1. METAGENOMICS OF CYANOBACTERIAL BLOOMS Phillip B Pope and Bharat K.C. Patel Microbial Gene Research and Resources Facility, School of Biomolecular and Biomedical Sciences, Faculty of Science, Griffith University, Brisbane, Queensland 4111, Australia, Eskitis Institute for cell and molecular therapies, Griffith University, Brisbane, Queensland 4111, Australia, and Cooperative Research Centre for Water Quality and Treatment, Australia. Extraction of high molecular weight DNA Construction of BAC library + Single gene polyketide- synthase library Single gene PKS library A high proportion of cyanobacterial secondary metabolites have been found to belong to groups of polyketides and peptides. A single- gene library constructed from PCR fragments amplified from cyano- bacterial bloom HMW DNA (Fig 4) revealed the presence of 9 nucleotide sequences that encoded regions of PKS genes (Table 1). Analysis suggests that the same gene diversity could be represented in the BAC library as the same HMW DNA was used to construct both libraries. Construction of BAC library High molecular weight (HMW) DNA was isolated from the toxic bloom sample and a BAC library consisting of 2850 clones constructed. The average insert size of CBNPD1 library determined after Bgl II digestion and pulsed field gel electrophoresis (PFGE) was determined to be 27 kb, with size ranging from 5kb to greater than 50kb (Fig 2 and 3). Greater than 60% of the clones contained inserts greater than 20kb in size. Fig 3. BAC insert size distribution. Table 1. Protein-coding genes of highest similarity to that of sequenced and analysed clone insert DNA originating from PCR screens (Fig 4). BACKGROUND & AIMS Cyanobacterial blooms are commonly associated with the production of secondary metabolites including numerous human health affecting toxins. Molecular analysis of large genomic fragments recovered directly from an environmental bloom sample represents an approach by which genes responsible for the formation and expression of secondary metabolites can be studied. Consequently, we have prepared a large-construct Bacterial Artificial Chromosome library (BAC) from the DNA of a natural toxin-producing cyanobacterial bloom. We have in tandem, from the same cyanobacterial bloom DNA, constructed and analysed PCR based single gene libraries of 16S rRNA and polyketide synthases (PKS). This three-fold experimental approach has the potential to provide a phylogenetic community snap shot of the cyanobacterial bloom community structure and their physiological functions within the bloom. Therefore molecular analysis of the BAC library together with the PCR single gene libraries provides a powerful tool for establishing a link between secondary metabolite production (e.g. PKS) and microbial diversity in situ. Single gene 16S rDNA library The diversity of bacterial communities associated with cyanobacterial blooms have been poorly studied despite their probable important role in cyanobacterial bloom structure and function. A phylogenetic comparison of 78 16S rDNA sequences has identified 5 new clusters which also may be characteristic of bloom events (one example shown in Fig 7) showing that bacterial communities associated with cyanobacterial blooms may have specific groups that are distinct for bloom events Library screening: sequence and expression 16S rRNA genes from pooled clones were amplified. Two clones identified (Fig 5) were presumed to contain 16S rRNA genes. Sequence analysis showed that they belonged to 2 phylogenetic groups (Table 2). One of the 2 BAC clones containing the 16S rRNA gene clone 578 was sequenced and is reported below (BAC clone insert sequencing and analysis). Evidence of functional expression of the cloned library was provided from the presence of amylase in clone 905 (Fig 6). Collection of cyanobacterial bloom sample Single gene 16S rDNA library BAC clone insert sequencing and analysis OUTCOMES & FUTURE DIRECTIONS In a bid to understand microbial community structure and composition as well as determine the mechanisms of blooming and toxin production in cyanobacterial blooms, we used a culture- independent approach and constructed a metagenomic library, designated CBNPD1. Preliminary molecular evidence suggests that the library contains valuable information on genes, gene clusters and partial or entire metabolic pathways of yet to be cultured microbes. Our single gene studies have demonstrated the presence of a diverse range of PKS genes present in the cyanobacterial bloom under investigation. We hope to screen for their presence in our metagenomic library in future. The marriage of our studies will provide a much-needed understanding of interactions within bacterial communities associated with cyanobacterial blooms and their distribution. It is hoped that this information will ultimately be used to predict bloom events and the data be included as part of water management strategies. Collection of bloom sample Lake Samsonvale (2716’S, 15256’E), a warm monomictic eutrophic reservoir experiences thermal stratification from September to May with toxic cyanobacterial blooms a frequent occurrence during this period. Water samples (0-3 m) were collected predefined locations, 10001S and 10006S (SEQwater cooperation, Brisbane, QLD), with an integrated depth sampler in mid-September 2003. Samples were kept at 4C for no more than 24 hours before use. Fig 4. Gel electrophoresis analysis of PCR reactions using cyanobacterial PKS specific primers DKF/DKR from two different DNA templates (DNA extraction (L2), and ligation mix (L3)) used in CBNPD1 BAC library construction. L2 L3 Fig 1. Lake Samsonvale (North Pine dam). Fig 2. PFGE of random BAC clones. Fig 6. Example of a starch hydrolysis screen detecting CBNPD1 clone 905 producing a metagenomic DNA encoded amylase by the clone host E.coli. Table 3. 16S rRNA sequences obtained from CBNPD1 BAC clones identified from 16S rDNA library screens. Fig 5. PCR screening of CBNPD1 clones 577- 588 to identify clones containing 16S rDNA. Lanes 3 and 6 represent amplified 16S rDNA products from individually examined clones (578 and 581). Sequence originating from lake Samsonvale Fig 7. Phylogenetic tree of partial 16S rDNA sequences obtained from Lake Samsonvale (CYN-) affiliating to sequences obtained from GenBank of the Domain Alpha-proteobacteria. Novel freshwater clusters identified in this study are designated as cyn. Sequences titled LiUU-, MCY- and CYN- are sequences from cyanobacterial bloom associated communities in Sweden and Australia. Cluster possibly characteristic for bloom events CloneBest Blast hitPutative FunctionSim. % pks-2Microcystis spp.PKS McyE98 pks-6Microcystis spp.PKS McyD84 pks-8Nostoc spp.PKS modules81 pks-12Anabaena spp.Hypothetical protein76 pks-14Nostoc spp.PKS modules75 pks-18Anabaena spp.PKS type 169 pks-19Microcystis spp.PKS McyE 62 pks-21Microcystis spp.PKS McyD61 pks-23Anabaena spp.Hypothetical protein81 97.0 Kb 48.5 Kb random BAC clones 23.1 Kb 9.0 Kb 6.0 Kb vector fragment Library screening: sequence and expression BAC clone insert sequencing and analysis BAC inserts sequenced to completion comprised 173 kb of a cyanobacterial bloom metagenome. 183 genes have been identified and assigned to COG functional categories, several of which were found to affiliate to proteins of interest both ecologically and in terms of potential in industrial processes. These include a putatuve RTX toxin and putative response regulators involved in quorum sensing and controlling expression of exoproteins, including toxins. Fig 8. Linear ORF maps of the 7 completely sequenced BAC clones from the Cyanobacterial bloom metagenome library. ORF’s are colour coded according to their COG affiliations and to highest ribosomal genes, where they exist CloneInsert size (Kb) Phylogenetic affiliation (Phylum Genus) Sim. % 54530 γ-Proteobacteria Pseudomonas 99 57822 β-Proteobacteria Roseateles 96 RTX toxin Response regulator Response regulator