Environmental and Taxonomic Drivers of Bacterial Extracellular Vesicle Production in Marine Ecosystems.

Extracellular vesicles are small (approximately 50 to 250 nm in diameter), membrane-bound structures that are released by cells into their surrounding environment. Heterogeneous populations of vesicles are abundant in the global oceans, and they likely play a number of ecological roles in these microbially dominated ecosystems. Here, we examine how vesicle production and size vary among different strains of cultivated marine microbes as well as explore the degree to which this is influenced by key environmental variables. We show that both vesicle production rates and vesicle sizes significantly differ among cultures of marine Proteobacteria, Cyanobacteria, and Bacteroidetes. Further, these properties vary within individual strains as a function of differences in environmental conditions, such as nutrients, temperature, and light irradiance. Thus, both community composition and the local abiotic environment are expected to modulate the production and standing stock of vesicles in the oceans. Examining samples from the oligotrophic North Pacific Gyre, we show depth-dependent changes in the abundance of vesicle-like particles in the upper water column in a manner that is broadly consistent with culture observations: the highest vesicle abundances are found near the surface, where the light irradiances and the temperatures are the greatest, and they then decrease with depth. This work represents the beginnings of a quantitative framework for describing extracellular vesicle dynamics in the oceans, which is essential as we begin to incorporate vesicles into our ecological and biogeochemical understanding of marine ecosystems. IMPORTANCE Bacteria release extracellular vesicles that contain a wide variety of cellular compounds, including lipids, proteins, nucleic acids, and small molecules, into their surrounding environment. These structures are found in diverse microbial habitats, including the oceans, where their distributions vary throughout the water column and likely affect their functional impacts within microbial ecosystems. Using a quantitative analysis of marine microbial cultures, we show that bacterial vesicle production in the oceans is shaped by a combination of biotic and abiotic factors. Different marine taxa release vesicles at rates that vary across an order of magnitude, and vesicle production changes dynamically as a function of environmental conditions. These findings represent a step forward in our understanding of bacterial extracellular vesicle production dynamics and provide a basis for the quantitative exploration of the factors that shape vesicle dynamics in natural ecosystems.

Dokdonia sinensis sp. nov., a flavobacterium isolated from surface seawater.

A taxonomic study was carried out on strain SH27T, which was isolated from seawater collected around Xiaoshi Island, PR China. Cells of strain SH27T were Gram-stain-negative, non-motile, rod-shaped, orange-pigmented and grew at 15-37 °C (optimum, 28 °C), at pH 6.0-8.0 (pH 7.0) and in 1.0-7.0 % (w/v) NaCl (2.0-3.0 %). The isolate was positive for catalase, but negative for nitrate reduction, oxidase, indole production and urease. Carotenoid pigment was produced. Phylogenetic analysis based on the 16S rRNA gene placed strain SH27T in the genus Dokdonia with the closest relative being Dokdonia donghaensis KCTC 12391T, exhibiting 96.7 % 16S rRNA gene pairwise similarity. The results of genomic comparisons, including average nucleotide identity and digital DNA-DNA hybridization, showed 72.9 and 19.2 % identity to D. donghaensis KCTC 12391T, respectively. The major cellular fatty acids were iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH. The major polar lipids were phosphatidylethanolamine and two unidentified lipids. Menaquinone-6 was the only respiratory quinone. The G+C content of the genomic DNA was 32.9 mol%. On the basis of the phenotypic and phylogenetic data, strain SH27T represents a novel species of the genus Dokdonia, for which the name Dokdonia sinensis sp. nov. is proposed, with the type strain SH27T (MCCC 1H00358T=CCTCC AB 2018323T=KCTC 62962T).

Identification of the key determinant of the transport promiscuity in Na+-translocating rhodopsins.

Bacterial Na+-transporting rhodopsins convert solar energy into transmembrane ion potential difference. Typically, they are strictly specific for Na+, but some can additionally transport H+. To determine the structural basis of cation promiscuity in Na+-rhodopsins, we compared their primary structures and found a single position that harbors a cysteine in strictly specific Na+-rhodopsins and a serine in the promiscuous Krokinobacter eikastus Na+-rhodopsin (Kr2). A Cys253Ser variant of the strictly specific Dokdonia sp. PRO95 Na+-rhodopsin (NaR) was indeed found to transport both Na+ and H+ in a light-dependent manner when expressed in retinal-producing Escherichia coli cells. The dual specificity of the NaR variant was confirmed by analysis of its photocycle, which revealed an acceleration of the cation-capture step by comparison with the wild-type NaR in a Na+-deficient medium. The structural basis for the dependence of the Na+/H+ specificity in Na+-rhodopsin on residue 253 remains to be determined.

Dokdonia aurantiaca sp. nov., isolated from seaweed Zostera marina.

A non-motile, orange-coloured and rod-shaped bacterial strain, designated strain ZOW29T, was isolated from a seaweed sample collected from the South Sea, Republic of Korea. Cells were Gram-stain-negative, aerobic and non-motile. The isolate required sea salts for growth. Carotenoid pigment was produced. A phylogenetic tree based on 16S rRNA gene sequences showed that strain ZOW29T forms an evolutionary lineage within the radiation enclosing the members of the genus Dokdonia with Dokdonia diaphoros MSKK-32T, Dokdonia eikasta PMA-26Tand Dokdonia donghaensis DSW-1T (97.1 % sequence similarity each) as its nearest neighbours. The DNA-DNA relatedness values between strain ZOW29T and these four type strains were 35-48 %. The major fatty acids were iso-C15:0, iso-C17 : 0 3-OH and iso-C15 : 1 G. Strain ZOW29T contained MK-6 and phosphatidylethanolamine, an unidentified aminolipid and an unidentified polar lipid as the only isoprenoid quinone and the major polar lipids, respectively. The DNA G+C content of strain ZOW29T was 38 mol%. On the basis of polyphasic characterization, it is suggested that the isolate represents a novel species of the genus Dokdonia, for which the name Dokdonia aurantiaca sp. nov. (type strain, ZOW29T=KCTC 52956T=JCM 32295T) is proposed.

Dokdonia flava sp. nov., isolated from the seaweed Zostera marina.

A non-motile, proteorhodopsin-containing, yellow and rod-shaped bacterial strain, designated ZODW10T, was isolated from the seaweed Zostera marina collected from the West Sea, Republic of Korea. Cells were Gram-stain-negative, aerobic and non-motile. The isolate required sea salts for growth. A carotenoid pigment was produced. A phylogenetic tree based on 16S rRNA gene sequences showed that strain ZODW10T forms an evolutionary lineage within the radiation enclosing members of the genus Dokdonia with Dokdoniadiaphoros CIP 108745T (96.7 % sequence similarity) as its nearest neighbour. The major fatty acids were iso-C15:0, iso-C17 : 0 3-OH and iso-C15 : 1 G. Strain ZODW10T contained menaquinone 6 (MK-6) and phosphatidylethanolamine, an unidentified aminolipid and an unidentified polar lipid as the only isoprenoid quinone and the major polar lipids, respectively. The DNA G+C content of strain ZODW10T was 36 mol%. On the basis of the present polyphasic characterization, it is suggested that the isolate represents a novel species of the genus Dokdonia, for which the name Dokdonia flava sp. nov. (type strain, ZODW10T=KCTC 52953T=JCM 32293T) is proposed.

Dokdonia lutea sp. nov., isolated from Sargassum fulvellum seaweed.

A non-motile, yellow-coloured and rod-shaped bacterium, designated strain SFD34T, was isolated from seaweed collected from the South Sea, Republic of Korea. Cells were Gram-stain-negative, aerobic, catalase-positive and oxidase-negative. The major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 1 G. Strain SFD34T contained MK-6 and phosphatidylethanolamine as the only isoprenoid quinone and major polar lipid, respectively. The DNA G+C content was 35 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain SFD34T formed evolutionary lineage within the radiation enclosing the members of the genus Dokdonia with Dokdoniapacifica SW230T (96.86 % sequence similarity) as its nearest neighbour. A number of phenotypic characteristics distinguished strain SFD34T from related members of the genus Dokdonia. On the basis of the evidence presented in this study, a novel species, Dokdonia lutea sp. nov., is proposed for strain SFD34T (=KCTC 52269T=JCM 31795T).

Genome characteristics of the proteorhodopsin-containing marine flavobacterium Polaribacter dokdonensis DSW-5.

Bacteria in the genus Polaribacter, belonging to the family Flavobacteriaceae, are typically isolated from marine environments. Polaribacter dokdonensis DSW-5, the type strain of the species, is a Gram-negative bacterium isolated from the East Sea of Korea. Whole genome shotgun sequencing was performed with the HiSeq 2000 platform and paired-end reads were generated at 188-fold coverage. The sequencing reads were assembled into two contigs with a total length of 3.08 Mb. The genome sequences of DSW-5 contain 2,776 proteincoding sequences and 41 RNA genes. Comparison of average nucleotide identities among six available Polaribacteria genomes including DSW-5 suggested that the DSW-5 genome is most similar to that of Polaribacter sp. MED152, which is a proteorhodopsin-containing marine bacterium. A phylogenomic analysis of the six Polaribacter strains and 245 Flavobacteriaceae bacteria confirmed a close relationship of the genus Polaribacter with Tenacibaculum and Kordia. DSW-5's genome has a gene encoding proteorhodopsin and genes encoding 85 enzymes belonging to carbohydrate-active enzyme families and involved in polysaccharide degradation, which may play important roles in energy metabolism of the bacterium in the marine ecosystem. With genes for 238 CAZymes and 203 peptidases, DSW-5 has a relatively high number of degrading enzymes for its genome size suggesting its characteristics as a free-living marine heterotroph.