Aspects of the interaction between the marine bacterium Alcanivorax DG881 and the toxic dinoflagellate Gymnodinium catenatum

The presence of a bacterial community is vital to the germination and growth of the toxic dinoflagellate Gymnodinium catenatum. Previous research has shown that the bacterium Alcanivorax DG881 is an important stimulatory member of the dinoflagellate-associated bacterial community, however the nature of the interaction between the two organisms, and the substances and mechanism involved in growth stimulation are unknown. This thesis uses a uni-bacterial G. catenatum experimental culture model to investigate elements of the interaction between the marine bacterium Alcanivorax DG881 with the dinoflagellate G. catenatum. In the first experiment, three treatments were used to determine whether the G. catenatum growth stimulating substances produced by Alcanivorax DG881 were extracellular or intracellular substances, and whether these substances need to be continuously provided to G. catenatum to support growth. Addition of extracellular filtrates from cultures of G. catenatum and it's associated bacteria showed increased growth stimulating activity in resting cyst germination experiments compared to treatments containing intracellular substances from Alcanivorax DG881 in absence of live Alcanivorax DG881 cells. Repeated addition of extracellular filtrates sustained G. catenatum growth after germination for a significantly longer period and to higher cell concentrations than a single addition of extracellular filtrate. These results indicated that the G. catenatum did not obtain growth stimulating substances by ingesting bacteria but requires one or more extracellular dissolved products produced by Alcanivorax DG881. The patterns of growth suggest that the products were either labile or utilised by the dinoflagellate during growth. It has been proposed that dinoflagellate-associated Alcanivorax DG881 benefits from the utilization of dissolved organic carbon (DOC) exuded from the dinoflagellate cell. To examine this idea, the single carbon utilization profile of Alcanivorax DG881 was compared with the closely related but no-stimulatory strain Alcanivorax borkumensis SK2 using the BIOLOG GN2 plate assay system. Alcanivorax DG881 was able to use a much wider range of carbon compounds for growth than Alcanivorax borkumensis SK2, particular a wider range of amino acids, known as an important component of the DOC exuded from algal cells. The data here suggest that Alcanivorax DG881 is relatively better adapted to a life associated with algal cells than Alcanivorax borkumensis SK2. Detection and sequence characterization of putative saxitoxin synthesis gene homologues was attempted. Degenerate PCR primers designed from sequence of three putative saxitoxin biosynthesis (Sxt) genes from cyanobacteria was used to screen G. catenatum total DNA extracts. PCR products of expected length were obtained for three Sxt genes and two products were sequenced and compared to the putative cyanobacterial homolog and other published DNA sequences available on Genbank. The putative G. catenatum SxtN gene sequence showed highest similarity with sulfurtransferase of bacteria Francisella philomiragia subsp. Philomiragia (84% similarity), and with a hypothetical protein the Arabidopsis thaliana (81% similarity). The putative G. catenatum SxtU gene sequence showed low similarity with a hypothetical protein of Peptostreptococcus micros (46% similarity) and hypothetical proteins from the fungi Aspergillus oryzae (46% similarity). Phylogenetic comparisons of the partial sequences of both candidate genes suggested that they were of bacterial rather than dinoflagellate origin, and bacteria associated with G. catenatum not involved in saxitoxin synthesis directly or indirectly.