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 x 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.

FIG. 1

Production and consumption of NO by M. luteus. (A) Growth (OD₆₆₀, ●), O₂ consumption (▾), and NO accumulation (○) during 7 days of growth in NMS medium under initially aerobic conditions. Each time point represents sacrificial sampling of one of eight replicate flasks, the samples being used for the assays reported in panel B. (B) Rates of production of NO at 0% O₂ (○) and 20% O₂ (▵), and calculated rates of consumption of 1 ppmv of NO in the presence of 20% O₂ (□). (Error bars represent the standard error of the mean for duplicate bottles and if not visible are contained within the symbols.) d, days.

FIG. 2

Production and consumption of NO by M. trichosporium. Details as in Fig. 1.

FIG. 3

Production and consumption of NO by strain T20. Details as in Fig. 1. Note that the scale of the ordinates for NO in this figure differs from that in Fig. 1 and 2.

FIG. 4

Production of NO by strain T20 at different O₂ concentrations. Assays of 1.75-h duration were done in the presence of 10% CH₄ using samples from cultures that had been incubated for 4 (●) and 7 (○) days.

FIG. 5

Consumption of NO₃⁻ and O₂ and production of NO₂⁻, NO, and N₂O in cultures of strain T20 growing initially aerobically in NMS medium in the presence of 30% CH₄. No more than half of the CH₄ was consumed during the experiment.