Nitrous Oxide Reduction Kinetics Distinguish Bacteria Harboring Clade I NosZ from Those Harboring Clade II NosZ

Abstract

Bacteria capable of reduction of nitrous oxide (N2O) to N2 separate into clade I and clade II organisms on the basis of nos operon structures and nosZ sequence features. To explore the possible ecological consequences of distinct nos clusters, the growth of bacterial isolates with either clade I (Pseudomonas stutzeri strain DCP-Ps1, Shewanella loihica strain PV-4) or clade II (Dechloromonas aromatica strain RCB, Anaeromyxobacter dehalogenans strain 2CP-C) nosZ with N2O was examined. Growth curves did not reveal trends distinguishing the clade I and clade II organisms tested; however, the growth yields of clade II organisms exceeded those of clade I organisms by 1.5- to 1.8-fold. Further, whole-cell half-saturation constants (Kss) for N2O distinguished clade I from clade II organisms. The apparent Ks values of 0.324 ± 0.078 μM for D. aromatica and 1.34 ± 0.35 μM for A. dehalogenans were significantly lower than the values measured for P. stutzeri (35.5 ± 9.3 μM) and S. loihica (7.07 ± 1.13 μM). Genome sequencing demonstrated that Dechloromonas denitrificans possessed a clade II nosZ gene, and a measured Ks of 1.01 ± 0.18 μM for N2O was consistent with the values determined for the other clade II organisms tested. These observations provide a plausible mechanistic basis for why the relative activity of bacteria with clade I nos operons compared to that of bacteria with clade II nos operons may control N2O emissions and determine a soil's N2O sink capacity.

Importance: Anthropogenic activities, in particular fertilizer application for agricultural production, increase N2O emissions to the atmosphere. N2O is a strong greenhouse gas with ozone destruction potential, and there is concern that nitrogen may become the major driver of climate change. Microbial N2O reductase (NosZ) catalyzes N2O reduction to environmentally benign dinitrogen gas and represents the major N2O sink process. The observation that bacterial groups with clade I nosZ versus those with clade II nosZ exhibit distinct affinities to N2O has implications for N2O flux models, and these distinct characteristics may provide opportunities to curb N2O emissions from relevant soil ecosystems.

FIG 1

Growth (●) and N₂O consumption (○) in cultures of P. stutzeri strain DCP-Ps1 (clade I) (A), S. loihica strain PV-4 (clade I) (B), D. aromatica strain RCB (clade II) (C), and A. dehalogenans strain 2CP-C (clade II) (D). The error bars represent the standard deviations of measurements made with samples extracted from three separate culture vessels.

FIG 2

Whole-cell Michaelis-Menten kinetics curves of P. stutzeri strain DCP-Ps1 (clade I) (A), S. loihica strain PV-4 (clade I) (B), D. aromatica strain RCB (clade II) (C), D. denitrificans strain ED-1 (clade II) (D), and A. dehalogenans strain 2CP-C (clade II) (E). The different symbols (○, ×, and ◆) represent the results from independent experiments.