotroph. Although the formation of nodules was slightly delayed, the mutant was able to form nodules on soybean and reduce atmospheric dinitrogen as well as the wild type, indicating that the plant was able to supply sufficient glutamate to permit infection. Combined with the results of other citric acid cycle mutants, these results suggest a role for the citric acid cycle in the infection and colonization stage of nodule development but not in the actual fixation of atmospheric dinitrogen.

In earlier studies from this laboratory, Xanthomonas campestris pv. glycines was found to exhibit a nutrition stress-related postexponential rapid cell death (RCD). The RCD was exhibited in protein-rich media but not in starch or other minimal media. This RCD in X. campestris pv. glycines was found to display features similar to those of the programmed cell death (PCD) of eukaryotes. Results of the present study showed that the observed RCD in this organism is both positively and negatively regulated by small molecules. The amino acids glycine and l -alanine as well as the D isomers of valine, methionine, and threonine were found to induce the synthesis of an active caspase-3-like protein that was associated with the onset of RCD. Addition of pyruvate and citrate to the culture medium induced both the synthesis of active caspase-3-like protein and RCD. Higher levels of intracellular accumulation of pyruvate and citrate were also observed under conditions favoring RCD. On the other hand, dextrin and maltose, the hydrolytic products of starch, inhibited the synthesis of the caspase-3-like protein. Addition of glucose and cyclic AMP (cAMP) to the RCD-favoring medium prevented RCD. Glucose, cAMP, caffeine (a known inhibitor of a phosphodiesterase that breaks down cAMP), and forskolin (from the herb Coleus forskholii , known to activate the enzyme adenylate cyclase that forms cAMP) inhibited the caspase enzyme activity in vivo and consequently the RCD process. The addition of glucose and other inhibitors of RCD enhanced intracellular cAMP accumulation. This is the first report demonstrating the involvement of small molecules in the regulation of nutrition stress-related stationary-phase rapid cell death in X. campestris pv. glycines, which is programmed.

Three leuA -like protein-coding sequences were identified in Leptospira interrogans . One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded α-isopropylmalate synthase (EC 4.1.3.12). Expressed in Escherichia coli , the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile Methanococcus jannaschii , the L. interrogans enzyme is temperature sensitive but exhibits a much lower K m (0.04 mM) for pyruvate. The reaction product was characterized as ( R )-citramalate, and the proposed β-methyl- d -malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and β-isopropylmalate dehydrogenase (EC 1.1.1.85) in E. coli extracts bearing the corresponding clones and in vivo by complementation of E. coli ilvA , leuC / D , and leuB mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L. interrogans under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L. interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes.

Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in gluconeogenesis. Although FBPase activity has been detected in several hyperthermophiles, no orthologs corresponding to the classical FBPases from bacteria and eukaryotes have been identified in their genomes. An inositol monophosphatase (IMPase) from Methanococcus jannaschii which displayed both FBPase and IMPase activities and a structurally novel FBPase (Fbp Tk ) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 have been proposed as the “missing” FBPase. For this study, using T. kodakaraensis , we took a genetic approach to elucidate which candidate is the major gluconeogenic enzyme in vivo. The IMPase/FBPase ortholog in T. kodakaraensis , Imp Tk , was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs. We therefore constructed Δ fbp and Δ imp strains by applying a gene disruption system recently developed for T. kodakaraensis and investigated their phenotypes. The Δ fbp strain could not grow under gluconeogenic conditions while glycolytic growth was unimpaired, and the disruption resulted in the complete abolishment of intracellular FBPase activity. Evidently, fbp Tk is an indispensable gene for gluconeogenesis and is responsible for almost all intracellular FBPase activity. In contrast, the endogenous imp Tk gene could not complement the defect of the fbp deletion, and its disruption did not lead to any detectable phenotypic changes under the conditions examined. These facts indicated that imp Tk is irrelevant to gluconeogenesis, despite the high FBPase activity of its protein product, probably due to insufficient transcription. Our results provide strong evidence that the true FBPase for gluconeogenesis in T. kodakaraensis is the Fbp Tk ortholog, not the IMPase/FBPase ortholog.

The nitrifying bacterium Nitrosomonas europaea can obtain all its carbon for growth from CO 2 and all its energy and reductant for growth from the oxidation of NH 3 and is considered an obligate chemolithoautotroph. Previous studies have shown that N. europaea can utilize limited amounts of certain organic compounds, including amino acids, pyruvate, and acetate, although no organic compound has been reported to support the growth of N. europaea . The recently completed genomic sequence of N. europaea revealed a potential permease for fructose. With this in mind, we tested if N. europaea could utilize fructose and other compounds as carbon sources to support growth. Cultures were incubated in the presence of fructose or other organic compounds in sealed bottles purged of CO 2 . In these cultures, addition of either fructose or pyruvate as the sole carbon source resulted in a two- to threefold increase in optical density and protein content in 3 to 4 days. Studies with [ 14 C]fructose showed that >90% of the carbon incorporated by the cells during growth was derived from fructose. Cultures containing mannose, glucose, glycerol, mannitol, citrate, or acetate showed little or no growth. N. europaea was not able to grow with fructose as an energy source, although the presence of fructose did provide an energy benefit to the cells. These results show that N. europaea can be grown in CO 2 -free medium by using fructose and pyruvate as carbon sources and may now be considered a facultative chemolithoorganotroph.

Genome-scale in silico metabolic networks of Escherichia coli have been reconstructed. By using a constraint-based in silico model of a reconstructed network, the range of phenotypes exhibited by E. coli under different growth conditions can be computed, and optimal growth phenotypes can be predicted. We hypothesized that the end point of adaptive evolution of E. coli could be accurately described a priori by our in silico model since adaptive evolution should lead to an optimal phenotype. Adaptive evolution of E. coli during prolonged exponential growth was performed with M9 minimal medium supplemented with 2 g of α-ketoglutarate per liter, 2 g of lactate per liter, or 2 g of pyruvate per liter at both 30 and 37°C, which produced seven distinct strains. The growth rates, substrate uptake rates, oxygen uptake rates, by-product secretion patterns, and growth rates on alternative substrates were measured for each strain as a function of evolutionary time. Three major conclusions were drawn from the experimental results. First, adaptive evolution leads to a phenotype characterized by maximized growth rates that may not correspond to the highest biomass yield. Second, metabolic phenotypes resulting from adaptive evolution can be described and predicted computationally. Third, adaptive evolution on a single substrate leads to changes in growth characteristics on other substrates that could signify parallel or opposing growth objectives. Together, the results show that genome-scale in silico metabolic models can describe the end point of adaptive evolution a priori and can be used to gain insight into the adaptive evolutionary process for E. coli .

Escherichia coli ketopantoate hydroxymethyltransferase (KPHMT) catalyzes the first step in the biosynthesis pathway of pantothenate (vitamin B 5 ), the transfer of a hydroxymethyl group onto α-ketoisovalerate. Here we describe a detailed comparative analysis of the KPHMT crystal structure and the identification of structural homologues, some of which have remarkable similarities in their active sites, modes of binding to substrates, and mechanisms. We show that KPHMT forms a family within the phosphoenolpyruvate/pyruvate superfamily. Based on the analysis, we propose that in this superfamily there should be a subdivision into two groups. This paper completes our structural analysis of the E. coli enzymes in the pantothenate pathway.

Replacement of the native fermentation pathway in Escherichia coli B with a homo-ethanol pathway from Zymomonas mobilis ( pdc and adhB genes) resulted in a 30 to 50% increase in growth rate and glycolytic flux during the anaerobic fermentation of xylose. Gene array analysis was used as a tool to investigate differences in expression levels for the 30 genes involved in xylose catabolism in the parent (strain B) and the engineered strain (KO11). Of the 4,290 total open reading frames, only 8% were expressed at a significantly higher level in KO11 ( P < 0.05). In contrast, over half of the 30 genes involved in the catabolism of xylose to pyruvate were expressed at 1.5-fold- to 8-fold-higher levels in KO11. For 14 of the 30 genes, higher expression was statistically significant at the 95% confidence level ( xylAB, xylE, xylFG, xylR, rpiA, rpiB, pfkA, fbaA, tpiA, gapA, pgk, and pykA ) during active fermentation (6, 12, and 24 h). Values at single time points for only four of these genes ( eno, fbaA, fbaB, and talA ) were higher in strain B than in KO11. The relationship between changes in mRNA (cDNA) levels and changes in specific activities was verified for two genes ( xylA and xylB ) with good agreement. In KO11, expression levels and activities were threefold higher than in strain B for xylose isomerase ( xylA ) and twofold higher for xylulokinase ( xylB ). Increased expression of genes involved in xylose catabolism is proposed as the basis for the increase in growth rate and glycolytic flux in ethanologenic KO11.

A number of unique microorganisms that thrive at temperatures of 90°C or higher have been isolated in the last 2 decades. One of the best studied of these so-called hyperthermophiles is the anaerobic archaeon Pyrococcus furiosus, which grows optimally at 100°C by the fermentation of carbohydrates and peptides (15). The organism uses a modified Embden-Meyerhof glycolytic pathway that contains several novel enzymes (13, 23). For example, both hexokinase and phosphofructokinase are ADP- rather than ATP-dependent enzymes (24, 48). In addition, the expected glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase enzymes, which would convert glyceraldehyde-3-phosphate to 3-phosphoglycerate with the concomitant phosphorylation of ADP to ATP and reduction of NAD+ to NADH, appear to be replaced by a single enzyme, glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAPOR). GAPOR oxidizes glyceraldehyde-3-phosphate directly to 3-phosphoglycerate and uses ferredoxin rather than NAD+ as the electron acceptor. Purified GAPOR is a unique tungsten-containing enzyme that has no known analog in mesophilic archaea or bacteria (31). Another unusual step in glucose catabolism includes the conversion of acetyl coenzyme A (acetyl-CoA) to acetate. In anaerobic bacteria, this is a two-step process via acetyl phosphate catalyzed by phosphotransacetylase and acetate kinase. In P. furiosus, however, these two enzymes are replaced by acetyl-CoA synthetase, which converts acetyl-CoA directly to acetate and phosphorylates ADP to ATP (29). Other enzymes involved in glucose oxidation that have been purified from Pyrococcus species include pyruvate:ferredoxin oxidoreductase (4), enolase (36), and triosephosphate isomerase (25). In contrast to the enzymes described previously, these are quite similar (except in thermostability) to their mesophilic counterparts. P. furiosus also synthesizes glucose during growth on peptide-derived amino acids (15), but less work has been done to explore the gluconeogenic pathway. This is thought to occur by a reversal of the conventional Embden-Meyerhof pathway, and the activities of the enzymes proposed to be involved have been measured in cell extracts (44). Of the gluconeogenic-specific enzymes, only phosphoglycerate kinase (19) has been purified from Pyrococcus species. Herein we focus on the enzyme that carries out the first step in the conversion of pyruvate to glucose, phosphoenolpyruvate (PEP) synthetase, which catalyzes the phosphorylation of pyruvate according to equation 1: 1 Transcriptional analyses indicated that the cellular concentration of PEP synthetase increased when a glycolytic substrate such as maltose was added to the growth medium of P. furiosus (38, 39). To explain this result, it was suggested that this enzyme functions in glycolysis (reverse of equation 1) as well as in gluconeogenesis (39). However, there are no kinetic data to support this contention. It was, therefore, of some relevance to determine the catalytic properties of PEP synthetase from this organism. Surprisingly, little is known about PEP synthetases from prokaryotes, in spite of their importance in controlling carbon flow during glucose metabolism (20, 28, 37, 45, 47). The only one to be studied extensively is that from Escherichia coli (10, 11, 32, 33, 34). It is a cofactorless homodimer with a molecular mass of 150 kDa and catalyzes the phosphorylation of pyruvate by using ATP to generate PEP, AMP, and phosphate. Only two other PEP synthetases have been purified and examined to any extent, and both are from archaea, the moderately thermophilic methanogen Methanobacterium thermoautotrophicum (14) and the heterotrophic hyperthermophile Staphylothermus marinus (8, 9, 16, 17). The quaternary structure of the former enzyme was not reported, but S. marinus PEP synthetase exists as a large homomultimeric complex containing 24 subunits (17). Conversely, while the kinetic properties of the latter enzyme are unknown, those of the M. thermoautotrophicum enzyme are similar to those of E. coli PEP synthetase (14). It was therefore of some interest to investigate the physical and catalytic properties of the enzyme from P. furiosus, especially as the enzyme has been reported to be sensitive to inactivation by oxygen (44). For example, did the enzyme exist as a large complex, did it function in both gluconeogenesis and glycolysis, did it utilize nucleotides other than ATP as the phosphate donor for PEP synthesis, and what may be the cause of its oxygen sensitivity?

The capacity of Escherichia coli to adapt its catabolism to prevailing redox conditions resides mainly in three catabolic branch points involving (i) pyruvate formate-lyase (PFL) and the pyruvate dehydrogenase complex (PDHc), (ii) the exclusively fermentative enzymes and those of the Krebs cycle, and (iii) the alternative terminal cytochrome bd and cytochrome bo oxidases. A quantitative analysis of the relative catabolic fluxes through these pathways is presented for steady-state glucose-limited chemostat cultures with controlled oxygen availability ranging from full aerobiosis to complete anaerobiosis. Remarkably, PFL contributed significantly to the catabolic flux under microaerobic conditions and was found to be active simultaneously with PDHc and cytochrome bd oxidase-dependent respiration. The synthesis of PFL and cytochrome bd oxidase was found to be maximal in the lower microaerobic range but not in a ΔArcA mutant, and we conclude that the Arc system is more active with respect to regulation of these two positively regulated operons during microaerobiosis than during anaerobiosis.

The porin composition of the Escherichia coli cell envelope was analyzed during growth at different external pHs (pHo) as a function of the acetyl phosphate (AcP) level (Δ ackA pta or ackA mutant, pyruvate or glucose as the carbon source) in the presence or absence of EnvZ. Our results indicate that the AcP level is influenced by the pHo, leading to modulation of the amount of OmpR-P and subsequent pHo-dependent expression of ompF and ompC . We also propose the existence of a specific signal, independent of EnvZ and AcP, leading to OmpR phosphorylation in response to pyruvate.

Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13 C labeling with a mixture of 90% unlabeled and 10% uniformly 13 C-labeled glycerol. The resulting 13 C-labeling patterns in the amino acids were analyzed by two-dimensional 13 C, 1 H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13 C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya.

Regulation of the katB catalase gene in the anaerobic bacterium Bacteroides fragilis was studied. Northern blot hybridization analyses revealed that katB was transcribed as an approximately 1.6-kb monocistronic mRNA. The levels of katB mRNA increased > 15-fold when anaerobic, mid-logarithmic-phase cultures were exposed to O2, O2 with paraquat, or hydrogen peroxide. Under anaerobic conditions, the low levels of katB mRNA increased in a growth-dependent manner, reaching maximum expression at late logarithmic or early stationary phase, followed by a decrease in stationary phase. Under anaerobic conditions, the expression of katB mRNA was strongly repressed by glucose and to a lesser extent by xylose. However, glucose repression was completely abolished upon exposure to oxygen. The nonfermentable carbon sources fumarate, succinate, acetate, and pyruvate did not significantly affect expression. Phosphate, nitrogen, and hemin limitation did not affect the expression of katB mRNA, suggesting that the nutritional control of katB expression is restricted to carbon and energy sources and not other forms of nutrient limitation. Primer extension analysis revealed that during both oxidative stress and carbon or energy limitation, katB utilized the same promoter region but transcription initiation occurred at two different nucleotides separated by 3 or 4 bases. Interestingly, a 6-bp inverted repeat sequence present in the katB regulatory region was also observed upstream of the B. fragilis superoxide dismutase gene sod. It is possible that this is a recognition site for a DNA binding protein involved in the regulation of oxidative stress genes in this organism.

Excretion of alpha-keto acids by clinical isolates and laboratory strains of Salmonella typhimurium was determined by high-performance liquid chromatography analysis of culture supernatants. The levels of excretion increased markedly with increasing iron stress imposed by the presence of alpha,alpha'-dipyridyl or conalbumin in the medium. The major product was pyruvic acid, but significant concentrations of alpha-ketoglutaric acid, alpha-ketoisovaleric acid, and alpha-ketoisocaproic acid were also observed. Maximal excretion occurred at iron stress levels that initially inhibited bacterial growth; the concentration of alpha,alpha'-dipyridyl at which this was observed differed between strains depending on their ability to secrete and utilize siderophores, suggesting that the intracellular iron status was important in determining alpha-keto acid excretion. However, prolonged incubation of the siderophore-deficient S. typhimurium strain enb-7 under conditions of high iron stress resulted in significant delayed bacterial growth, promoted by tonB-dependent uptake of iron complexed with the high accumulated levels of pyruvic acid and other alpha-keto acids. Strain RB181, a fur derivative of enb-7, excreted massive amounts of alpha-keto acids into the culture medium even in the absence of any iron chelators (the concentration of pyruvic acid, for example, was >25 mM). Moreover, RB181 was able to grow and excrete alpha-keto acids in the presence of alpha,alpha'-dipyridyl at concentrations threefold greater than that which inhibited the growth of enb-7.

The synthesis of pyruvate carboxylase (PC) was studied by using quantitative immunoblot analysis with an antibody raised against PC purified from Rhodobacter capsulatus and was found to vary 20-fold depending on the growth conditions. The PC content was high in cells grown on pyruvate or on carbon substrates metabolized via pyruvate (lactate, D-malate, glucose, or fructose) and low in cells grown on tricarboxylic acid (TCA) cycle intermediates or substrates metabolized without intermediate formation of pyruvate (acetate or glutamate). Under dark aerobic growth conditions with lactate as a carbon source, the PC content was approximately twofold higher than that found under light anaerobic growth conditions. The results of incubation experiments demonstrate that PC synthesis is induced by pyruvate and repressed by TCA cycle intermediates, with negative control dominating over positive control. The content of PC in R. capsulatus cells was also directly related to the growth rate in continuous cultures. The analysis of intracellular levels of pyruvate and TCA cycle intermediates in cells grown under different conditions demonstrated that the content of PC is directly proportional to the ratio between pyruvate and C4 dicarboxylates. These results suggest that the regulation of PC synthesis by oxygen and its direct correlation with growth rate may reflect effects on the balance of intracellular pyruvate and C4 dicarboxylates. Thus, this important enzyme is potentially regulated both allosterically and at the level of synthesis.

The fermentative capacities of the acetogenic bacterium Peptostreptococcus productus U-1 (ATCC 35244) were examined. Although acetate was formed from all the substrates tested, additional products were produced in response to CO2 limitation. Under CO2-limited conditions, fructose-dependent growth yielded high levels of lactate as a reduced end product; lactate was also produced under CO2-enriched conditions when fructose concentrations were elevated. In the absence of supplemental CO2, xylose-dependent growth yielded lactate and succinate as major reduced end products. Although supplemental CO2 and acetogenesis stimulated cell yields on fructose, xylose-dependent cell yields were decreased in response to CO2 and acetogenesis. In contrast, glycerol-dependent growth yielded high levels of ethanol in the absence of supplemental CO2, and pyruvate was subject to only acetogenic utilization independent of CO2. CO2 pulsing during the growth of CO2-limited fructose cultures stopped lactate synthesis immediately, indicating that CO2-limited cells were nonetheless metabolically poised to respond quickly to exogenous CO2. Resting cells that were cultivated at the expense of fructose without supplemental CO2 readily consumed fructose in the absence of exogenous CO2 and formed only lactate. Although the specific activity of lactate dehydrogenase was not appreciably influenced by supplemental C02 during cultivation, cells cultivated on fructose under CO2-enriched conditions displayed minimal capacities to consume fructose in the absence of exogenous CO2. These results demonstrate that the utilization of alternative fermentations for the conservation of energy and growth of P. productus U-1 is augmented by the relative availability of CO2 and growth substrate.

Enzymes involved in (methyl)phenol degradation of Pseudomonas putida H are encoded by the catabolic operon (phlA-L) on plasmid pPGH1. Transcription of this operon by the sigma54 (RpoN)-containing RNA polymerase is positively controlled by the gene product of the divergently transcribed phlR in response to the availability of the respective substrate. Additionally, phenol degradation is subject to carbon catabolite repression induced by organic acids (e.g., succinate, lactate, and acetate) or carbohydrates (e.g., glucose and gluconate). Analysis of lacZ fusion to the catabolic promoter and quantified primer extension experiments indicate that carbon catabolite repression also occurs at the transcriptional level of the catabolic operon. In this study, it is furthermore shown that carbon catabolite repression is a negative control. Titration of the postulated negative controlling factor was exclusively observed when extra copies of functional phlR gene were present in the cell. We therefore conclude that PhlR is the target and that carbon catabolite repression of phenol degradation occurs by interfering with the activating function of PhlR.

We report the cloning of the pykA and pykF genes from Escherichia coli, which code for the two pyruvate kinase isoenzymes (ATP:pyruvate 2-O-phosphotransferases; EC 2.7.1.40) in this microorganism. These genes were insertionally inactivated with antibiotic resistance markers and utilized to interrupt one or both pyk genes in the E. coli chromosome. With these constructions, we were able to study the role of these isoenzymes in pyruvate biosynthesis.

The putative glycine dehydrogenase of Mycobacterium tuberculosis catalyzes the reductive amination of glyoxylate to glycine but not the reverse reaction. The enzyme was purified and identified as the previously characterized alanine dehydrogenase. The Ald enzyme was expressed in Escherichia coli and had both pyruvate and glyoxylate aminating activities. The gene, ald, was inactivated in M. tuberculosis, which resulted in the loss of all activities. Both enzyme activities were found associated with the cell and were not detected in the extracellular filtrate. By using an anti-Ald antibody, the protein was localized to the cell membrane, with a smaller fraction in the cytosol. None was detected in the extracellular medium. The ald knockout strain grew without alanine or glycine and was able to utilize glycine but not alanine as a nitrogen source. Transcription of ald was induced when alanine was the sole nitrogen source, and higher levels of Ald enzyme were measured. Ald is proposed to have several functions, including ammonium incorporation and alanine breakdown.

13C nuclear magnetic resonance spectroscopy was used to study the metabolism of [2-13C]pyruvate in intact cells of Halobacterium salinarium. The spectra of these cells show that pyruvate is reduced to lactic acid and transaminated to alanine. The intensity of C-2 lactate is higher under anaerobic conditions than under aerobic conditions. When cells are grown in the absence of glucose, the level of C-2 lactate intensity is lower. In extracts of these cells, the level of NADH-dependent lactate dehydrogenase activity is lower than that of cells grown in the presence of glucose. A C-5 glutamate resonance suggests the entry of pyruvate into the tricarboxylic acid cycle through acetyl-coenzyme A. In addition, the label is also observed at C-3 and C-4 of glutamate, signifying a pyruvate carboxylase-type reaction and scrambling of label at the fumarate-succinate stage plus malic enzyme operation, respectively. Citrate synthase and malic enzyme activity appear to be controlled by the growth conditions of H. salinarium.

The expression of the pyruvate formate-lyase gene (pfl) is induced by anaerobic growth, and this is increased further by growth in pyruvate. Previous work has shown that anaerobic induction is strongly dependent on the activator FNR and partially dependent on a second transcription factor, ArcA, while pyruvate induction only required FNR. Anaerobic and pyruvate regulation both require the presence of a 5' nontranslated regulatory sequence which spans approximately 500 bp of DNA. A mobility shift assay was developed to identify proteins that bind to this regulatory region. Several binding activities were separated by heparin agarose chromatography, and one of these activities was characterized and shown to be integration host factor (IHF). Mobility shift and DNase I footprinting experiments defined a single IHF binding site in the pfl promoter-regulatory region. With pfl-lacZ fusions, it could be shown that introduction of a himD mutation abolished pyruvate-dependent induction of anaerobic expression in vivo. The same result was observed when the pfl IHF binding site was mutated. In addition, the partial anaerobic induction of expression found in an fnr strain was completely blocked in an fnr himD double mutant and in an fnr IHF binding site double mutant. Taken together, these data suggest that IHF is necessary for both pyruvate induction and the anaerobic induction mediated by ArcA.

Nitrogen fixation in legume bacteroids is energized by the metabolism of dicarboxylic acids, which requires their oxidation to both oxaloacetate and pyruvate. In alfalfa bacteroids, production of pyruvate requires NAD + malic enzyme (Dme) but not NADP + malic enzyme (Tme). However, we show that Rhizobium leguminosarum has two pathways for pyruvate formation from dicarboxylates catalyzed by Dme and by the combined activities of phosphoenolpyruvate (PEP) carboxykinase (PckA) and pyruvate kinase (PykA). Both pathways enable N 2 fixation, but the PckA/PykA pathway supports N 2 fixation at only 60% of that for Dme. Double mutants of dme and pckA/pykA did not fix N 2 . Furthermore, dme pykA double mutants did not grow on dicarboxylates, showing that they are the only pathways for the production of pyruvate from dicarboxylates normally expressed. PckA is not expressed in alfalfa bacteroids, resulting in an obligate requirement for Dme for pyruvate formation and N 2 fixation. When PckA was expressed from a constitutive nptII promoter in alfalfa dme bacteroids, acetylene was reduced at 30% of the wild-type rate, although this level was insufficient to prevent nitrogen starvation. Dme has N-terminal, malic enzyme (Me), and C-terminal phosphotransacetylase (Pta) domains. Deleting the Pta domain increased the peak acetylene reduction rate in 4-week-old pea plants to 140 to 150% of the wild-type rate, and this was accompanied by increased nodule mass. Plants infected with Pta deletion mutants did not have increased dry weight, demonstrating that there is not a sustained change in nitrogen fixation throughout growth. This indicates a complex relationship between pyruvate synthesis in bacteroids, nitrogen fixation, and plant growth.

Fifteen-fold overexpression of phosphoenolpyruvate synthase (Pps) (EC 2.7.9.2) in Escherichia coli stimulated oxygen consumption in glucose minimal medium. A further increase in Pps overexpression to 30-fold stimulated glucose consumption by approximately 2-fold and resulted in an increased excretion of pyruvate and acetate. Insertion of two codons at the PvuII site in the pps gene abolished the enzymatic activity and eliminated the above-described effects. Both the active and the inactive proteins were detected at the predicted molecular weight by polyacrylamide gel electrophoresis. Therefore, the observed physiological changes were due to the activity of Pps. The higher specific rates of consumption of oxygen and glucose indicate a potential futile cycle between phosphoenolpyruvate (PEP) and pyruvate. A model for the stimulation of glucose uptake is presented; it involves an increased PEP/pyruvate ratio caused by the overexpressed Pps activity, leading to a stimulation of the PEP:sugar phosphotransferase system.

The levels of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Rhodobacter sphaeroides were found to depend on the concentration of ammonia supplied to photolithoautotrophically grown cultures. Under conditions in which the cells rapidly depleted the available ammonia, the level of in situ RubisCO activity decreased to less than 5% maximum activity; even at its maximum level under these conditions, the RubisCO activity was only 5% of the activity obtained from cultures supplied with saturating levels of ammonia. When cells were incubated with somewhat higher but not saturating amounts of ammonia, in situ RubisCO activity decreased immediately after the cells depleted the cultures of ammonia. The decrease in activity was not due to any detectable degradation of RubisCO protein, indicative of some mechanism to regulate the activity of the enzyme in response to the intracellular levels of assimilated ammonia. Furthermore, under conditions optimum for RubisCO inactivation, in situ RubisCO activity in permeabilized whole cells greatly exceeded the levels of enzymatic activity determined in vitro in cell extracts. Blockage of ammonia assimilation by inhibition of glutamine synthetase with methionine sulfoximine prevented the recovery of form I RubisCO from pyruvate-mediated inactivation, suggesting the presence of regulatory mechanisms common to both CO2 fixation and ammonia assimilation.

To better understand the global effects of “natural” lesions in genes involved in the pyruvate metabolism in Mycobacterium bovis , null mutations were made in the Mycobacterium tuberculosis H37Rv ald and pykA genes to mimic the M. bovis situation. Like M. bovis , the M. tuberculosis Δ pykA mutant yielded dysgonic colonies on solid medium lacking pyruvate, whereas colony morphology was eugonic on pyruvate-containing medium. Global effects of the loss of the pykA gene, possibly underlying colony morphology, were investigated by using proteomics on cultures grown in the same conditions. The levels of Icd2 increased and those of Icl and PckA decreased in the Δ pykA knockout. Proteomics suggested that the synthesis of enzymes involved in fatty acid and lipid biosynthesis were decreased, whereas those involved in β-oxidation were increased in the M. tuberculosis Δ pykA mutant, as confirmed by direct assays for these activities. Thus, the loss of pykA from M. tuberculosis results in fatty acids being used principally for energy production, in contrast to the situation in the host when carbon from fatty acids is conserved through the glyoxylate cycle and gluconeogenesis; when an active pykA gene was introduced into M. bovis , the opposite effects occurred. Proteins involved in oxidative stress—AhpC, KatG, and SodA—showed increased synthesis in the Δ pykA mutant, and iron-regulated proteins were also affected. Ald levels were decreased in the Δ pykA knockout, explaining why an M. tuberculosis Δ pykA Δ ald double mutant showed little additional phenotypic effect. Overall, these data show that the loss of the pykA gene has powerful, global effects on proteins associated with central metabolism.

Escherichia coli has several elaborate sensing mechanisms for response to availability of oxygen and other electron acceptors, as well as the carbon source in the surrounding environment. Among them, the CreBC and ArcAB two-component signal transduction systems are responsible for regulation of carbon source utilization and redox control in response to oxygen availability, respectively. We assessed the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions by means of 13C-labeling experiments in chemostat cultures of a wild-type strain, ΔcreB and ΔarcA single mutants, and a ΔcreB ΔarcA double mutant. Continuous cultures were conducted at D = 0.1 h-1 under carbon-limited conditions with restricted oxygen supply. Although all experimental strains metabolized glucose mainly through the Embden-Meyerhof- Parnas pathway, mutant strains had significantly lower fluxes in both the oxidative and the nonoxidative pentose phosphate pathways. Significant differences were also found at the pyruvate branching point. Both pyruvate-formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis from pyruvate, and their activity seemed to be modulated by both ArcAB and CreBC. Strains carrying the creB deletion showed a higher biomass yield on glucose compared to the wild-type strain and its ΔarcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate shunt and lactate dehydrogenase were active mainly in the ΔarcA strain. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in the mutant strains. Copyright © 2009, American Society for Microbiology. All Rights Reserved. Fil: Nikel, Pablo Ivan. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Rice University; Estados Unidos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Universidad Nacional de San Martín; Argentina Fil: Zhu, Jiangfeng. Rice University; Estados Unidos Fil: San, Ka-Yiu. Rice University; Estados Unidos Fil: Mendez, Beatriz Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Bennett, George N.. Rice University; Estados Unidos

Strain DCB-1 is a strict anaerobe capable of reductive dehalogenation. We elucidated metabolic processes in DCB-1 which may be related to dehalogenation and which further characterize the organism physiologically. Sulfoxy anions and CO2 were used by DCB-1 as catabolic electron acceptors. With suitable electron donors, sulfate and thiosulfate were reduced to sulfide. Sulfate and thiosulfate supported growth with formate or hydrogen as the electron donor and thus are probably respiratory electron acceptors. Other electron donors supporting growth with sulfate were CO, lactate, pyruvate, butyrate, and 3-methoxybenzoate. Thiosulfate also supported growth without an additional electron donor, being disproportionated to sulfide and sulfate. In the absence of other electron acceptors, CO2 reduction to acetate plus cell material was coupled to pyruvate oxidation to acetate plus CO2. Pyruvate could not be fermented without an electron acceptor. Carbon monoxide dehydrogenase activity was found in whole cells, indicating that CO2 reduction probably occurred via the acetyl coenzyme A pathway. Autotrophic growth occurred on H2 plus thiosulfate or sulfate. Diazotrophic growth occurred, and whole cells had nitrogenase activity. On the basis of these physiological characteristics, DCB-1 is a thiosulfate-disproportionating bacterium unlike those previously described.

Pyruvate-dependent reduction of NADP has been demonstrated in cell extracts of the human gastric pathogen Helicobacter pylori. However, NADP is not a substrate of purified pyruvate:ferredoxin oxidoreductase (PFOR), suggesting that other redox active enzymes mediate this reaction. Here we show that fqrB (HP1164), which is essential and highly conserved among the epsilonproteobacteria, exhibits NADPH oxidoreductase activity. FqrB was purified by nickel interaction chromatography following overexpression in Escherichia coli . The protein contained flavin adenine dinucleotide and exhibited NADPH quinone reductase activity with menadione or benzoquinone and weak activity with cytochrome c , molecular oxygen, and 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB). FqrB exhibited a ping-pong catalytic mechanism, a k cat of 122 s −1 , and an apparent K m of 14 μM for menadione and 26 μM for NADPH. FqrB also reduced flavodoxin (FldA), the electron carrier of PFOR. In coupled enzyme assays with purified PFOR and FldA, FqrB reduced NADP in a pyruvate- and reduced coenzyme A (CoA)-dependent manner. Moreover, in the presence of NADPH, CO 2 , and acetyl-CoA, the PFOR:FldA:FqrB complex generated pyruvate via CO 2 fixation. PFOR was the rate-limiting enzyme in the complex, and nitazoxanide, a specific inhibitor of PFOR of H. pylori and Campylobacter jejuni , also inhibited NADP reduction in cell-free lysates. These capnophilic (CO 2 -requiring) organisms contain gaps in pathways of central metabolism that would benefit substantially from pyruvate formation via CO 2 fixation. Thus, FqrB provides a novel function in pyruvate metabolism and, together with production of superoxide anions via quinone reduction under high oxygen tensions, contributes to the unique microaerobic lifestyle that defines the epsilonproteobacterial group.

The archaeon Methanocaldococcus jannaschii uses three different 2-oxoacid elongation pathways, which extend the chain length of precursors in leucine, isoleucine, and coenzyme B biosyntheses. In each of these pathways an aconitase-type hydrolyase catalyzes an hydroxyacid isomerization reaction. The genome sequence of M. jannaschii encodes two homologs of each large and small subunit that forms the hydrolyase, but the genes are not cotranscribed. The genes are more similar to each other than to previously characterized isopropylmalate isomerase or homoaconitase enzyme genes. To identify the functions of these homologs, the four combinations of subunits were heterologously expressed in Escherichia coli , purified, and reconstituted to generate the iron-sulfur center of the holoenzyme. Only the combination of MJ0499 and MJ1277 proteins catalyzed isopropylmalate and citramalate isomerization reactions. This pair also catalyzed hydration half-reactions using citraconate and maleate. Another broad-specificity enzyme, isopropylmalate dehydrogenase (MJ0720), catalyzed the oxidative decarboxylation of β-isopropylmalate, β-methylmalate, and d -malate. Combined with these results, phylogenetic analysis suggests that the pyruvate pathway to 2-oxobutyrate (an alternative to threonine dehydratase in isoleucine biosynthesis) evolved several times in bacteria and archaea. The enzymes in the isopropylmalate pathway of leucine biosynthesis facilitated the evolution of 2-oxobutyrate biosynthesis through the introduction of a citramalate synthase, either by gene recruitment or gene duplication and functional divergence.