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Achromobacter denitrificans strain YD35 pyruvate dehydrogenase controls NADH production to allow tolerance to extremely high nitrite levels.

ABSTRACT: We identified the extremely nitrite-tolerant bacterium Achromobacter denitrificans YD35 that can grow in complex medium containing 100 mM nitrite (NO2(-)) under aerobic conditions. Nitrite induced global proteomic changes and upregulated tricarboxylate (TCA) cycle enzymes as well as antioxidant proteins in YD35. Transposon mutagenesis generated NO2(-)-hypersensitive mutants of YD35 that had mutations at genes for aconitate hydratase and ?-ketoglutarate dehydrogenase in the TCA cycle and a pyruvate dehydrogenase (Pdh) E1 component, indicating the importance of TCA cycle metabolism to NO2(-) tolerance. A mutant in which the pdh gene cluster was disrupted (?pdh mutant) could not grow in the presence of 100 mM NO2(-). Nitrite decreased the cellular NADH/NAD(+) ratio and the cellular ATP level. These defects were more severe in the ?pdh mutant, indicating that Pdh contributes to upregulating cellular NADH and ATP and NO2(-)-tolerant growth. Exogenous acetate, which generates acetyl coenzyme A and then is metabolized by the TCA cycle, compensated for these defects caused by disruption of the pdh gene cluster and those caused by NO2(-). These findings demonstrate a link between NO2(-) tolerance and pyruvate/acetate metabolism through the TCA cycle. The TCA cycle mechanism in YD35 enhances NADH production, and we consider that this contributes to a novel NO2(-)-tolerating mechanism in this strain.

SUBMITTER: Doi Y

PROVIDER: S-EPMC3957653 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Achromobacter denitrificans strain YD35 pyruvate dehydrogenase controls NADH production to allow tolerance to extremely high nitrite levels.

Doi Yuki Y Shimizu Motoyuki M Fujita Tomoya T Nakamura Akira A Takizawa Noboru N Takaya Naoki N

Applied and environmental microbiology 20140110 6

We identified the extremely nitrite-tolerant bacterium Achromobacter denitrificans YD35 that can grow in complex medium containing 100 mM nitrite (NO2(-)) under aerobic conditions. Nitrite induced global proteomic changes and upregulated tricarboxylate (TCA) cycle enzymes as well as antioxidant proteins in YD35. Transposon mutagenesis generated NO2(-)-hypersensitive mutants of YD35 that had mutations at genes for aconitate hydratase and α-ketoglutarate dehydrogenase in the TCA cycle and a pyruva ...[more]

PMID: 24413603

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Project description:Monospecific antibody to the respiratory NADH dehydrogenase from Paracoccus denitrificans was prepared by using as antigen specific immunoprecipitates containing NADH dehydrogenase which were excised from crossed-immunoelectrophoresis plates. The latter were run with selectively solubilized plasma membranes and antibodies against plasma membranes. The antibody immunoprecipitated NADH dehydrogenase from P. denitrificans membranes biosynthetically labelled with 14C and solubilized with a wide range of detergents. All immunoprecipitates contained the two subunits of Mr 48,000 and 25,000, in an approximate 1:1 stoichiometry, that had previously been assigned to NADH dehydrogenase. A polypeptide of Mr 46,000 in P. denitrificans membranes, previously shown to cross-react with a subunit-specific antibody to mitochondrial NADH dehydrogenase (complex I), was not detected in any immunoprecipitate. Under some conditions a third polypeptide, of Mr 31,000, was also detected, but in variable and non-stoichiometric amounts relative to the two other subunits. It was concluded that this polypeptide was incorporated into the immunoprecipitates as an artefact and that the polypeptides of Mr 48,000 and 25,000 are the sole polypeptides firmly identified in the NADH dehydrogenase. Flavoproteins were specifically radiolabelled by growth of P. denitrificans in the presence of [14C]riboflavin. Crossed immunoelectrophoresis of membranes from such cells showed that succinate dehydrogenase contained flavin, but that there was no detectable flavin in NADH dehydrogenase under these conditions. Analysis of excised immunoprecipitates of succinate dehydrogenase showed that flavin was covalently bound to a polypeptide of Mr 56,000. Flavin was retained by NADH dehydrogenase under mild conditions of detergent solubilization. Subsequent immunoprecipitation, followed by analysis of the acid-extracted flavin, established that FMN is a cofactor, in common with mitochondrial NADH-ubiquinone oxidoreductase (complex I).

Dataset Information

Achromobacter denitrificans strain YD35 pyruvate dehydrogenase controls NADH production to allow tolerance to extremely high nitrite levels.

Publications

Achromobacter denitrificans strain YD35 pyruvate dehydrogenase controls NADH production to allow tolerance to extremely high nitrite levels.

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