Culture-independent diversity of actinobacteria associatedwith two marine sponges Hymeniacidon perlevis and Sponge sp.

A culture-independent nested PCR approach was employed to\ninvestigate the phylogenetic diversity of actinobacteria communities\nassociated with two marine sponges Hymeniacidon perleve\nand Sponge sp. (Stach et al., 2003). The phylogenetic affiliation of\nsponge-derived actinobacteria was assessed by restriction fragment\nlength polymorphism (RFLP) analysis (Zhang et al., 2006)\nand 16S rDNA sequencing of cloned DNA fragments. Of 196\npositive clones screened by RFLP, a total of 46 representative\ncloneswere selected for sequencing. Phylogenetic analysis of these\nclones revealed representatives of 10 actinobacteria genera including\nAcidimicrobium, Gorynebacterium, Propionibacterium, Gordonia,\nMicrococcus, Microbacterium, Streptomyces, Mycobacterium, Cellulosimicrobium\nSporichthya, and unidentified actinobacteria clones.\nAcidimicrobium was the most abundant group in H. perleve clone\nlibrary, representing 33% of the clones, with the second abundant\ngroup being Sporichthya, representing 20% of the clones. There was\na high predominance of Mycobacteria group in Sponge sp. clone\nlibrary, which accounted for 70% of all clones. The second abundant\ngroup was Acidimicrobium, representing 24%. These results\nindicated the presence of a high diversity of actinobacteria phylotypes\nwithin the sponges H. perleve and Sponge sp., belonging to\n11 main groups. Actinobacteria community grouped within subclass\nAcidimicrobidae was found in both clone libraries, which\nwas closely affiliated with previous reports (Taylor et al., 2007).\nThe information obtained by the cultural-independentmethod will\nbe useful for designing isolation and cultivation protocols of unculturable\nactinobacteria.