Production and Consumption of Nitric Oxide by Three Methanotrophic Bacteria

Abstract

We studied nitrogen oxide production and consumption by methanotrophs Methylobacter luteus (group I), Methylosinus trichosporium OB3b (group II), and an isolate from a hardwood swamp soil, here identified by 16S ribosomal DNA sequencing as Methylobacter sp. strain T20 (group I). All could consume nitric oxide (nitrogen monoxide, NO), and produce small amounts of nitrous oxide (N 2 O). Only Methylobacter strain T20 produced large amounts of NO (>250 parts per million by volume [ppmv] in the headspace) at specific activities of up to 2.0 ؋ 10 ؊17 mol of NO cell ؊1 day ؊1, mostly after a culture became O 2 limited. Production of NO by strain T20 occurred mostly in nitrate-containing medium under anaerobic or nearly anaerobic conditions, was inhibited by chlorate, tungstate, and O 2 , and required CH 4 . Denitrification (methanol-supported N 2 O production from nitrate in the presence of acetylene) could not be detected and thus did not appear to be involved in the production of NO. Furthermore, cd 1 and Cu nitrite reductases, NO reductase, and N 2 O reductase could not be detected by PCR amplification of the nirS, nirK, norB, and nosZ genes, respectively. M. luteus and M. trichosporium produced some NO in ammonium-containing medium under aerobic conditions, likely as a result of methanotrophic nitrification and chemical decomposition of nitrite. For Methylobacter strain T20, arginine did not stimulate NO production under aerobiosis, suggesting that NO synthase was not involved. We conclude that strain T20 causes assimilatory reduction of nitrate to nitrite, which then decomposes chemically to NO. The production of NO by methanotrophs such as Methylobacter strain T20 could be of ecological significance in habitats near aerobic-anaerobic interfaces where fluctuating O 2 and nitrate availability occur.Nitric oxide (nitrogen monoxide, NO) is an important trace gas that plays important roles in tropospheric chemistry and is a factor in acid precipitation and ozone turnover (12, 13, 38). Soils are a major source of NO, accounting for up to 40% of the global budget (43), most coming from arable land (38). Processes of microbial metabolism are responsible for most of the NO source or sink strength of terrestrial and aquatic systems.Biological production and consumption of NO occur via a variety of reductive and oxidative processes involving heterotrophic and autotrophic microorganisms. The detailed mechanisms of these processes are not yet fully understood but are further considered in the Discussion section.Methane-oxidizing bacteria are important in reducing global emissions of methane from terrestrial and aquatic systems. They also participate in various nitrogen cycle processes: nitrate and ammonium assimilation, nitrogen fixation (15), and nitrification (6). They produce nitrous oxide (N 2 O) during nitrification in ammonium-containing medium (24,26,47). Methylomonas agile and Methylosinus trichosporium, group I and II methanotrophs, respectively, consume NO, but aerobic cultures in ammonium-containing medium were no...

The stability of microbially reduced U(IV); impact of residual electron donor and sediment ageing

“…For example, heterotrophic methane cycling bacteria closely related to Methylobacter comprised at least 16% of the detected sequences fifteen days after nitrate addition. Methylobacter species have been associated with denitrification under anaerobic conditions in previous studies (Ren et al, 2000) and also have been associated previously with marine anoxic zones (Lloyd et al, 2006). The increase in the relative abundance of methane cycling bacteria in the four month aged sample is perhaps unsurprising given that significant quantities of methane were detected in microcosm headspace, and that the electron equivalent calculations estimated that up to 90% of the added electron donor may have been consumed by methanogenesis.…”

Nitric Oxide Formation by Escherichia coli

“…NO production from nitrite, measured by the nitrosation of 2,3-diaminonaphthalene (DAN) (6) was proposed to involve enzymatic reduction of nitrite to NO followed by oxygen-dependent DAN nitrosation. NO production has also been shown in Serratia marcescens (7), Bacillus cereus (8), three species of methanotrophic bacteria (9), and the green micro alga Scenedesmus obliquus (10). Ji and Hollocher (11) concluded that nitrite-dependent NO production by E. coli was due to the activity of the membrane-associated (dissimilatory) nitrate reductase.…”

Genome Analysis Coupled with Physiological Studies Reveals a Diverse Nitrogen Metabolism in Methylocystis sp. Strain SC2

“…Another possibility may be the presence of an active/functional nitrous oxide reductase produced from the plasmid-borne nos operon, thereby resulting in the rapid conversion of N 2 O to N 2 . To examine the second possibility, we checked N 2 O production after blocking the nos activity with purified acetylene [93], [94], [95]. As acetylene is also a potent inhibitor of methane monooxygenase [90], we performed this experiment either by adding methanol or without a carbon source.…”

“…The acetylene inhibition method [93], [94], [95] was used to verify the production of N 2 O by strain SC2. Briefly, NMS-grown log-phase cells (3.4 mg dry weight) were resuspended in 5 ml of fresh NMS medium supplemented with methanol (0.1% [vol/vol]) or without a carbon source in 25-ml serum bottles that were capped with butyl rubber stoppers.…”