Isolation And Multilocus Sequence Analysis Of Actinbacteria Isolated From A Coastal Wetland Of The Yellow River Delta And Identification Of A Novel Aceinomycete Species

The Yellow River Delta is the largest, youngest and most complete Chinesecoastal wetland ecosystems, which is located in warm temperate zone of China. It hasa wealth of natural biological resources. However, the action of land and sea togetherand insufficiency of the fresh water result in a seriously imbalance of salt and water.Because unique geographical location and ecological environment of the YellowRiver Delta wetland, there is a wealth of actinomycetes group, many of whom arespecial actinomycetes group. Meanwhile, these actinomycetes may have uniquemetabolic pathways and potential abilities of synthesizing novel metabolites duringtheir long adaption in such specific ecosystem, these are worthy of our study andexploration in the further.Eight media were used to isolate actinomycetes strains from12samples and80strains were obtained. According to the morphological characteristics,80strains wereselected to sequence the16S rDNA. Sequence analysis and blast in the EzBioClouddata showed that these strains belong to20different species, which indicate theabundant and huge resources diversity of the Yellow River Delta. The maximumhomology identity of the OAct353strain with the known type strain is97.43%，whichmeans it is a potential novel species. However, the physiologic and biochemical indexand the chemical identification index need to be determined to confirm these strains.The Neighbor-Joining phylogenetic tree was constructed from the alignments of thesequences between all these strains and maximum homology strains, Strrptomycesand Nocardiopsis clustered into a big and independent branch, which illustrated thatStreptomyces and Nocardiopsis were the dominant population.Actinobacterialpopulation structures of the sediments in the Yellow River Delta is different fromwhich of the terrestrial environment. The actinobacterial diversity is abundant and massive new speices resources are contained.A Gram-positive, non-endospore-forming, dirty white-coloured strain, OAct353was isolated.Phylogenetic analysis based on16S rDNA gene sequences showed strainOAct353was most closely related to members of established species in the genusAgromyces with sequence similarities of approximately94.%–97.43%, particularlyAgromyces humatus CD5T,Agromyces subbeticus Z33T�'�Agromyces flavus CPCC202695T. DNA–DNA hybridization results showed none of the DNA–DNArelatedness values between strain OAct353and members of the genus Agromycesexceeded40%. The genomic DNA G+C content of the novel strain was68%. Thechemotaxonomic characteristics of strain OAct353, which had MK-11（21%）、MK-12（74%） and MK13（5%） as its major menaquinones and anteiso-C15:0（35%）、iso-C16:（036%）and anteiso-C17:（011%）as its predominant fatty acid were consistentwith classification in the genus Agromyces. Peptidoglycan in the novel straincontained alanine, glycine and glutamic acid. The polar lipid profile of strainOAct353comprised diphosphatidyl glycerol, phosphatidyly glycerol, phosphatidylyethanolamine and an unknown glycolipid. The novel strain’s major cell-wall sugarswere rhamnose, glucose and xylose. Based on the phylogenetic, DNA–DNAhybridization, chemotaxonomic and phenotypic data, strain OAct353represents anovel species within the genus Agromyces for which the name Agromycesbinzhouensis sp.nov is proposed; the type strain is OAct353(DSM28305).Multilocus sequence analysis (MLSA), a high resolution molecular classificationmethod, carries lots of advantages such as lateral comparable, simple and repeatability,which makes it an advanced approach to conclude the evolutionary relationships.According to the literature and public databases, five house keeping genes encodingproteins (atpD, gyrB, recA, rpoB and trpB) are chosen to research StreptomycesMLSA, meanwhile, gene amplification primers are identified.6pairs of strain wereselected as the experimental materials, and their genome DNA are extracted, theneach gene fragment was amplified and sequenced. By analyzing each of the single site,the compatibility of the sites is determined and the single gene trees are overallconsistent. Finally phylogenetic trees based on the five genes is constructed. A series of multiple gene phylogenetic tree analysis showed that Streptomyces from themangrove wetlands and coastal wetlands which has similar16S rRNA sequences hasclose phylogenetic relationship indeed. Combination of MLSA and DNA-DNAhybridization technology can distinguish Streptomyces which has quite similar16SrRNA sequences relationship.It has a certain academic significance to geographytaxonomy and development in chemistry and ecology.