Chemical diversity of the marine actinobacterial genus Salinispora isolated from sponges of the Australian Great Barrier Reef

Abstract

This thesis studies the microbiology, chemical ecology, evolution and secondary metabolite production of the actinobacterial genus Salinispora, isolated from the Australian Great Barrier Reef (GBR) from Bundaberg to Horn Island, using liquid chromatography-mass spectrometry (LC-MS) based metabolomics. Untargeted and targeted metabolomics methodologies were used to explore the similarities and differences between the two species: Salinispora arenicola and Salinispora pacifica. The secondary metabolite profiles of these two species were obtained by ultra high pressure liquid chromatography-quadrupole time of flight- mass spectrometry (UHPLC-QToF-MS). The resultant data were interrogated using multivariate data analysis methods to compare their (bio)chemical profiles. Factors responsible for the chemotype variability in the bacterial secondary metabolite production were then identified, followed by a thorough analysis of the evidence for novel patterns of chemotypic differences. The results demonstrate that the main differences between these species depend on the host specificity defining the pattern of secondary metabolite production and show that metabolite production patterns are dependent upon: species, intra-species, sponge association and latitudinal progression along the GBR. Inter-species studies revealed consistent production of rifamycins and saliniketals in S. arenicola species; these appear to be exclusive to S. arenicola strains only and are therefore the principal secondary metabolites differentiating the two species. Furthermore, the tentative discovery of 57 compounds not previously recorded in both species substantially increases the small number of secondary metabolites previously known to be produced. A number of compounds were identified as being specific to particular sponge species and multiple location sites in a large-scale investigation of secondary metabolome. The application of the metabolomics approach, coupled with database searching and data visualization of chromatograms, revealed, for the first time, complete identification of eight new compounds from these two species, which were previously known from other bacterial and fungal species. During this study, rifamycins (antibiotics) were only found in Dercitus and Hyatella sponge-associated S. arenicola species, however, most of the Salinispora isolates were sourced from Cinachyrella sponge species and fewer were isolated from Dercitus and Hyatella. Production of rifamycins in these two sponge-associated bacterial species demonstrates that the production of this antibiotic compound is likely to provide some information as to how the bacterial species can be controlled in particular host species. The final experimental chapter of this thesis describes the adaptation of S. arenicola species to its marine environment and secondary metabolite production profile across a time course. This latter study also used metabolomics as a tool to investigate the targeted production of rifamycin derivatives at two defined salt concentrations when analysed three different time points. LC-MS based metabolomics study shows lchemical profiler link between the days of incubation and different salt concentration of the growth medium was shown to exist and reliably represents a strong critical point for selection of growth medium and harvest time. This thesis has demonstrated that certain species of the marine actinobacteria are able to produce a much wider spectrum of secondary metabolites than previously shown, and this knowledge contribute to the field for biodiscovery as well as a database for understanding relationships between speciation, evolution and chemical ecology.n