Environmental Microbiology (2006) 8(8), 1460–1470 doi:10.1111/j.1462-2920.2006.01040.x © 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd Blackwell Publishing LtdOxford, UKEMIEnvironmental Microbiology 1462-2912© 2006 The Authors; Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd ? 20068814601470Original Article Polyketide synthase diversity of sponge symbiontsT. K. Kim and J. A. Fuerst Received 4 November, 2005; accepted 17 February, 2006. *For correspondence. E-mail j.fuerst@mailbox.uq.edu.au; Tel. (+1) 617 3365 4643; Fax (+1) 617 3365 4699. Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata : culture-dependent and culture-independent approaches Tae Kyung Kim and John A. Fuerst* School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Qld 4072, Australia. Summary Diverse ketosynthase (KS) genes were retrieved from the microbial community associated with the Great Barrier Reef sponge Pseudoceratina clavata . Bacterial isolation and metagenomic approaches were employed. Phylogenetic analysis of 16S rRNA of cul- turable sponge-associated bacterial communities comprised eight groups over four phyla. Ten KS domains were amplified from four genera of isolates and phylogenetics demonstrated that these KS domains were located in three clusters (actinobacte- rial, 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 screen- ing. Five KS domains were retrieved from polymerase chain reaction (PCR) amplification using sponge metagenome DNA as template and five fosmid clones containing KS domains found using multiplex PCR screening. Analysis of selected polyketide synthase (PKS) from one fosmid showed that the PKS consists of two modules. Open reading frames located up- and downstream of the PKS displayed similarity with mem- brane synthesis-related proteins such as cardiolipin synthase. Metagenome approaches did not detect KS domains found in sponge isolates. All KS domains from both metagenome approaches formed a single cluster with KS domains originating from metage- nomes derived from other sponge species from other geographical regions. Introduction Marine sponges have been used as a major source of new natural bioactive compounds for decades. The origin of bioactive compounds in marine sponges has been pro- posed to be from either the sponge itself, their symbiotic microorganisms, or a cooperative interaction between sponge and symbiotic microorganisms (Faulkner et al ., 1993; Uriz et al ., 1996; Flowers et al ., 1998). These den- sity gradient centrifugation-based studies have failed to establish the location of the source of bioactive com- pounds with respect to different cell types or regions within the sponge. In situ hybridization techniques for targeting biosynthesis genes of interest have been introduced and have provided strong evidence for the bacterial origin of marine invertebrate bioactive compounds and expression of related genes for their synthesis (Davidson et al ., 2001; Flatt et al ., 2005). In addition, the increasing data regard- ing bacterial diversity associated with marine sponges enhances the possibility of finding novel compounds and their related genes or gene clusters from the marine sponge-associated bacterial communities (Webster et al ., 2001; Hentschel et al ., 2002; Kim et al ., 2005; Montalvo et al ., 2005). Both cultural and direct gene retrieval approaches may be needed to explore the diversity of the bacteria associated with sponges. Approaches which iso- late biosynthetic genes or gene clusters from such micro- bial communities with the aim of eventually providing symbiont-generated bioactive compounds under con- trolled conditions have been proposed (Hildebrand et al ., 2004). Polyketides are well-studied and include many commer- cially produced biopharmaceuticals. The diversity of their structures includes macrolides and polyenes and the diversity of applications includes antibiotics, antitumour agents, immunosuppressants and the statin class of cho- lesterol-controlling agents. Type I (modular) polyketide synthases (PKSs) have been of special interest for their modularity and suitability for the creation of customized polyketides (Hopwood, 1997; Pfeifer et al ., 2001; McDaniel et al ., 2005). A multiplasmid approach employ- ing plasmids carrying type I PKS genes has a potential to create thousands of combinations of PKS modules to produce novel polyketides (Xue et al ., 1999). Recent advances using synthetic PKS modules have indicated the importance of a wide database of PKS sequences