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PubMed1982

Mapping nonselectable genes of Escherichia coli by using transposon Tn10: location of a gene affecting pyruvate oxidase.

Mutants of Escherichia coli K-12 deficient in pyruvate oxidase were isolated by screening for the production of 14CO2 from [1-14C]pyruvate by the method of Tabor et al. (J. Bacteriol. 128:485-486, 1976). One of these lesions (designated poxA) decreased the pyruvate oxidase activity to 10 to 15% of the normal level but grew well. To map this nonselectable mutation, we isolated strains having transposon Tn10 inserted into the chromosome close to the poxA locus and mapped the transposon. These insertions were isolated by the following procedure: (i) pools of Tn10 insertions into the chromosomes of two different Hfr strains were prepared by transposition from a lambda::Tn10 vector; (ii) these Tn10-carrying strains were then mated with a poxA recipient strain, and tetracycline-resistant (Tetr) recombinants were selected; (iii) the Tetr recombinants were then screened for 14CO2 production from [1-14C]pyruvate. This method was shown to give a greater than 40-fold enrichment of insertions of Tn10 near the poxA gene as compared with transduction. Calculations indicate that a similar enrichment should be expected for other genes. The enrichment is due to the much greater map interval over which strong linkage between selected and unselected markers is found in conjugational crosses as compared with transductional crosses. The use of Hfr conjugative transfer allows isolation of transposon insertions closely linked to a nonselectable gene by scoring hundreds rather than thousands of colonies. Using a Tn10 insertion greater than 98% cotransduced with the poxA locus, we mapped the poxA gene on the E. coli genetic map. The poxA locus is located at 94 min, close to the psd locus. The clockwise gene order is ampA, poxA, psd, purA. The poxA mutation is recessive and appears to be a regulatory gene.

Chromosome Mapping↗

PubMed1984

Molecular cloning of the gene (poxB) encoding the pyruvate oxidase of Escherichia coli, a lipid-activated enzyme.

The pyruvate oxidase structural gene (poxB) of Escherichia coli was cloned into derivatives of plasmid pBR322. The gene was first cloned into a cosmid vector by selection for the tetracycline resistance determinant of a closely linked Tn10 insertion (no direct selection for the gene was available). Subsequent subcloning resulted in localization of the gene to a 3.1-kilobase-pair DNA segment. Two of the smaller poxB plasmids were shown to cause the overproduction of oxidase activity (by six- to eightfold), and one of these plasmids was shown to encode a protein having the size and antigenic determinants of pyruvate oxidase. Introduction of poxB plasmids into strains (aceEF) lacking pyruvate dehydrogenase activity relieved the aerobic growth requirement of the strains for exogenous acetate.

Alleles↗

PubMed1986

Molecular cloning, DNA sequencing, and enzymatic analyses of two Escherichia coli pyruvate oxidase mutants defective in activation by lipids.

Two Escherichia coli pyruvate oxidase (EC 1.2.2.2) mutant genes, poxB3 and poxB4, were cloned on plasmid pBR322. The poxB3 mutant oxidase which was described previously (Y. Y. Chang and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. USA 81:4348-4352, 1984) was deficient in lipid activation but retained full catalytic activity. The poxB3 mutation was located in the C-terminal half of the gene, and the nucleotide alteration has been determined by DNA sequencing of this part of the gene and by comparing the sequence with that of the wild-type strain (C. Grabau and J. E. Cronan, Jr., submitted for publication). The poxB3 oxidase mutation is the substitution of a serine residue for Pro-536. poxB4, another pyruvate oxidase mutant gene, was also deficient in lipid activation. The major difference between the poxB3 and poxB4 oxidase was in the binding of Triton detergents. The poxB4 mutation was also located in the C-terminal half of the gene, and sequence analysis has shown that only one nucleotide base was altered, which resulted in Ala-467 being converted to a threonine residue. The results of the amino acid substitutions in the mutant proteins, leading to the functional alteration of the enzyme, are discussed.

Alleles↗

PubMed1982

Reconstitution of native Escherichia coli pyruvate oxidase from apoenzyme monomers and FAD.

Pyruvate oxidase, a tetrameric enzyme consisting of 4 identical subunits, dissociates into apoenzyme monomers and free FAD when treated with acid ammonium sulfate in the presence of high concentrations of potassium bromide. Reconstitution of the native enzymatically active protein can be accomplished by incubating equimolar concentrations of apomonomers and FAD at pH 6.5. The kinetics of the reconstitution reaction have been measured by 1) enzyme activity assays, 2) spectrophotometric assays to measure FAD binding, and 3) high performance liquid chromatography analysis measuring the distribution of monomeric, dimeric, and tetrameric species during reconstitution. The kinetic analysis indicates that the second order reaction of apomonomers with FAD to form an initial monomer-FAD complex is fast. The rate-limiting step for enzymatic reactivation appears to be the folding of the polypeptide chain in the monomer-FAD complex to reconstitute the three-dimensional FAD binding site prior to subunit reassociation. The subsequent formation of native tetramers appears to proceed via an essentially irreversible dimer assembly pathway.

Apoenzymes↗

PubMed2002

[Construction of the pyruvate oxidase gene deficiency variant strain of Streptococcus oralis].

OBJECTIVE: The purpose of this study is to construct a pyruvate oxidase gene deficiency variant strain of Streptococcus oralis (S. oralis). METHODS: The sopox gene, which was got using polymerase chain reaction (PCR), and the 130-basepair segment of which was cut down with endonuclease BamHI, and transferred into S. oralis (ATCC10557) by using electrotransformation. The authors obtained a variant strain of S. oralis, and then the catalase activity of the first culture and 3-4 subcultures was examined. RESULTS: The authors obtained a pyruvate oxidase gene deficiency variant strain of S. orlis. The catalase activity examination showed that the ability of producing H2 O2 of the variant strain of S. orlis declined, whose catalase activity was between those of the positive control (ATCC10557) and the negative control (Escherichia coli, JM109). But the produced H2 O2 quantity of their subcultures was less than that of the negative control. CONCLUSION: The construction of the pyruvate oxidase gene deficiency variant strain of Streptococcus oralis is successful.

Cloning, Molecular↗

PubMed1988

Common ancestry of Escherichia coli pyruvate oxidase and the acetohydroxy acid synthases of the branched-chain amino acid biosynthetic pathway.

A number of enzymes require flavin for their catalytic activity, although the reaction catalyzed involves no redox reaction. The best studied of these enigmatic nonredox flavoproteins are the acetohydroxy acid synthases (AHAS), which catalyze early steps in the synthesis of branched-chain amino acids in bacteria, yeasts, and plants. Previously, work from our laboratory showed strong amino acid sequence homology between these enzymes and Escherichia coli pyruvate oxidase, a classical flavoprotein dehydrogenase that catalyzes the decarboxylation of pyruvate to acetate. We have now shown this homology (i) to also be present in the DNA sequences and (ii) to represent functional homology in that pyruvate oxidase has AHAS activity and a protein consisting of the amino-terminal half of pyruvate oxidase and the carboxy-terminal half of E. coli AHAS I allows native E. coli AHAS I to function without added flavin. The hybrid protein contains tightly bound flavin, which is essential for the flavin substitution activity. These data, together with the sequence homologies and identical cofactors and substrates, led us to propose that the AHAS enzymes are descended from pyruvate oxidase (or a similar protein) and, thus, that the flavin requirement of the AHAS enzymes is a vestigial remnant, which may have been conserved to play a structural rather than a chemical function.

Acetolactate Synthase↗

PubMed2003

[DNA sequence analysis for the promoter of pyruvate oxidase gene from Streptococcus oralis].

OBJECTIVE: To elucidate the molecular structure of pyruvate oxidase gene promoter. METHODS: The 1.30 kb fragment with promoter activity, amplified from upstream of Streptococcus oralis pyruvate oxidase gene (Sopox), was cloned into vector PBK-CMV. The positive transformed E. coli JM109 clone was selected, the recombinant plasmid was further identified with restriction mapping analysis. The positive recombinant plasmid was studied with sequence analysis. RESULTS: After digesting the recombinant plasmid with Hind III, 1% agarose electrophoresis showed 1.30 kb fragment, which was consistent with predicted size. Sequence analysis revealed 1,350 bp. CONCLUSION: The Sopox promoter region is sequenced. Further characterization of the Sopox promoter region will elucidate the molecular mechanism of H2O2 production of streptococcus oralis.

Base Sequence↗

PubMed2001

[Pyruvate oxidase gene from Streptococcus sanguis: molecular cloning and sequence analysis of the gene].

OBJECTIVE: To clone and sequence the gene of pyruvate oxidase (Sopox) from Streptococcus sanguis. METHODS: The PCR primers for Sopox gene were designed and synthesized according to the sequence of pyruvate oxidase (spxB) gene of S. pneumonia. The amplified PCR product was cloned into pUC18 and then subcloned into M13mp18 and M13mp19. The DNA sequence of the gene was analyzed. RESULTS: Sopox gene was successfully amplified from S. sanguis ATCC10557. The nucleotide sequence of the whole gene was revealed to be 1788 base pairs with one open reading frame coding pyruvate oxidase with 591 amino acid residuals. CONCLUSION: The clone and DNA sequence of Sopox gene were obtained which could serve as a foundation on which to elucidate the molecular mechanisms of hydrogen peroxide production and its regulation by oral streptococci.

Base Sequence↗

PubMed1982

Proximity of reactive cysteine residue and flavin in Escherichia coli pyruvate oxidase as estimated by fluorescence energy transfer.

Pyruvate oxidase of Escherichia coli possesses a reactive cysteine residue believed to be associated with the thiamin pyrophosphate (TPP) binding site. This residue is not reactive in the presence of TPP. Exposure of the enzyme to cysteine-directed fluorescent reagents results in the formation of fluorescent protein conjugates. Although these reagents do not react solely with the TPP-protectable cysteine residue, the fluorescence emission spectrum of a probe attached to this residue can be obtained by a difference technique. It was determined that the fluorescence emission of probes at the TPP-protectable site is very low due to energy transfer to the FAD coenzyme and that this fluorescence is greatly enhanced upon reduction or extraction of the flavin. Application of fluorescence energy transfer theory enabled the determination of an upper limit for the distance between the probes at the TPP-protectable site and the flavin adenine dinucleotide (FAD) (roughly 20 A). Thus, the TPP binding site and the FAD coenzyme are likely in close proximity.

Anilino Naphthalenesulfonates↗

PubMed1986

Nucleotide sequence and deduced amino acid sequence of Escherichia coli pyruvate oxidase, a lipid-activated flavoprotein.

The entire nucleotide sequence of the poxB (pyruvate oxidase) gene of Escherichia coli K-12 has been determined by the dideoxynucleotide (Sanger) sequencing of fragments of the gene cloned into a phage M13 vector. The gene is 1716 nucleotides in length and has an open reading frame which encodes a protein of Mr 62,018. This open reading frame was shown to encode pyruvate oxidase by alignment of the amino acid sequences deduced for the amino and carboxy termini and several internal segments of the mature protein with sequences obtained by amino acid sequence analysis. The deduced amino acid sequence of the oxidase was not unusually rich in hydrophobic sequences despite the peripheral membrane location and lipid binding properties of the protein. The codon usage of the oxidase gene was typical of a moderately expressed protein. The deduced amino acid sequence shares homology with the large subunits of the acetohydroxy acid synthase isozymes I, II, and III, encoded by the ilvB, ilvG, and ilvI genes of E. coli.

Acetolactate Synthase↗

PubMed2000

Mechanism of elementary catalytic steps of pyruvate oxidase from Lactobacillus plantarum.

Single steps in the catalytic cycle of pyruvate oxidase from Lactobacillus plantarum have been characterized kinetically and mechanistically by stopped-flow in combination with kinetic solvent isotope effect studies. Reversible substrate binding of pyruvate occurs with an on-rate of 6.5 x 10(4) M(-1) s(-1) and an off-rate of pyruvate of 20 s(-1). Decarboxylation of the intermediate lactyl-ThDP and the reduction of FAD which consists of two consecutive single electron-transfer steps from HEThDP to FAD occur with rates of about k(dec) = 112 s(-1) and k(red) = 422 s(-1). Flavin radical intermediates are not observed during reduction, and kinetic solvent isotope effects are absent, indicating that electron transfer and protonation processes are not rate limiting in the overall reduction process. Reoxidation of FADH(2) by O(2) to yield H(2)O(2) takes place at a pseudo-first-order rate of about 35 s(-1) in air-saturated buffer. A comparable value of about 35 s(-1) was estimated for the phosphorolysis of the acetyl-ThDP intermediate at phosphate saturation. In competition with phosphorolysis, enzyme-bound acetyl-ThDP is hydrolyzed with a rate k = 0.03 s(-1). This is the first report in which the reaction of enzyme-bound acetyl-ThDP with phosphate and OH(-) is monitored directly by FAD absorbance changes using the sequential stopped-flow technique.

2,6-Dichloroindophenol↗

PubMed1985

Spectroscopic studies of pyruvate oxidase flavoprotein from Escherichia coli trapped in the lipid-activated form by cross-linking.

Pyruvate oxidase is a flavoprotein dehydrogenase isolated from Escherichia coli which catalyzes the oxidative decarboxylation of pyruvate to acetate plus CO2. The maximal turnover of the enzyme, measured using a ferricyanide reductase assay, is increased 20-to 30-fold by either of two methods. Proteolysis in the presence of the substrate (pyruvate) and cofactor (Mg2+-thiamin pyrophosphate) results in cleavage at a single locus near the carboxyl terminus and concomitant activation. Phospholipids and detergents can bind to the enzyme and result in a similar activation, which is presumed to be physiologically relevant, since the enzyme functions as a peripheral membrane enzyme. Previous studies showed that proteolytic activation of pyruvate oxidase results in substantial changes in the absorption spectrum of the oxidized form of the bound flavin. Up to this time, similar studies of the lipid-activated form of the enzyme have not been feasible, since it is necessary to reduce the flavoprotein in order to induce binding to the lipids. In this paper, glutaraldehyde cross-linking of the lipid-activated enzyme is used to trap the enzyme in this form. Spectroscopic studies show alterations of the flavin spectrum similar to those observed upon proteolytic activation. This alteration in the flavin binding site is consistent with kinetic studies which suggest that activation results from an acceleration in the rates of electron transfer both into and out of the bound flavin, which appears to be more "accessible" in the activated forms of the enzyme.

Cross-Linking Reagents↗

PubMed1982

Role of arginine in the binding of thiamin pyrophosphate to Escherichia coli pyruvate oxidase.

The mode of interaction between Escherichia coli pyruvate oxidase and its cofactor, thiamin pyrophosphate (TPP), was studied with the aid of arginine-directed reagents. The enzyme is rapidly inactivated by either phenylglyoxal or 2,3-butanedione, with the cofactor, TPP, offering partial protection against these reagents. The inactivation by phenylglyoxal was found to be reversible. Experiments with [7-14C]phenylglyoxal showed that while several arginine residues react with this reagent, TPP can prevent the labeling of one such residue. Furthermore, inactivation by 2,3-butanedione is attended by at least a 100-fold decrease in affinity of the enzyme for TPP. These results suggest a direct role for arginine in the binding of the cofactor.

Arginine↗

PubMed1996

Pyruvate oxidase, as a determinant of virulence in Streptococcus pneumoniae.

Pneumococcus has been shown to bind to epithelial cells of the nasopharynx and lung, and to endothelial cells of the peripheral vasculature. To characterize bacterial elements required for attachment to these cell types, a library of genetically altered pneumococci with defects in exported proteins was screened for the loss of attachment to glycoconjugates representative of the nasopharyngeal cell receptor, type II lung cells (LC) and human endothelial cells (EC). A mutant was identified which showed a greater than 70% loss in the ability to attach to all cell types. This mutant also showed decreased adherence to the glycoconjugates containing the terminal sugar residues GalNAcbeta1-3Gal, GalNAcbeta1-4Gal and the carbohydrate GlcNAc, which are proposed components of the pneumococcal receptors specific to the surfaces of LC and EC. Analysis of the locus altered in this mutant revealed a gene, spxB, that encodes a member of the family of bacterial pyruvate oxidases which decarboxylates pyruvate to acetyl phosphate plus H2O2 and CO2. This mutant produced decreased concentrations of H2O2 and failed to grow aerobically in a chemically defined medium, unless supplemented with acetate which presumably restores acetyl phosphate levels by the action of acetate kinase, further suggesting that spxB encodes a pyruvate oxidase. The addition of acetate to the growth medium restored the adherence properties of the mutant indicating a link between the enzyme and the expression of bacterial adhesins. A defect in spxB corresponded to impaired virulence of the mutant in vivo. Compared to the parent strain, an spxB mutant showed reduced virulence in animal models for nasopharyngeal colonization, pneumonia, and sepsis. We propose that a mutation in spxB leads to down-regulation of the multiple adhesive properties of pneumococcus which, in turn, may correlate to diminished virulence in vivo.

Animals↗

PubMed2004

Role of pyruvate oxidase in Escherichia coli strains lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system.

We report a study to determine the role of pyruvate oxidase among Escherichia coli isogenic strains with active and inactive phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). Strain PB11, displaying a specific growth rate (mu) in glucose minimal medium of 0.1 h(-1) is a ptsHI, crr operon deletion derivative of wild-type JM101 (displaying a mu of 0.70 h(-1)). Strain PB12 is a spontaneous mutant obtained from PB11 after selection for its capacity to grow on glucose with a mu of 0.40 h(-1). In minimal medium cultures supplemented with glucose plus acetate, strain JM101 displayed preferential consumption of glucose, whereas strains PB11 and PB12 did not display glucose catabolic repression of acetate consumption. Inactivation of poxB caused a severe reduction in growth rate in strain PB11 when grown in the fermentor with medium containing glucose or glucose plus acetate, whereas under the same conditions poxB(-)derivative strains of JM101 and PB12 were not affected. Relative transcript levels for 29 genes related to poxB transcriptional regulation and central metabolism were determined using RT-PCR. This analysis revealed 2-fold lower transcript levels for genes encoding subunits of the pyruvate dehydrogenase complex (Pdh) in strain PB11 and 4- to 6-fold higher transcript levels for poxB in strains PB11 and PB12, when compared to JM101. In addition, in the PTS(-) strains, upregulation of the poxB transcription factors rpoS, soxS and marA, was detected. The results presented here strongly suggest that AcCoA is mainly synthesized from acetate produced by pyruvate oxidase in strain PB11, whereas in strains JM101 and PB12, AcCoA is synthesized preferentially from pyruvate by Pdh.

Acetic Acid↗

PubMed2004

Pyruvate oxidase is a determinant of Avery's rough morphology.

In pioneering studies, Avery et al. identified DNA as the hereditary material (A. T. Avery, C. M. MacLeod, and M. McCarty, J. Exp. Med. 79:137-158, 1944). They demonstrated, by means of variation in colony morphology, that this substance could transform their rough type 2 Streptococcus pneumoniae strain R36A into a smooth type 3 strain. It has become accepted as fact, from modern textbook accounts of these experiments, that smooth pneumococci make capsule, while rough strains do not. We found that rough-to-smooth morphology conversion did not occur in rough strains R36A and R6 when the ability to synthesize native type 2 capsule was restored. The continued rough morphology of these encapsulated strains was attributed to a second, since-forgotten, morphology-affecting mutation that was sustained by R36A during strain development. We used a new genome-PCR-based approach to identify spxB, the gene encoding pyruvate oxidase, as the mutated locus in R36A and R6 that, with unencapsulation, gives rise to rough colony morphology, as we know it. The variant spxB allele of R36A and R6 is associated with increased cellular pyruvate oxidase activity relative to the ancestral strain D39. Increased pyruvate oxidase activity alters colony shape by mediating cell death. R36A requires a wild-type spxB allele for the expression of smooth type 2 morphology but not for the expression of smooth type 3 morphology, the phenotype monitored by Avery et al. Thus, the mutated spxB allele did not impact their use of smooth morphology to identify the transforming principle.

Bacterial Capsules↗

PubMed2001

Pyruvate oxidase contributes to the aerobic growth efficiency of Escherichia coli.

The metabolic importance of pyruvate oxidase (PoxB), which converts pyruvate directly to acetate and CO(2), was assessed using an isogenic set of genetically engineered strains of Escherichia coli. In a strain lacking the pyruvate dehydrogenase complex (PDHC), PoxB supported acetate-independent aerobic growth when the poxB gene was expressed constitutively or from the IPTG-inducible tac promoter. Using aerobic glucose-limited chemostat cultures of PDH-null strains, it was found that steady-states could be maintained at a low dilution rate (0.05 h(-1)) when PoxB is expressed from its natural promoter, but not at higher dilution rates (up to at least 0.25 h(-1)) unless expressed constitutively or from the tac promoter. The poor complementation of PDH-deficient strains by poxB plasmids was attributed to several factors including the stationary-phase-dependent regulation of the natural poxB promoter and deleterious effects of the multicopy plasmids. As a consequence of replacing the PDH complex by PoxB, the growth rate (mu(max)), growth yield (Y(max)) and the carbon conversion efficiency (flux to biomass) were lowered by 33%, 9-25% and 29-39% (respectively), indicating that more carbon has to be oxidized to CO(2) for energy generation. Extra energy is needed to convert PoxB-derived acetate to acetyl-CoA for further metabolism and enzyme analysis indicated that acetyl-CoA synthetase is induced for this purpose. In similar experiments with a PoxB-null strain it was shown that PoxB normally makes a significant contribution to the aerobic growth efficiency of E. coli. In glucose minimal medium, the respective growth rates (mu(max)), growth yields (Y(max)) and carbon conversion efficiencies were 16%, 14% and 24% lower than the parental values, and correspondingly more carbon was fluxed to CO(2) for energy generation. It was concluded that PoxB is used preferentially at low growth rates and that E. coli benefits from being able to convert pyruvate to acetyl-CoA by a seemingly wasteful route via acetate.

Aerobiosis↗

PubMed1986

In vivo function of Escherichia coli pyruvate oxidase specifically requires a functional lipid binding site.

The pyruvate oxidase of Escherichia coli is a peripheral membrane flavoprotein that is dramatically activated by lipids. The enzyme strongly binds to phospholipid vesicles in vitro. In vivo, in addition to enzyme activation, binding is thought to be important to provide access of the enzyme to ubiquinone dissolved in the lipid bilayer. It was unclear if both or either of these attributes is needed for enzyme function in vivo. To differentiate between activation and lipid binding, we have constructed, using recombinant DNA techniques, a mutant gene that produces a truncated protein. The truncated protein lacks the last 24 amino acids of the C-terminus of the oxidase (due to introduction of a translation termination codon) and thus is closely analogous to the activated species produced in vitro by limited chymotrypsin cleavage [Recny, M.A., Grabau, C., Cronan, J.E., Jr., & Hager, L.P. (1985) J. Biol. Chem. 260, 14287-14291]. The truncated protein (like the protease-derived species) is fully active in vitro in the absence of lipid, and its activity is not further increased by addition of lipid activators. Moreover, the truncated enzyme fails to bind Triton X-114, a detergent that binds to and activates the wild-type oxidase. Strains producing the truncated protein were devoid of oxidase activity in vivo. This result indicates that binding to membrane lipids is specifically required for function of the oxidase in vivo; activation alone does not suffice.

Amino Acid Sequence↗