Bioactive compound-producing symbiotic bacteria from marine sponge species: cultural, gene retrieval and metagenomic studies

Abstract

A total of 142 strains were isolated from the microbial community associated with the Great Barrier Reef sponge Pseudoceratina clavata and 62 representatives were chosen after grouping on the basis of colony characteristics. 16S rRNA genes were amplified and partial sequences derived from the representatives. The BLASTN search result showed that these strains were composed of four phyla of bacteria which comprised Alphaproteobacteria (23 strains), Gammaproteobacteria (15 strains), Firmicutes (11 strains), and Actinobacteria (12 strains). Ten isolates were identified as members of the Salinispora group previously reported only from marine sediments. The relationship of the isolates to Salinispora was confirmed by phylogenetic analysis of 16S rRNA gene sequences. Colony morphology and pigmentation, occurrence and position of spores, and salinity requirements for growth were all consistent with this relationship. Phylogenetic analysis of the ketosynthase (KS) gene sequences of marine sponge-derived Salinispora isolates indicated that the polyketide synthase (PKS) gene sequence most closely related to that of Salinispora was the rifamycin B synthase of Amycolatopsis mediterranei. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis was applied to one sponge-derived Salinispora strain to test the hypothesis of rifamycin synthesis. The analysis demonstrated that this Salinispora isolate produced compounds of the rifamycin class including rifamycin B and rifamycin SV.Diverse KS genes were retrieved from the microbial community associated with the Great Barrier Reef sponge P. clavata. Bacterial isolation and metagenomic approaches were employed. Ten KS domains were amplified from four genera of isolates and phylogenetics demonstrated that these KS domains were located in three clusters (actinobacterial, cyanobacterial, and trans-AT type). Metagenomic DNA of the sponge microbial community was extracted to explore community KS genes by two approaches: direct amplification of KS domains and construction of fosmid libraries for KS domain screening. Five KS domains were retrieved from PCR amplification using sponge metagenome DNA as template and five fosmid clones containing KS domains were found using multiplex PCR screening. Analysis of a selected PKS from one fosmid showed that the PKS consists of two modules. Orfs located up- and downstream of the PKS displayed similarity with membrane synthesis-related proteins such as cardiolipin synthase. Metagenome approaches did not detect the same KS domains as those found in sponge isolates. All KS domains from both metagenome approaches formed a single cluster with the KS domains originating from metagenomes derived from other sponge species from other geographical regions.