The Effects of Methanobactin Secreted by Methylosinus trichosporium Strain OB3b on N2O Reduction in Pseudomonas stutzeri

Abstract

Background: Methanobactin (Mb) is a Cu chelator secreted by methanotrophs for scavenging of Cu from Cu-deficient environments. Cu scavenging may result in deprivation of Cu from the environment and thus, may have a negative impact on the biogeochemical reactions that require Cu for their activities. N2O reduction mediated by nitrous oxide reductase (N2OR) is one of such Cu-dependent reactions, which has an environmentally significant function of reducing N2O emissions. This study investigated the effect of methanobactin secretion by Methylosinus trichosporium strain OB3b on N2O reduction activity in Pseudomonas stutzeri strain DCP-Ps1, a denitrifying bacteria isolated from agricultural soil. Methods: N2O evolution from strain DCP-Ps1 was monitored upon incubation in a nitrate mineral salts (NMS) medium amended with 500 µmoles NO3-, 500 µmoles acetate, 0.2 µM CuCl2 and varying concentrations of purified Mb-OB3b (0, 0.5, 1, 5 µM). In a separate set of experiments, strain DCP-Ps1 was grown in O2-depleted cultures or spent media of strain OB3b wildtype or mbnA- mutant grown (to OD600~0.3) in NMS medium amended with 0.2 µM CuCl2. In order to examine whether this regulatory mechanism is actually significant in the environment, a soil enrichment was used as the inoculum in place of axenic culture of strain DCP-Ps1. Results: In absence of Mb, transient N2O accumulation during reduction of NO3- strain DCP-Ps1 maxed at 70 ± 4 nmoles N2O-N/bottle, while cultures amended with 0.5, 1, and 5 µM of Mb-OB3b permanently accumulated 435.7 - 453.3 µmoles of N2O-N. The total Cu concentration in the spent media decreased to 0.043 ± 0.01 µM in strain DCP-Ps1 cultures incubated without Mb, while the changes in Cu concentrations in the cultures incubated with Mb were insignificant (0.189 ± 0.03 µM after incubation), suggesting that Mb-bound Cu was unavailable to strain DCP-Ps1. A permanent N2O production of 305.1 ± 30.2 µmoles was observed when strain DCP-Ps1 was incubated in the O2-depleted culture of strain OB3b, while no significant N2O accumulation was observed when mbnA- mutants were used, supporting that the inhibition of N2OR was due to Cu abstraction by secreted methanobactin. Use of soil enrichment inoculum in place of the axenic culture yielded similar results, as permanent accumulation of >80% of NO3--N as N2O-N was observed. Conclusions: Our results suggest that proliferation of methanotrophic activity may have significant implications in soil nitrogen cycling and greenhouse gas emissions in CH4 and N-rich environments such as ricefields and landfill cover soils.