Diversity And Functional Gene Mining Of Deep-sea Microorganisms

Deep-sea microorganisms,due to their adaption to extreme conditions,have evolved a completely different regulatory mechanism and unique metabolic capacity from those in terrestrial environment,which provides great theory significance and application value.However,most of deep-sea microorganisms resist cultivation in the laboratory limited to current technologies,which largely restricts the further utilization of deep-sea microorganisms.In recent years,the combination of enrichment culture strategies and high-throughput separation technologies enable us to achieve a high isolation rate for uncultured specific strains and realize the research on the diversity and function of deep-sea microorganisms.Planctomycetes,which is widely distributed in nature,has the characterised by unique genomes and physiological characteristics such as intracellular septum and many others similar to eukaryotes.Among them,marine Planctomycetes plays an important role in marine carbon and nitrogen cycle,which is an important model microorganism for studying microbial evolution and ecology.However,it has been difficult to obtain a large number of pure cultures in the laboratory for a long time This study aimed to isolate and culture deep-sea culturable bacteria and Planctomycetes,by using high-throughput sequencing and enrichment culture.Then,the isolates were identified based on the morphological,physiological characteristics and whole genome sequencing and biological activities analysis.The main research results are as follows:(1)Isolation,identification and diversity of deep-sea microorganisms:A total of 138 culturable bacteria were obtained from 26 deep-sea samples located at South China Sea and Indian Ocean,by using a special enrichment strategy was designed for Planctomycetes combined with high-throughput sequencing and 16 S rRNA gene specific detection.A unknown group OTU835 appeared in the enriched culture by high-throughput sequencing.The 16 S rRNA sequencig of OTU835 showed low similarity(84.17%)with the known relative of Planctomycetes.After 40 days of culture,relative abundance of OTU835 significantly increased.Finally,the 138 isolates belonged to 4 phyla,5 classes and 19 genera,and the dominant group was Erythrobacter of Proteobacteria.45 strains of culturable Planctomycetes were successfully isolated,belonging to Bremerella,Blastopirellula and Gimesia genus,and the dominant taxa was Gimesia(23 strains).At one time,deep-sea sediments was enriched and cultured using common medium,and a total of 242 culturable bacteria were obtained.According to phylogenetic analysis based on 16 SrRNA gene sequence,they belong to 4 phyla and 19 genera,among which the dominant groups are Firmicutes(85.54%)and Bacillus(81.8%).The whole genome analysis of representative strains of Bremerella and Blastopirellula genus showed that the 30T1 stain was similar(16SrRNA gene similiarity 99.43%)with its closest strain Bremerella volcania Pan97,but the DNADNA hybridization is only 20.20%,indicating that 30T1 could be a new species of Bremerella.The 30T3 stain was similar(16SrRNA gene similiarity 99.00%)with its closest strain Blastopirellula cremea LHWP2,the DNA-DNA hybridization is 85.10%,indicating that 30T3 is a known species of Blastopirellula.The physiological characteristics analysis showed that Bremerella sp.30T1 and Blastopirellula sp.30T3 are similar.They were Gram-negative,light brown,oval,with crater on the cell wall surface,strict aerobic and flagella.It could grow at 4?40?(optimum 35?37?),p H range was 6?9(optimum 7?8),NaCl range are respectively1?6%(w/v,optimum 3%)and 1?7%(w/v,optimum 3%).(2)Functional gene mining of deep-sea microorganisms:Genome analysis of the isolated bacteria revealed that there were many genes related to biotoxins and pesticide degradation enzymes(such as glucosyltransferase UDP and carboxylesterase),polysaccharide degradation enzymes(such as pullulanase,chitinase,chitosanase and cellulase),and enzymes(such as trehalose phosphorylase and mannosyltransferase)that improved plant stress resistance.The biological toxin degradation activity of the strains was preliminarily screened by high performance liquid chromatography.The results showed that four strains of Bacillus had the ability to degrade vomitoxin,zearalenone and aflatoxin.The maximum degradation rate of zearalenone by Bacillus wiedmannii RB44-7 could reach 80%.Whole genome analysis of 30T1,30T3 and Gimesia benthica E7 showed that three domains(named nagB1,nagB2,nagB3 genes)of glucosamine-6-phosphate deaminase,a key enzyme in the metabolic pathway of n-acetylglucosamine(GlcNAc),according to the gene annotation.Phylogenetic analysis indicated that the gene of this domain was homologous to the nagB gene with a strain of Gimesia benthica E7 which we were studying.GlcNAC was used as the only carbon and nitrogen source to culture strains 30T1 and 30T3,and the expressions of nagB1 and nagB2 in strain 30T1 were significantly up-regulated by real-time quantitative PCR,indicating that nagB1 and nagB2 genes are involved in the regulation of N-acetylglucosamine metabolism in strains 30T1.These results provide a basis for the enrichment culture,physiological characteristics study and functional gene exploration of deep-sea microorganisms,and provide a scheme for the isolation and culture of rare Marine microorganisms and the analysis of N-acetylglucosamine metabolism pathway,which lays a theoretical foundation for the exploitation and utilization of deep-sea microbial resources.