Microbial Diversity Investigation And Metagenomic Library Analysis In The Sediment From Zhongshan Station, Antarctica

Microbe play a key role in Antarctic ecological system.Microorganisms have many characteristics including physiological structure,metabolism mechanism, substances with special function due to the condition of the extreme environments.So, the research of Antarctic microorganisms is important not only in understanding the diversity of life on earth and the global ecology process,but also recovering the bioactive materials and metabolism mechanism.The sediment within the treated sewage effluent to the sea was influenced frequently by human activities.Foreign materials which were introduced to the sediment probably resulted in changing microbial communities.And,the indigenous microbes under environmental pressure maybe produce novel bioactive compounds.In this study,we applied PCR-DGGE in examining and comparing the phylogenetic diversity of microbial communities in different layers of a sediment sample core from ZSS and ZSN.The samples ZSS and ZSN were collected from the coast near the China Zhongshan Station.The site ZSS was the sediment within the treated sewage effluent to the sea,while the ZSN site,200 meters away from ZSS site, was sediment of normal Antarctic seashore.Phylogenetic of bacterial communities analysis revealed that the bacterial communities present in two sites primarily clustered closely together within theα-,γ-,β-,δ- andθ-subdivisions of Proteobacteria,Cytophaga-Flavobacterium-Bacterioides(CFB)group, Actinobacteria,Acidobacteria,Gemmatimonadetes,low G+C gram-positive bacteria, Planctomycete,Chloroflexi,and an unknown group.Phylogenetic analysis of the partial 16S rRNA gene sequences showed that at least five clusters of Archaea inhabited ZSS and ZSN sediment,i.e.,four groups of Euryarchaeota and one of Crenarchaeota.Within the Euryarchaeota,at least three clusters of methanogens were detected.The structure of Archaea community in ZSS was relatively simple.Most 16S rRNA sequences of archaea were not closely related to any known isolates.The modular polyketide synthase(PKS)and nonribosomal peptide synthetase (NRPS)have been found to be involved in natural product synthesis in many microorganisms.The study on their diversities in natural environment may provide important ecological insights,in addition to opportunities for antibacterial drugs development.In this study,the PKS and NRPS gene diversities in ZSS and ZSN were studied.The phylogenetic analysis of amino acid(AA)sequences indicated that the identified KS domains were clustered with that from diverse bacterial group, including Proteobacteria,low G+C gram positive bacteria,Planctomycetes, Cyanobacteria,Actinobacteria,and uncultured sponge marine-associated microorganisms.Five unknown clades and one new branch belonging to hybrid PKS/NRPS enzyme complexes were found on the phylogenetic tree.The obtained A domains were mainly clustered within the Cyanobacteria and Proteobacteria group. Almost all of the identified KS and A domains showed below 80%and 60%identities at the AA level to the reference sequences in GenBank,respectively.Both of the KS and A domains in natural environmental sample were different.These results revealed the great diversity and novelty of both PKS and NRPS genes in Antarctic sediment.A metagenomic cosmid library containing about 6500 clones was constructed using the ZSS sample.The library contained at least 228 Mbp of the genomic resource of environmental microbes and provided more information about the microbial assemblages.Using the methods of PCR and Southern dot hybridization, we screened four cosmids carrying the gene cluster associated with secondary metabolites production clones.Among these clones,ZSK-90 was selected out to sequence.The length of the inserted DNA fragment in ZSK-90 was 41,263kb of which about 11kb was closely related with NRPS gene cluster.Sequence analysis of the 11kb fragment revealed there existed gene sections coding for adenylation domain (A),condensation domain(C),thiolation(T)(or Peptidyl Carrier Protein,PCP)and thioesterase domain(TE),and the gene sections of this fragment formed 4 modules involved in a nonribosmal peptide biosynthesis.It indicated the 11kb sequence was a intact gene cluster sequence coding for a novel nonribosmal peptide synthetase due to 43%identity to known NRPSs gene sequences.By analysis of active sites of A domain,this domain maybe specially select and activate Val. The different DNA repair test(DDRT)could be firstly applied to the screening of antitumor substances from environmental metagenomic cosmid library at the first time.About 10 cosmid clones with potential anti-tumor activity were obtained.Then, MTT assay confirmed the activity with the supernatant derived form liquid culture of the potential anti-tumor cosmid clones.Among these clones,ZSE-3 showed higher tumor inhibition in rates of 55%.ZSE-3 also showed the obvious capacities of anti-microbes against Staphylococcus aureus,Bacillus subtilis and Candida albicans. By selective medium,some cosmid clones which produce protease,amylase,lipase and cellulase were screened.One of them was confirmed to grow and produce protease in pH13 agar.In this study,we investigated microbial diversity and functional gene diversity (PKS I-KS and NRPS-A)of the two coast sediments near China Zhongshan Station and found abundant microbial resource.The cultured-independent metagenomic library provides many information for studying the novel metabolic pathways and new bioactive compounds synthesis of uncultured and unidentified microorganisms. More stirring results might be obtained from the ZS cosmid library for subsequent further research.