Achromobacter denitrificans Strain YD35 Pyruvate Dehydrogenase Controls NADH Production To Allow Tolerance to Extremely High Nitrite Levels

Abstract

bWe identified the extremely nitrite-tolerant bacterium Achromobacter denitrificans YD35 that can grow in complex medium containing 100 mM nitrite (NO 2 ؊ ) under aerobic conditions. Nitrite induced global proteomic changes and upregulated tricarboxylate (TCA) cycle enzymes as well as antioxidant proteins in YD35. Transposon mutagenesis generated NO 2 ؊ -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 NO 2 ؊ tolerance. A mutant in which the pdh gene cluster was disrupted (⌬pdh mutant) could not grow in the presence of 100 mM NO 2 ؊ . 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 NO 2 ؊ -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 NO 2؊ . These findings demonstrate a link between NO 2 ؊ 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 NO 2 ؊ -tolerating mechanism in this strain. Microorganisms in nature are exposed to various stimuli, such as temperature, osmotic pressure, pH, and reactive chemicals, which in excess cause stress through damage to cellular components (1). Microorganisms have diversified detoxification and tolerance mechanisms during the course of evolution to fight such stressors and adapt to various environments, including extremes (2, 3). Nitrite (NO 2 Ϫ ) is in equilibrium with protons in aqueous solution, and its protonated nitrous acid (HNO 2 ) is a powerful oxidant and mutagen that inhibits bacterial activities (4-6). Further protonation and dismutation generate highly unstable nitrosonium cations (NO ϩ ) that in turn generate reactive nitrogen species (RNS) (7, 8). Thus, NO 2 Ϫ causes more severe oxidative stress under acidic conditions. Nitric oxide (NO) is a toxic RNS which the immune systems of vertebrates produce to kill infective bacteria (9, 10). Microorganisms have developed various RNSdetoxifying proteins, such as flavohemoglobin, flavorubredoxin, and peroxiredoxin, to escape the damage caused by RNS (11-13).The microbial denitrification mechanism dissimilates nitrate (NO 3 Ϫ ), which is significant in anaerobic respiration (14). Bacteria denitrify NO 3 Ϫ to gaseous dinitrogen through successive reduction steps to generate NO 2 Ϫ , NO, and nitrous oxide as reaction intermediates that might accumulate, especially if environmental conditions abruptly change. Denitrifying bacteria that can grow in the presence of high NO 2 Ϫ concentrations were isolated from tropical fish aquariums, where NO 3 Ϫ removal is often inadequate, resulting in high levels of accumulated NO 2 ...

A Novel A3 Group Aconitase Tolerates Oxidation and Nitric Oxide

“…Strains, Culture, and Media-Supplemental Table 1 shows the bacterial strains and plasmids used in this study, and they were cultured and propagated in DM (0.5% Polypeptone, 0.3% nutrient broth, 0.4% NaCl, 0.1% K 2 HPO 4 , and 0.2% trace element solution (25), pH 7.0), LB (1% Tryptone, 0.5% yeast extract, and 0.5% NaCl), YENB (0.75% yeast extract and 0.8% nutrient broth), and Super Optimal broth with catabolite repression (2% Tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 ⅐6H 2 O, 10 mM MgSO 4 ⅐7H 2 O, and 20 mM glucose, pH 7.0) media. Bacterial strains were cultured at 120 rpm and 30°C under aerobic conditions.…”

“…After digesting pAcnB::tet with XbaI and SacI, the acnB::tet gene fragment was cloned into pHSG396 to generate pAcnB KO . These plasmids transformed A. denitrificans YD35 by electroporation (25), and transformants were cultured on YENB plates containing 20 mg/liter tetracycline sulfate. Gene disruptions were confirmed by PCR, and we designated the strains AcnA4⌬ and AcnB⌬ (Fig.…”

“…AcnA3, AcnA4, and AcnB were produced using plasmids pAcnA3, pAcnA4, and pAcnB, respectively. Recombinant aconitases were prepared and purified as described (25).…”

“…Intracellular NADH/ NAD ϩ ratios and ATP concentrations were quantified as described (25). Cellular citrate was extracted from cells using cold methanol as described (27) and analyzed using a Roche Yellow line kit (R-Biopharm AG, Darmstadt, Germany) or a GC/MS-QP2010 Plus gas chromatograph-mass spectrometer (Shimadzu Co., Kyoto, Japan).…”

“…When protonated under acidic conditions, NO 2 Ϫ generates RNS (22,23). To determine bacterial responses to RNS, we isolated Achromobacter denitrificans YD35 from a water treatment tank as a strain that thrives in the presence of NO 2 Ϫ concentrations as high 100 mM, and we investigated its RNS tolerance mechanisms (24,25 which provides acetyl-CoA, increases turnover of the TCA cycle, and hence generates NADH, which could be a substrate for enzymes that detoxify NO (25). This constitutes a possible RNS tolerance mechanism of YD35 and implies that the TCA cycle is important for bacterial adaptation to RNS.…”