Estimating The Actinobacterial Diversity And Activity Potentiality In A Coastal Wetland Of The Yellow River Delta

The Yellow River delta is located in warm temperate zone of China, which has awealth of natural resources and biological resources. However, insufficiency of thefresh water and the action of land and sea together result in a seriously imbalance ofsalt and water. It is believed that there are diverse actinomycetes resource adapting tothe environment which have unique metabolic pathways and potential abilities ofsynthesizing novel metabolites during their long adaption in such specific ecosystem.It is very important to investigate the diversity of actinomycetes from the YellowRiver delta, to screen functional genes and explore its potential resources.Culture-independent methods was used to estimate the diversity ofactinomycetes of four samples from the Yellow River delta based on16S rRNA geneclone library and RFLP analysis. Site BZ10exhibited the best actinomycetes diversityincluding15genera; actinomycetes diversity of site DY14was less than BZ10,comprising12genera; genera were obtained from site DY40; site BZ25was found tohave the least actinomycetes diversity, just3genera. The actinomycetes communitiesin the soils of coastal wetland in the Yellow River delta displayed a highly diversitycomposition including members from22genera belonging to9suborders whichincluding10families totally from classes Actinobacteria and Acidimicrobiia, most ofthe actinomycetes were new genera. There were also some unclassified sequences,which may represent some new actinomycetes.Based on culture-dependent method,165actinomycetes were isolated from8different media, including GS1medium, ISP5medium, GW1medium, M3medium,glycerol-Arg medium, GS1improved medium, starch-casein improved medium andsodium propionate medium. Nystafungin, nalidixate sodium and potassiumdichromate were supplemented in all the media to inhibit the growth of fungi and bacteria. According to the morphological characteristics and cultural features, allstrains were divided into10groups: white spore class group, ash brown classgroup,yellow class group, pink spore class group, ash red purple class group, ashesash class group, green class group, blue class group, cyan class group and goldenclass group. Analysis of the16S rDNA sequences showed that these strains belong to12different genera of Streptomyces, Agromyces, Arthrobacter, Brachybacterium,Brevibacterium, Kocuria, Rhodococcus, Paracoccus, Tsukamurella, Micrococcus,Nesterenkonia, Nocardiopsis, including a potential new species of Agromyces. Theactinomycetes diversity obtained during culture-independent method were more thanthat obtained from culture-independent method, because of the limitation of isolationtechnology, most actinomycetes in the environment can not be cultured currently.Therefore, culture-dependent method combing with culture-independence method canestimate microorganism of some environment comprehensively.Sequence Guided Genetic screening was performed to evaluate the potentialability and diversity of synthesizing natural products. Three pairs of degenerateprimers K1F/M6R, FW/RV, A3F/A7R were used to amplify the type I polyketidesynthetase(PKS-I) gene, the type II polyketide synthetase(PKS-II) gene,non-ribosomal peptide synthetase(NRPS) gene respectively. The result showed thatthere were9strains containing PKS-I gene, there were11strains containing PKS-IIgene, there were6strains containing NRPS gene. Some gene fragments had a lowsimilarity with existing sequences submitted into Gene Bank, which could illustratethat there were new compounds produced from some strains.Anti-microbial activity selection was carried out during59strains.22activestrains were selected finally, including9Streptomyces,8Nocardiopsis and5Bacillus.Strain OAct388showed the best anti-microbial activity, can inhibit6pathogenbacteria, and5bacteria were inhibited by strain OAct363.The present studies revealed that abundant actinomycetes resources existed in thesoil sample in coastal wetland of the Yellow River delta, and anti-microbial activityselection showed a high activity of compounds produced from actinomycetes isolatedfrom the Yellow River delta. Functional gene screening and combination method between culture-dependent and culture-independent could be powerful exploring thepotential microbial resources.