Surface structures and taxonomy of marine Flavobacteriia

Flavobacteria play a major role in the turnover of organic matter in the ocean. Their importance in the degradation of algal biomass was revealed by the dynamics of heterotrophic bacterial populations during the degradation of an algal bloom in the North Sea near Helgoland. Isolation of bacterial strains from seawater of the bloom allowed pure culture studies and visual examination of the bacteria, with strain Hel3 A1 48 showing unexpected surface extensions. In this study, the cell appendages of the flavobacterial strain Hel3 A1 48 were investigated for their morphology, composition and conditions of formation to reveal the unique properties these surface extensions may provide for the cells. All applied electron microscopic methods (SEM, TEM, thin section TEM and cryo-EM) showed the appendages as membrane tubes or vesicle chains of outer membrane origin. These up to 10 micrometer long chains were composed of vesicles being 80-100 nm long and 50-80 nm wide. Sometimes larger vesicles (160-250 x 130-200 nm) were present that included parts of the inner membrane and cytoplasm (O-IMVs). A model for formation was proposed starting with the outward-bulging of outer membrane and subsequent elongation as tube which later transformed into a chain of vesicles by an abiotic process called 'pearling'. In some cases the inner membrane was pulled together with the outer membrane tube and an O-IMV was formed. Electron microscopic visualization at different stages of growth and culture conditions revealed an increased production towards the stationary growth phase and did not show a condition without appendage production. Fluorescein-labeled laminarin uptake into the periplasm of the cells according to the 'selfish' uptake mechanism was visualized by SR-SIM. Staining was also observed in the vesicle chains. Proteomic analyses of total membrane fractions and of vesicle fractions confirmed the outer membrane origin by enrichment of outer membrane and extracellular surface-associated proteins in the vesicle fractions. Especially enriched were porins, TonB-dependent receptors and type IX secretion system components and secreted LPS anchored surface proteins. The appendages of strain Hel3 A1 48 enlarge the surface area of the cell and allow a larger number of surface-associated degradation enzymes and an additional periplasmic storage space. Strain Hel3 A1 48 was described taxonomically together with three other North Sea isolates. Two of the Helgoland strains (Hel3 A1 48 and Hel1 33 131) were isolated from the summer or spring bloom 2010, respectively. They differed in biochemical and physiological traits from each other and other type strains of related genera and were clustering together as a separate branch in phylogenetic trees, thus forming a new genus in the Flavobacteriaceae. The names Fragilitenera margaritagerula gen. nov., sp. nov., and Fragilitenera forsetii sp. nov., are tentatively proposed. Strain Hel1 31 208, isolated from the spring bloom 2010 near Helgoland, has a 98.6% 16S rRNA gene sequence identity to the newly described genus Arcticiflavibacter. Due to differences in biochemical and physiological characteristics, a new species, Arcticiflavibacter funis sp. nov., is tentatively proposed. Strain TFI002, isolated from Sylt seawater in autumn 2013, belongs to the family of Cytophagaceae and formed in the 16S rRNA tree a separate branch, clustering next to the genera Jiulongibacter and Taeseokella. The strain represents a new genus for which the name Simonia syltensis gen. nov., sp. nov., is tentatively proposed. All three flavobacterial strains isolated from Helgoland possess extracellular structures and all four have the TonB-dependent transport system and the major components of the type IX secretion system. Within the guild of bacteria that degrade polymers with surface enzymes (selfish mode), strain Hel3 A1 48 and other bacteria with similar surface appendages are expected to have an advantage in their nutrient acquisition.