Novel Denitrifying Bacterium Ochrobactrum anthropi YD50.2 Tolerates High Levels of Reactive Nitrogen Oxides

Abstract

Most studies of bacterial denitrification have used nitrate (NO 3؊ ) as the first electron acceptor, whereas relatively less is understood about nitrite (NO 2 ؊ ) denitrification. We isolated novel bacteria that proliferated in the presence of high levels of NO 2 ؊ (72 mM T proliferated even in the presence of higher levels of NO 2 ؊ (100 mM), and both were considerably more resistant to acidic NO 2 ؊ than were the other strains noted above. These results indicated that O. anthropi YD50.2 is a novel denitrifier that has evolved reactive nitrogen oxide tolerance mechanisms.Environmental bacteria maintain the global nitrogen cycle by metabolizing organic and inorganic nitrogen compounds. Denitrification is critical for maintenance of the global nitrogen cycle, through which nitrate (NO 3 Ϫ ) or nitrite (NO 2 Ϫ ) is reduced to gaseous nitrogen forms such as N 2 and nitrous oxide (N 2 O) (19, 47). Decades of investigations into denitrifying bacteria have revealed their ecological impact (9), their molecular mechanisms of denitrification (13,25,47), and the industrial importance of removing nitrogenous contaminants from wastewater (31, 36). Bacterial denitrification is considered to comprise four successive reduction steps, each of which is catalyzed by NO 3 Ϫ reductase (Nar), NO 2 Ϫ reductase (Nir), nitric oxide (NO) reductase (Nor), and N 2 O reductase (Nos). The reaction of each enzyme is linked to the electron transport chain on the cellular membrane and accompanies oxidative phosphorylation, implying that bacterial denitrification is of as much physiological significance as anaerobic respiration (25, 47). Most denitrifying bacteria are facultative anaerobes and respire with oxygen under aerobic conditions. Because denitrification is induced in the absence of oxygen, it is considered an alternative mechanism of energy conservation that has evolved as an adaptation to anaerobic circumstances (13, 47).Nitrite and NO are hazardous to bacteria, since they generate highly reactive nitrogen species (RNS) under physiological conditions and damage cellular DNA, lipid, and proteins (28,37). Denitrifying bacteria are thought to be threatened by RNS since they reduce NO 3 Ϫ to generate NO 2 Ϫ and NO as denitrifying intermediates. Furthermore, denitrifying bacteria often inhabit environments where they are exposed to NO 2 Ϫ and NO and hence high levels of RNS. Recent reports suggest that pathogenic bacteria invading animal tissues are attacked by NO generated by macrophages (12). Such bacteria involve denitrifiers, and some of them, for example, Neisseria meningitidis (1) and Pseudomonas aeruginosa, acquire resistance to NO by producing Nor (44). The utilization (reduction) of NO by Brucella increases the survival of infected mice (2). These examples suggest that production of a denitrifying mechanism affects bacterial survival of threats from both endogenous and extracellular RNS. However, the mechanism of RNS tolerance induced by denitrifying bacteria is not fully understood.Ubiquitous gram-negative Ochrobactrum strains ar...

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

“…We induced denitrification activity in 200 ml of DM containing 10 mM NaNO 2 or NaNO 3 , replaced the headspace air in the flasks with argon gas by purging for 15 min, and then sealed the flasks with butyl rubber stoppers. We measured NO 3 Ϫ , NO 2 Ϫ , N 2 O, and N 2 as described previously (15).…”

“…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 Ϫ (15). One isolate, Ochrobactrum anthropi YD50.2, converts NO 2 Ϫ to dinitrogen under anaerobic conditions, whereas it grows under extremely high levels of NO 2 Ϫ under aerobic conditions without producing denitrification activity (15). Thus, this isolate can tolerate high levels of NO 2 Ϫ , although the mechanism(s) involved remains unknown.…”

“…Transformants were selected on LB agar plates containing 10 mg liter Ϫ1 of tetracycline sulfate. Plasmids were isolated using QIAprep spin miniprep kits (Qiagen), and nucleotides were sequenced as described previously (15). Table S1 in the supplemental material lists the primers used.…”

A Novel A3 Group Aconitase Tolerates Oxidation and Nitric Oxide

“…Some of them notably encode pyruvate dehydrogenase, as well as ␣-ketoglutarate dehydrogenase and cytochrome bd-type terminal oxidase, which are both involved in energy conservation processes. Together with the role of high level of NO 2 Ϫ contamination, and it is a denitrifier (24,25). The AcnA3 generated by this species of bacteria contributes to the ability of the bacteria to thrive in very high NO 2 Ϫ concentrations, against which conventional bacteria have no resistance.…”

“…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.…”