Suggested role of NosZ in preventing N2O inhibition of dissimilatory nitrite reduction to ammonium.

IMPORTANCE: Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is a microbial energy-conserving process that reduces NO3 - and/or NO2 - to NH4 +. Interestingly, DNRA-catalyzing microorganisms possessing nrfA genes are occasionally found harboring nosZ genes encoding nitrous oxide reductases, i.e., the only group of enzymes capable of removing the potent greenhouse gas N2O. Here, through a series of physiological experiments examining DNRA metabolism in one of such microorganisms, Bacillus sp. DNRA2, we have discovered that N2O may delay the transition to DNRA upon an oxic-to-anoxic transition, unless timely removed by the nitrous oxide reductases. These observations suggest a novel explanation as to why some nrfA-possessing microorganisms have retained nosZ genes: to remove N2O that may otherwise interfere with the transition from O2 respiration to DNRA.

Enhancement of Nitrous Oxide Emissions in Soil Microbial Consortia via Copper Competition between Proteobacterial Methanotrophs and Denitrifiers.

Unique means of copper scavenging have been identified in proteobacterial methanotrophs, particularly the use of methanobactin, a novel ribosomally synthesized, post-translationally modified polypeptide that binds copper with very high affinity. The possibility that copper sequestration strategies of methanotrophs may interfere with copper uptake of denitrifiers in situ and thereby enhance N2O emissions was examined using a suite of laboratory experiments performed with rice paddy microbial consortia. Addition of purified methanobactin from Methylosinus trichosporium OB3b to denitrifying rice paddy soil microbial consortia resulted in substantially increased N2O production, with more pronounced responses observed for soils with lower copper content. The N2O emission-enhancing effect of the soil's native mbnA-expressing Methylocystaceae methanotrophs on the native denitrifiers was then experimentally verified with a Methylocystaceae-dominant chemostat culture prepared from a rice paddy microbial consortium as the inoculum. Finally, with microcosms amended with various cell numbers of methanobactin-producing Methylosinus trichosporium OB3b before CH4 enrichment, microbiomes with different ratios of methanobactin-producing Methylocystaceae to gammaproteobacterial methanotrophs incapable of methanobactin production were simulated. Significant enhancement of N2O production from denitrification was evident in both Methylocystaceae-dominant and Methylococcaceae-dominant enrichments, albeit to a greater extent in the former, signifying the comparative potency of methanobactin-mediated copper sequestration, while implying the presence of alternative copper abstraction mechanisms for Methylococcaceae. These observations support that copper-mediated methanotrophic enhancement of N2O production from denitrification is plausible where methanotrophs and denitrifiers cohabit. IMPORTANCE Proteobacterial methanotrophs-groups of microorganisms that utilize methane as a source of energy and carbon-have been known to employ unique mechanisms to scavenge copper, namely, utilization of methanobactin, a polypeptide that binds copper with high affinity and specificity. Previously the possibility that copper sequestration by methanotrophs may lead to alteration of cuproenzyme-mediated reactions in denitrifiers and consequently increase emission of potent greenhouse gas N2O has been suggested in axenic and coculture experiments. Here, a suite of experiments with rice paddy soil slurry cultures with complex microbial compositions were performed to corroborate that such copper-mediated interplay may actually take place in environments cohabited by diverse methanotrophs and denitrifiers. As spatial and temporal heterogeneity allows for spatial coexistence of methanotrophy (aerobic) and denitrification (anaerobic) in soils, the results from this study suggest that this previously unidentified mechanism of N2O production may account for a significant proportion of N2O efflux from agricultural soils.

Involvement of NO3- in Ecophysiological Regulation of Dissimilatory Nitrate/Nitrite Reduction to Ammonium (DNRA) Is Implied by Physiological Characterization of Soil DNRA Bacteria Isolated via a Colorimetric Screening Method.

Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) has recently regained attention as a nitrogen retention pathway that may potentially be harnessed to alleviate nitrogen loss resulting from denitrification. Until recently, the ecophysiology of DNRA bacteria inhabiting agricultural soils has remained largely unexplored, due to the difficulty in targeted enrichment and isolation of DNRA microorganisms. In this study, >100 DNRA bacteria were isolated from NO3--reducing anoxic enrichment cultures established with rice paddy soils using a newly developed colorimetric screening method. Six of these isolates, each assigned to a different genus, were characterized to improve the understanding of DNRA physiology. All the isolates carried nrfA and/or nirB, and the Bacillus sp. strain possessed a clade II nosZ gene conferring the capacity for N2O reduction. A common prominent physiological feature observed in the isolates was NO2- accumulation before NH4+ production, which was further examined with Citrobacter sp. strain DNRA3 (possessing nrfA and nirB) and Enterobacter sp. strain DNRA5 (possessing only nirB). Both isolates showed inhibition of NO2--to-NH4+ reduction at submillimolar NO3- concentrations and downregulation of nrfA or nirB transcription when NO3- was being reduced to NO2- In batch and chemostat experiments, both isolates produced NH4+ from NO3- reduction when incubated with excess organic electron donors, while incubation with excess NO3- resulted in NO2- buildup but no substantial NH4+ production, presumably due to inhibitory NO3- concentrations. This previously overlooked link between NO3- repression of NO2--to-NH4+ reduction and the C-to-N ratio regulation of DNRA activity may be a key mechanism underpinning denitrification-versus-DNRA competition in soil.IMPORTANCE Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is an anaerobic microbial pathway that competes with denitrification for common substrates NO3- and NO2- Unlike denitrification, which leads to nitrogen loss and N2O emission, DNRA reduces NO3- and NO2- to NH4+, a reactive nitrogen compound with a higher tendency to be retained in the soil matrix. Therefore, stimulation of DNRA has often been proposed as a strategy to improve fertilizer efficiency and reduce greenhouse gas emissions. Such attempts have been hampered by lack of insights into soil DNRA bacterial ecophysiology. Here, we have developed a new screening method for isolating DNRA-catalyzing organisms from agricultural soils without apparent DNRA activity. Physiological characteristics of six DNRA isolates were closely examined, disclosing a previously overlooked link between NO3- repression of NO2--to-NH4+ reduction and the C-to-N ratio regulation of DNRA activity, which may be a key to understanding why DNRA activity is rarely observed at substantial levels in nitrogen-rich agricultural soils.