Study On The Functional Genes Related To Denitrification And Sulfur Reduction Of Sponge-associated Microorganism

Sponges harbor phylogenetically complex and abundant microbialcommunities, however the role of sponge-associated microorganisms innitrogen cycling remains largely unknown, particularly, we hardly knowdenitrification and sulfur reduction process in sponges.Nitrogen-cycle is a critical biogeochemical process of the oceans. Marinenitrogen cycle is the main driving force of Earth’s carbon cycle; it has animportant role in regulating the Earth’s climate system. Marine nitrogen cyclestudy become marine geoscience and marine biodiversity research focus.Microorganisms from ocean involved in biological nitrogen fixation,nitrification and denitrification can be exploited to the most important sourceof nitrogen, therefore, studies of marine microorganisms has become thefocus center. Denitrification is a dissimilatory process in which nitrate andnitrite are reduced to gaseous nitric oxide, nitrous oxide and molecularnitrogen when oxygen is limited, which consists of four reaction stepscatalyzed by nitrate reductase (napA or narG), nitrite reductase (nirK or nirS), nitric oxide reductase (qnorB or cnorB) and nitrous oxide reductase (nosZ).Nitrite reductase is the key enzyme of this respiratory process since itcatalyzes the reduction of soluble nitrite into gas NO. Nitrous oxide reductasecatalyses the last step in the complete denitrification pathway. Greenhousegas N2O, which contribute not only to global warming but also directly to thedestruction of the stratospheric ozone layer, will go to the atmosphere withoutthe further reduction by nitrous oxide reductase. Therefore nitrite reductase(cytochrome cd1-dependent nirK or copper-containing nirS) genes andnitrous oxide reductase (nosZ) gene are usually used as genetic markers toinvestigate denitrifying communityThe nitrogen fixation by sponge cyanobacteria was early observed. Untilrecently, sponges were found to be able to release nitrogen gas. However thegene-level evidence for the role of bacterial symbionts from different speciessponges in nitrogen gas release is limited. And meanwhile, the quanitativeanalysis of nitrogen cycle-related genes of sponge microbial symbionts isrelatively lacking.The nirK gene encoding nitrite reductase which catalyzessoluble nitrite into gas NO and nosZ gene encoding nitrous oxide reductasewhich catalyzes N2O into N2are two key functional genes in the completedenitrification pathway. In this study, using nirK and nosZ genes as markers, the potential of bacterial symbionts in six species of sponges in the release ofN2was investigated by phylogenetic analysis and real-time qPCR. As a result,totally,2OTUs of nirK and5OTUs of nosZ genes were detected by genelibrary-based saturated sequencing. Difference phylogenetic diversity of nirKand nosZ genes were observed at OTU level in sponges. Meanwhile,real-time qPCR analysis showed that Xestospongia testudinaria had thehighest abundance of nosZ gene, while Cinachyrella sp. had the greatestabundance of nirK gene. Phylogenetic analysis showed that the nirK andnosZ genes were probably of Alpha-, Beta-, and Gammaproteobacteria origin.The results from this study suggest that the denitrification potential ofbacteria varies among sponges because of the different phylogenetic diversityand relative abundance of nosZ and nirK genes in sponges. Totally, both thequalitative and quantitative analyses of nirK and nosZ genes indicated thedifferent potential of sponge bacterial symbionts in the release of nitrogengas.The sulfur cycle is complex, because sulphur has a broad range ofoxidation states, from–2(completely reduced) to+6(completely oxidized),and can be transformed both chemically and biologically. The sulfur cyclehas multiple ties to the cycles of other elements, most notably those of carbon, nitrogen, phosphorous, and iron. Sulfate-reducing bacteria (SRB) andsulfate-oxidizing bacteria (SOB) are biogeochemically important organismsin the sulfur transformation in the marine environment. Different techniqueshave been used to detect SRB and SOB, study their diversity and activity.Cloning or denaturing gradient gel electrophoresis of PCR-amplified16SrRNA，dsrAB or aprA gene fragments has been used to determine thediversity of SRB and SOB in many different habitats. Apr reductase(adenosine5’-phosphosulfate reductase) consists of an alpha and betasubunit, encoded by the genes aprA and aprB, respectively. The aprA genehas been studied in SRB and SOB by specific PCR. Homologous proteinshave been shown to exist in several anoxygenic phototrophic andchemotrophic, facultative anaerobic sulfur-oxidizing bacteria (SOB) in whichthey were suggested to operate in the reverse direction oxidizing sulfide tosulfate.The global sulfur cycle depends on the activities of metabolically andphylogenetically diverse microorganisms, most of which reside in the ocean.Marine sponges often harbour dense and diverse microbial communities, withmany of the microorganisms being specific to sponge hosts. The spongeassociated microbes may play important role in ocean sulfur cycle. In this study, functional gene aprA was used as a marker to investagate symbionts ofsix sponges from two sites in China coast. By phylogenetic analysis andquantitative study by RT-qPCR, this study enriched the diversity of microbiesrelated to sulfur-cycle in sponges. In particular, a large group of unknownSOBs in Apr lineage I was detected, which may be new group of SOB insulfur metabolism. Moreover, it is the first quantitative study on the sulfurcycle-related functional gene of sponge assicatied microorganism.