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Denitrifying alphaproteobacteria from the Arabian Sea that express nosZ, the gene encoding nitrous oxide reductase, in oxic and suboxic waters.

ABSTRACT: Marine ecosystems are significant sources of the powerful greenhouse gas nitrous oxide (N2O). A by-product of nitrification and an intermediate in the denitrification pathway, N2O is formed primarily in oxygen-deficient waters and sediments. We describe the isolation of a group of alphaproteobacteria from the suboxic waters of the Arabian Sea that are phylogenetically affiliated with Labrenzia spp. and other denitrifiers. Quantitative PCR assays revealed that these organisms were very broadly distributed in this semienclosed ocean basin. Their biogeographical range extended from the productive, upwelling region off the Omani shelf to the clear, oligotrophic waters that are found much further south and also included the mesotrophic waters overlying the oxygen minimum zone (OMZ) in the northeastern sector of the Arabian Sea. These organisms actively expressed NosZ (N2O reductase, the terminal step in the denitrification pathway) within the OMZ, an established region of pelagic denitrification. They were found in greatest numbers outside the OMZ, however, and nosZ mRNAs were also readily detected near the base of the upper mixed layer in nutrient-poor, oxic regions. Our findings provide firm molecular evidence of a potential sink for N2O within well-ventilated, oceanic surface waters in this biogeochemically important region. We show that the Labrenzia-like denitrifiers and their close relatives are habitual colonizers of the pseudobenthic environment provided by Trichodesmium spp. We develop the conjecture that the O2-depleted microzones that occur within the colonies of these filamentous, diazotrophic cyanobacteria might provide unexpected niches for the reduction of nitrogen oxides in tropical and subtropical surface waters.

SUBMITTER: Wyman M

PROVIDER: S-EPMC3623181 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Project description:The effect of standard agricultural management on the genetic heterogeneity of nitrous oxide reductase (nosZ) fragments from denitrifying prokaryotes in native and cultivated soil was explored. Thirty-six soil cores were composited from each of the two soil management conditions. nosZ gene fragments were amplified from triplicate samples, and PCR products were cloned and screened by restriction fragment length polymorphism (RFLP). The total nosZ RFLP profiles increased in similarity with soil sample size until triplicate 3-g samples produced visually identical RFLP profiles for each treatment. Large differences in total nosZ profiles were observed between the native and cultivated soils. The fragments representing major groups of clones encountered at least twice and four randomly selected clones with unique RFLP patterns were sequenced to verify nosZ identity. The sequence diversity of nosZ clones from the cultivated field was higher, and only eight patterns were found in clone libraries from both soils among the 182 distinct nosZ RFLP patterns identified from the two soils. A group of clones that comprised 32% of all clones dominated the gene library of native soil, whereas many minor groups were observed in the gene library of cultivated soil. The 95% confidence intervals of the Chao1 nonparametric richness estimator for nosZ RFLP data did not overlap, indicating that the levels of species richness are significantly different in the two soils, the cultivated soil having higher diversity. Phylogenetic analysis of deduced amino acid sequences grouped the majority of nosZ clones into an interleaved Michigan soil cluster whose cultured members are alpha-Proteobacteria. Only four nosZ sequences from cultivated soil and one from the native soil were related to sequences found in gamma-Proteobacteria. Sequences from the native field formed a distinct, closely related cluster (D(mean) = 0.16) containing 91.6% of the native clones. Clones from the cultivated field were more distantly related to each other (D(mean) = 0.26), and 65% were found outside of the cluster from the native soil, further indicating a difference in the two communities. Overall, there appears to be a relationship between use and richness, diversity, and the phylogenetic position of nosZ sequences, indicating that agricultural use of soil caused a shift to a more diverse denitrifying community.

Project description:UNLABELLED:Microbial activities in soils, such as (incomplete) denitrification, represent major sources of nitrous oxide (N2O), a potent greenhouse gas. The key enzyme for mitigating N2O emissions is NosZ, which catalyzes N2O reduction to N2. We recently described "atypical" functional NosZ proteins encoded by both denitrifiers and nondenitrifiers, which were missed in previous environmental surveys (R. A. Sanford et al., Proc. Natl. Acad. Sci. U. S. A. 109:19709-19714, 2012, doi:10.1073/pnas.1211238109). Here, we analyzed the abundance and diversity of both nosZ types in whole-genome shotgun metagenomes from sandy and silty loam agricultural soils that typify the U.S. Midwest corn belt. First, different search algorithms and parameters for detecting nosZ metagenomic reads were evaluated based on in silico-generated (mock) metagenomes. Using the derived cutoffs, 71 distinct alleles (95% amino acid identity level) encoding typical or atypical NosZ proteins were detected in both soil types. Remarkably, more than 70% of the total nosZ reads in both soils were classified as atypical, emphasizing that prior surveys underestimated nosZ abundance. Approximately 15% of the total nosZ reads were taxonomically related to Anaeromyxobacter, which was the most abundant genus encoding atypical NosZ-type proteins in both soil types. Further analyses revealed that atypical nosZ genes outnumbered typical nosZ genes in most publicly available soil metagenomes, underscoring their potential role in mediating N2O consumption in soils. Therefore, this study provides a bioinformatics strategy to reliably detect target genes in complex short-read metagenomes and suggests that the analysis of both typical and atypical nosZ sequences is required to understand and predict N2O flux in soils. IMPORTANCE:Nitrous oxide (N2O) is a potent greenhouse gas with ozone layer destruction potential. Microbial activities control both the production and the consumption of N2O, i.e., its conversion to innocuous dinitrogen gas (N2). Until recently, consumption of N2O was attributed to bacteria encoding "typical" nitrous oxide reductase (NosZ). However, recent phylogenetic and physiological studies have shown that previously uncharacterized, functional, "atypical" NosZ proteins are encoded in genomes of diverse bacterial groups. The present study revealed that atypical nosZ genes outnumbered their typical counterparts, highlighting their potential role in N2O consumption in soils and possibly other environments. These findings advance our understanding of the diversity of microbes and functional genes involved in the nitrogen cycle and provide the means (e.g., gene sequences) to study N2O fluxes to the atmosphere and associated climate change.

Project description: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 ?M for D. aromatica and 1.34 ± 0.35 ?M for A. dehalogenans were significantly lower than the values measured for P. stutzeri (35.5 ± 9.3 ?M) and S. loihica (7.07 ± 1.13 ?M). Genome sequencing demonstrated that Dechloromonas denitrificans possessed a clade II nosZ gene, and a measured Ks of 1.01 ± 0.18 ?M 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.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.

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Denitrifying alphaproteobacteria from the Arabian Sea that express nosZ, the gene encoding nitrous oxide reductase, in oxic and suboxic waters.

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