| Popis: |
The isolation and cultivation of heterotrophic marine bacteria opens possibilities to study their physiology and genomes with respect to their function in the marine environment. In the pelagic marine realm bacteria remineralize more than half of the photosynthetically produced biomass, and thus play an important role in the biogeochemical cycling of elements. Flavobacteria are abundant of up to 30% in the North Sea. In previous studies marine Gammaproteobacteria, Alphaproteobacteria and Actinobacteria were predominantly cultivated, but cultures of Flavobacteria were infrequently obtained. This thesis addresses the isolation of phylogenetically diverse marine Flavobacteria using three new approaches. First, samples were retrieved from various pelagic and benthic habitats of the North Sea. Second, a new marine artificial seawater HaHa medium was developed to facilitate the growth of Flavobacteria. This medium was supplemented with saccharides and proteins as carbon sources at a concentration of 2 g/L. Third, a specific 16S rRNA gene PCR assay was applied to identify Flavobacteria-Cytophagia among the colonies. The molecular screen was preferred over the identification by cell and colony morphology, since the latter has predominantly resulted in the isolation of strains of the genera Arenibacter, Cellulophaga and Maribacter. The 375 Flavobacteriaceae strains isolated on agar plates comprised (i) seven presumably novel genera, (ii) 42 presumably novel species in 22 validly described Flavobacteriaceae genera and (iii) many isolates that were so far not distinguishable from 37 type strains in 16 genera. Thus, in contrast to previous studies, we could show that phylogenetically diverse Flavobacteria from the North Sea can be cultivated on solid medium. The isolation of representative strains of the genera Formosa, Polaribacter, and Reinekea from the North Sea was attempted. In previous studies these bacterial populations were proposed to be of importance during coastal diatom-dominated phytoplankton blooms, based on their high abundance of 15% to 25% of the bacterioplankton and their potential capability to decompose algae derived polysaccharides. A new medium was devised which had the same composition as the marine HaHa medium, but with environmental-like micromolar carbon, nitrogen, and phosphate concentrations. Aerobic dilution cultivation in the HaHa medium led to a high culturability of 35% of the bacterioplankton in spring 2010 and 27% of the bacterioplankton in summer 2010. 23 strains of Flavobacteria, Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria were obtained directly by dilution cultivation of single cell inocula. One strain that belonged to the genus Reinekea was isolated by generating co-cultivatures of randomly mixed bacterial populations which potentially had a positive effect on the growth of Reinekea. Strains that affiliated with Polaribacter , Formosa, Gillisia (Flavobacteria), the Roseobacter clade associated (RCA) lineage (Alphaproteobacteria), Reinekea, and the OM182 clade (Gammaproteobacteria) had 16S rRNA gene sequence identities of >99.9% with 16S rRNA clones of the bacterioplankton from the North Sea in spring 2009. In addition, draft genomes of Formosa, Polaribacter , and Reinekea strains were used to recruit reads of metagenomes of the bacterioplankton in spring 2009. Thereby, reads of >95% nucleotide identity covered the draft genomes of the Formosa clade B strain to 94%, of Reinekea sp. to 90% and of Polaribacter sp. to 50%. Based on these results we argue that the novel species of Formosa, Polaribacter , and Reinekea are representatives of ecologically relevant clades catalyzing the remineralization of coastal diatom-dominated phytoplankton biomass. The physiological characteristics of the strains were investigated focusing on the growth on different mono- and polysaccharides, to provide further evidence that Formosa, Polaribacter and Reinekea species could prevail in different ecological niches during algae decay. Interestingly, Polaribacter strains grew heterotrophically on all tested sulfated (e.g. agar, carrageenan) and non-sulfated polysaccharides (e.g. cellulose, laminarin), whereas Formosa strains grew only on non-sulfated polysaccharides. In contrast, Reinekea sp. did not grow on polysaccharides but on all tested mono-, di-, and trisaccharides including N-acetylneuraminic acid. Finally, I proposed for these novel species the names ’Formosa flavarachnoidea’, ’Formosa forsetii ’, ’Polaribacter forsetii ’, ’Polaribacter frigidimaris’, ’Polaribacter adhaesivus’, and ’Reinekea forsetii ’. |
