The Different Potential of Sponge Bacterial Symbionts in N2 Release Indicated by the Phylogenetic Diversity and Abundance Analyses of Denitrification Genes, nirK and nosZ

Xia Zhang,Zhiyong Li,Liming He,Wei Sun,Fengli Zhang,Jack Anthony Gilbert

doi:10.1371/journal.pone.0065142

Abstract

Nitrogen cycle is a critical biogeochemical process of the oceans. The nitrogen fixation by sponge cyanobacteria was early observed. Until recently, sponges were found to be able to release nitrogen gas. However the gene-level evidence for the role of bacterial symbionts from different species sponges in nitrogen gas release is limited. And meanwhile, the quanitative analysis of nitrogen cycle-related genes of sponge microbial symbionts is relatively lacking. The nirK gene encoding nitrite reductase which catalyzes soluble nitrite into gas NO and nosZ gene encoding nitrous oxide reductase which catalyzes N2O into N2 are two key functional genes in the complete denitrification pathway. In this study, using nirK and nosZ genes as markers, the potential of bacterial symbionts in six species of sponges in the release of N2 was investigated by phylogenetic analysis and real-time qPCR. As a result, totally, 2 OTUs of nirK and 5 OTUs of nosZ genes were detected by gene library-based saturated sequencing. Difference phylogenetic diversity of nirK and nosZ genes were observed at OTU level in sponges. Meanwhile, real-time qPCR analysis showed that Xestospongia testudinaria had the highest abundance of nosZ gene, while Cinachyrella sp. had the greatest abundance of nirK gene. Phylogenetic analysis showed that the nirK and nosZ genes were probably of Alpha-, Beta-, and Gammaproteobacteria origin. The results from this study suggest that the denitrification potential of bacteria varies among sponges because of the different phylogenetic diversity and relative abundance of nosZ and nirK genes in sponges. Totally, both the qualitative and quantitative analyses of nirK and nosZ genes indicated the different potential of sponge bacterial symbionts in the release of nitrogen gas.

Highlights

The oceans are a central feature of the biosphere with biogeochemical links to atmosphere
As a result, based on the 23 and 34 clones sequenced, 2 and 5 operational taxonomic unit (OTU) were obtained for nirK and nosZ genes, respectively, at 97% identity level (Table 2; Figure S1).The mean estimations of OTU richness using Chao estimators were 2 and 5.5 OTUs for nirK and nosZ genes, respectively
For nosZ genes, A. queenslandica and S. vesparium had OTU1 and OTU2, X. testudinaria had OTU1, 2 and OTU4, Cinachyrella sp. had OTU2 and OTU5, whereas OTU3 was observed in C. australiensis

Summary

Introduction

The oceans are a central feature of the biosphere with biogeochemical links to atmosphere. Marine sponges (Porifera) are thought to have evolved approximately ,580 million years and are among the most ancient metazoans [2,3]. They represent a significant component of the marine biosphere throughout the coral reefs and benthic ecosystem. Many achievements in nitrification mediated by sponge microbial symbionts have been made, for example ammonia-oxidizing archaea (AOA) [17,18,19,20], ammonia-oxidizing Gamma- and Betaproteobacteria, nitrite-oxidizing Nitrospira and anaerobic ammonia-oxidizing bacteria Planctomycetales [16,18]

Methods

Results

Conclusion