1 National Food Institute, Technical University of Denmark2 Division of Industrial Food Research, National Food Institute, Technical University of Denmark3 Department of Systems Biology, Technical University of Denmark4 Bacterial Ecophysiology and Biotechnology, Department of Biotechnology and Biomedicine, Technical University of Denmark
The purpose of this Ph.D. project was to evaluate a global collection of marine Pseudoalteromonas bacteria as a source of novel bioactive compounds, and to investigate the distribution and production of such compounds among different species within the Pseudoalteromonas genus. The strain collection was obtained during the research cruise “Galathea 3”, which circumnavigated the Earth while screening marine bacteria for the ability to inhibit Vibrio anguillarum 90-11-287. Pseudoalteromonas strains were one of the most frequently isolated genera. The Pseudoalteromonas strains were evaluated for their ability to repeatedly inhibit the fish pathogen Vibrio anguillarum 90-11-287 or Staphylococcus aureus 8325. Based on previous work, a hypothesis that antagonistic Pseudoalteromonas strains primarily were pigmented and surface associated was investigated. This Ph.D. work confirmed that surface-associated strains were significantly more likely to possess stable antibacterial activity and be pigmented. Pseudoalteromonas strains are known as prolific producers of bioactive secondary metabolites; hence screening the global strain collection for production of novel antibiotics was initiated. Novel quinolone-related compounds were described, but were not antibacterial. Several antibacterial compounds known from other sources were identified, for instance indolmycin which was hitherto only known from terrestrial Streptomycetes. Genome sequencing of P. luteoviolacea S4054 revealed up to 11 biosynthetic pathways with unknown products, confirming the potential for discovery of new secondary metabolites from Pseudoalteromonas strains. The elaborate secondary metabolite production led me to speculate whether it was possible to use secondary metabolites to assist in species identification within this genus. This would also provide information on the use of 16S rRNA gene sequences to dereplicate strain collections in biodiscovery efforts. A phylogenetic study of 16S rRNA gene sequences of the Pseudoalteromonas strains confirmed the division into two clades; one consisted of bioactive pigmented strains and one predominantly of inactive nonpigmented strains. Correlating this to a dendrogram based on the secondary metabolites in each strain showed that some strains clustered together in a species-specific way, whereas other strains did not cluster near strains of the same species. Hence, secondary metabolite production was not unequivocally reflected in the secondary metabolite profile, possibly due to the limited resolving power of the 16S rRNA gene. The species P. luteoviolacea showed an interesting pattern indicative of phylotype specific antibiotic production strains. Detailed phylogenetic analysis of an expanded collection of P. luteoviolacea strains showed confirmed that production of antibiotics was related to phylogeny within this species, which indicates that the underlying biosynthetic pathways are maintained under selective pressure and hence are important traits for the organism. One strain stood out during work with the strain collection, in part because of its production of an intense black pigment in contrast to its phylogenetic placement within the non-pigmented clade. This strain was subsequently shown to represent a new bacterial species named Pseudoalteromonas galatheae. Initial studies revealed the potential production of regulatory compounds involved in cell to cell signaling within some strains of the species P. luteoviolacea. Since such mechanisms are known to govern antibiotic production in some bacteria, this was investigated. A quorum sensing system controlling a putative novel biosynthetic pathway with high homology to the lux system of Vibrio fischeri was identified in P. luteoviolacea S4054. The signal molecule was potentially a new acylated homoserine lactone (AHL) like compound, and the AHL synthethase was phylogenetically distinct from related synthethases. This expands our knowledge of bacterial signaling and lux homologue system, and further work will resolve is this system has implications for antibiotic production. In summary, this Ph.D. work explored the phylogeny and chemical diversity of the genus Pseudoalteromonas. Novel compounds were discovered but they possessed no antibiotic activity. However, analysis of the genome sequence of P. luteoviolacea S4054 revealed genetic potential for discovery of secondary metabolites not known within this species. Secondary metabolites were not unequivocally representative of species assignments, but on an intra-species level the use of detailed phylogenetic analysis showed phylotype specific production of antibiotics within the species P. luteoviolacea. These findings validate the genus Pseudoalteromonas as a potential source of novel secondary metabolites and may be useful when designing future biodiscovery strategies. The novel species P. galatheae was described which contributed to resolving the taxonomy of the genus. This thesis also provides evidence of a quorum sensing system related to the lux system of Vibrio fischeri but relying on putatively novel signaling molecule encoded by a distinct synthethase, which might be involved in the regulation of antibiotics production.