Exploring bacterioplankton communities and their temporal dynamics in the rearing water of a biofloc-based shrimp (Litopenaeus vannamei) aquaculture system.

Biofloc technology (BFT) has recently gained considerable attention as a sustainable method in shrimp aquaculture. In a successful BFT system, microbial communities are considered a crucial component in their ability to both improve water quality and control microbial pathogens. Yet, bacterioplankton diversity in rearing water and how bacterioplankton community composition changes with shrimp growth are rarely documented. In this study, the Pacific white shrimp, Litopenaeus vannamei was cultivated in a greenhouse-enclosed BFT system. Rearing water samples were collected on a weekly basis for 5 months (152 days) and water quality variables such as physicochemical parameters and inorganic nutrients were monitored. In parallel, 16S rRNA gene pyrosequencing was employed to investigate the temporal patterns of rearing-water microbiota. The productivity, survival rate, and feed conversion ratio were 3.2-4.4 kg/m3, 74%-89%, and 1.2-1.3, respectively, representing successful super-intensive cultures. The metataxonomic results indicated a highly dynamic bacterioplankton community, with two major shifts over the culture. Members of the phylum Planctomycetes dominated in rearing water during the early stages, while Actinobacteria dominated during the middle stages, and Chloroflexi and TM7 dominated during the late stages of culture. The bacterioplankton community fluctuated more in the beginning but stabilized as the culture progressed. Intriguingly, we observed that certain bacterioplankton groups dominated in a culture-stage-specific manner; these groups include Rhodobacteraceae, Flavobacteriaceae, Actinobacteria, and Chloroflexi, which either contribute to water quality regulation or possess probiotic potential. Altogether, our results indicate that an operationally successful BFT-based aquaculture system favors the growth and dynamics of specific microbial communities in rearing water. Our study expands the scientific understanding of the practical utilization of microbes in sustainable aquaculture. A thorough understanding of rearing-water microbiota and factors influencing their dynamics will help to establish effective management strategies.

A sulfate-reducing bacterial genus, Desulfosediminicola gen. nov., comprising two novel species cultivated from tidal-flat sediments.

Tidal-flat sediments harbor a diverse array of sulfate-reducing bacteria. To isolate novel sulfate-reducing bacteria and determine their abundance, a tidal-flat sediment sample collected off Ganghwa Island (Korea) was investigated using cultivation-based and culture-independent approaches. Two Gram-stain-negative, strictly anaerobic, rod-shaped, sulfate-reducing bacteria, designated IMCC35004T and IMCC35005T, were isolated from the sample. The two strains reduced sulfate, sulfite, elemental sulfur, thiosulfate, Fe(III) citrate, and Mn(IV) oxide by utilizing several carbon sources, including acetate. The 16S rRNA gene amplicon sequencing revealed that the tidal-flat sediment contained diverse members of the phylum Desulfobacterota, and the phylotypes related to IMCC35004T and IMCC35005T were < 1%. The two strains shared 97.6% similarity in 16S rRNA gene sequence and were closely related to Desulfopila aestuarii DSM 18488T (96.1-96.5%). The average nucleotide identity, level of digital DNA-DNA hybridization, average amino acid identity, and percentages of conserved proteins determined analyzing the whole-genome sequences, as well as the chemotaxonomic data showed that the two strains belong to two novel species of a novel genus. Additionally, genes related to dissimilatory sulfate reduction were detected in the genomes of the two strains. Unlike the genera Desulfopila and Desulfotalea, IMCC35004T and IMCC35005T contained menaquinone-5 as the major respiratory quinone. Collectively, IMCC35004T and IMCC35005T were concluded to represent two novel species of a novel genus within the family Desulfocapsaceae, for which the names Desulfosediminicola ganghwensis gen. nov., sp. nov. (IMCC35004T = KCTC 15826T = NBRC 114003T) and Desulfosediminicola flagellatus sp. nov. (IMCC35005T = KCTC 15827T = NBRC 114004T) are proposed.

Uliginosibacterium aquaticum sp. nov., Isolated from a Freshwater Lake.

A Gram-stain-negative, aerobic, chemoheterotrophic, rod-shaped bacterium motile by a polar flagellum, designated IMCC34675T, was isolated from Chungju Lake, an artificial freshwater reservoir in Republic of Korea. The 16S rRNA gene sequence analysis indicated that strain IMCC34675T belongs to the genus Uliginosibacterium, sharing ≤ 97.1% sequence similarities with the type strains of the genus. Whole genome sequencing of strain IMCC34675T revealed a 4.1 Mbp of genome size with 62.4% of the DNA G + C content. The IMCC34675T genome shared 73.3% of average nucleotide identity and 19.9% of digital DNA-DNA hybridization values to the genome of Uliginosibacterium gangwonense DSM 18521T, the type species of the genus. The major fatty acids of strain IMCC34675T were summed feature 3 (comprising C16:1ω6c and/or C16:1ω7c) and C16:0. The respiratory quinone detected in the strains was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified aminophospholipid, one aminolipid, and five unidentified lipids. Based on the phylogenetic and phenotypic characterization, strain IMCC34675T was considered to represent a novel species within the genus Uliginosibacterium, for which the name Uliginosibacterium aquaticum sp. nov. is proposed with IMCC34675T (= KACC 21758T = NBRC 114418T) as the type strain.

Permianibacter fluminis sp. nov., isolated from a freshwater stream.

A Gram-stain-negative, aerobic, non-motile and rod-shaped bacterium, designated as IMCC34836T, was isolated from a freshwater stream. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain IMCC34836T was most closely related to Permianibacter aggregans HW001T (of the family Pseudomonadaceae) with 95.6 % sequence similarity and formed a robust clade with P. aggregans HW001T. The draft genome sequence of strain IMCC34836T was 4.4 Mbp in size with 59.1 mol% DNA G+C content. Average nucleotide identity and digital DNA-DNA hybridization values between strain IMCC34836T and P. aggregans HW001T were 71.2 and 22.0 %, respectively, indicating that the new strain represents a novel species. The strain contained iso-C15 : 0, summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) and summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 1 10-methyl) as the major fatty acids and harboured phosphatidylethanolamine, two unidentified aminophospholipids and three unidentified lipids as major polar lipids. The isoprenoid quinone detected in the strain was ubiquinone-8. Based on the phylogenetic and phenotypic characteristics, strain IMCC34836T is considered to represent a novel species of the genus Permianibacter, for which the name Permianibacter fluminis sp. nov. is proposed. The type strain is IMCC34836T (=KACC 21755T=NBRC 114416T).

Development of an antibody-like T-cell engager based on VH-VL heterodimer formation and its application in cancer therapy.

Following the clinical success of immunotherapeutic antibodies, bispecific antibodies for cytotoxic effector cell redirection, tumor-targeted immunomodulation and dual immunomodulation, have received particular attentions. Here, we developed a novel bispecific antibody platform, termed Antibody-Like Cell Engager (ALiCE), wherein the Fc domain of each heavy chain of immunoglobulin G (IgG) is replaced by the VH and VL domains of an IgG specific to a second antigen while retaining the N-terminal Fab of the parent antibody. Because of specific interactions between the substituted VH and VL domains, the C-terminal stem Fv enables ALiCE to assemble autonomously into hetero-tetramers, thus simultaneously binding to two distinct antigens but with different avidities. This design strategy was used to generate ACE-05 (two anti-PD-L1 Fab × anti-CD3 Fv) and ACE-31 (two anti-CD3 Fab × anti-PD-L1 Fv), both of which bound PD-L1 and CD3. However, ACE-05 was more effective than ACE-31 in reducing off-target toxicity caused by the indiscriminate activation of T cells. Moreover, in cell-based assays and PBMC-reconstituted humanized mice harboring human non-small-cell lung cancer tumors, ACE-05 showed marked antitumor efficacy, causing complete tumor regression at a dose of 0.05 mg/kg body weight. The dual roles of ACE-05 in immune checkpoint inhibition and T-cell redirection, coupled with reduced off-target toxicity, suggest that ACE-05 may be a promising anti-cancer therapeutic agent. Moreover, the bispecific ALiCE platform can be further used for tumor-targeted or multiple immunomodulation applications.

High-throughput cultivation based on dilution-to-extinction with catalase supplementation and a case study of cultivating acI bacteria from Lake Soyang.

Multi-omics approaches, including metagenomics and single-cell amplified genomics, have revolutionized our understanding of the hidden diversity and function of microbes in nature. Even in the omics age, cultivation is an essential discipline in microbial ecology since microbial cultures are necessary to assess the validity of an in silico prediction about the microbial metabolism and to isolate viruses infecting bacteria and archaea. However, the ecophysiological characteristics of predominant freshwater bacterial lineages remain largely unknown due to the scarcity of cultured representatives. In an ongoing effort to cultivate the uncultured majority of freshwater bacteria, the most abundant freshwater Actinobacteria acI clade has recently been cultivated from Lake Soyang through catalase-supplemented high-throughput cultivation based on dilution-to-extinction. This method involves physical isolation of target microbes from mixed populations, culture media simulating natural habitats, and removal of toxic compounds. In this protocol, we describe detailed procedures for isolating freshwater oligotrophic microbes, as well as the essence of the dilution-to-extinction culturing. As a case study employing the catalase-supplemented dilution-to-extinction protocol, we also report a cultivation trial using a water sample collected from Lake Soyang. Of the 480 cultivation wells inoculated with a single lake-water sample, 75 new acI strains belonging to 8 acI tribes (acI-A1, A2, A4, A5, A6, A7, B1, B4, C1, and C2) were cultivated, and each representative strain per subclade could be revived from glycerol stocks. These cultivation results demonstrate that the protocol described in this study is efficient in isolating freshwater bacterioplankton harboring streamlined genomes.

Sphingobacterium chungjuense sp. nov., isolated from a freshwater lake.

A Gram-stain-negative, rod-shaped, aerobic, non-flagellated, chemoheterotrophic bacterium, designated strain IMCC25678T, was isolated from an artificial freshwater reservoir, Chungju Lake, in the Republic of Korea. The 16S rRNA gene sequence analysis indicated that strain IMCC25678T belongs to the genus Sphingobacterium with ≤98.7 % sequence similarities to Sphingobacterium species. Whole genome sequencing of strain IMCC25678T revealed a 3.9 Mbp genome size with a DNA G+C content of 42.2 mol%. The IMCC25678T genome shared ≤89.7 % average nucleotide identity and ≤21.4 % digital DNA-DNA hybridization values with closely related species of the genus Sphingobacterium, indicating that the strain represents a novel species. Summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), iso-C15 : 0 and iso-C17 : 0 3-OH were found to be the predominant cellular fatty acid constituents in the strain. The major respiratory quinone was MK-7. The major polar lipids were phosphatidylethanolamine, one unidentified phosphoglycolipid, one unidentified sphingolipid and three unidentified polar lipids. Based on the phylogenetic and phenotypic characteristics, strain IMCC25678T was considered to represent a novel species within the genus Sphingobacterium, for which the name Sphingobacterium chungjuense sp. nov. is proposed. The type strain is IMCC25678T (=KACC 19485T=NBRC 113130T).

Leeia aquatica sp. nov., isolated from freshwater.

A Gram-stain-negative, rod-shaped, obligately aerobic, motile by a single polar flagellum, chemoheterotrophic bacterium, designated strain IMCC25680T, was isolated from surface water in Chungju Lake, Republic of Korea. 16S rRNA gene sequence analysis revealed that strain IMCC25680T was most closely related to Leeia oryzae HW7T with 95.5% sequence similarity and formed a robust clade with L. oryzae HW7T. Whole genome sequencing showed that strain IMCC25680T had a genome 3.6 Mbp long with 60.7 mol% DNA G+C content. Average nucleotide identity and digital DNA-DNA hybridization values between strain IMCC25680T and L. oryzae HW7T were 72.4% and 18.5%, respectively, indicating that the novel strain represents a novel species of the genus Leeia. The major cellular fatty acids of strain IMCC25680T were iso-C16:0 and summed feature 3 (comprising C16:1 ω6c and/or C16:1 ω7c). The respiratory quinone detected in the strain was ubiquinone-8. The major polar lipids were found to be phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and two unidentified polar lipids. On the basis of the phylogenetic and phenotypic characterization, strain IMCC25680T was considered to represent a novel species within the genus Leeia, for which the name Leeia aquatica sp. nov. is proposed. The type strain is IMCC25680T (=KACC 19487T =NBRC 113132T).

Ferrimonas sediminicola sp. nov. and Ferrimonas aestuarii sp. nov., Fe(III)-reducing bacteria isolated from marine environments.

Two Gram-stain-negative, Fe(III)-reducing, facultatively anaerobic, motile via a single polar flagellum, rod-shaped bacterial strains, designated IMCC35001T and IMCC35002T, were isolated from tidal flat sediment and seawater, respectively. Results of 16S rRNA gene sequence analysis showed that IMCC35001T and IMCC35002T shared 96.6 % sequence similarity and were most closely related to Ferrimonas futtsuensis FUT3661T (98.6 %) and Ferrimonas kyonanensis Asr22-7T (96.8 %), respectively. Draft genome sequences of IMCC35001T and IMCC35002T revealed 4.0 and 4.8 Mbp of genome size with 61.0 and 51.8 mol% of DNA G+C content, respectively. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the two strains were 73.1 and 19.8 %, respectively, indicating that they are separate species. The two genomes showed ≤84.4 % ANI and ≤27.8 % dDDH to other species of the genus Ferrimonas, suggesting that the two strains each represent novel species. The two strains contained both menaquinone (MK-7) and ubiquinones (Q-7 and Q-8). Major fatty acids of strain IMCC35001T were iso-C15 : 0, C18 : 1 ω9c, C17 : 1 ω8c and C16 : 0 and those of strain IMCC35002 T were C18 : 1 ω9c, C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). Major polar lipids in both strains were phosphatidylethanolamine, phosphatidylglycerol, unidentified phospholipid, unidentified aminophospholipid and unidentified lipids. The two strains reduced Fe(III) citrate, Fe(III) oxyhydroxide, Mn(IV) oxide and sodium selenate but did not reduce sodium sulfate. They were also differentiated by several phenotypic characteristics. Based on the polyphasic taxonomic data, IMCC35001T and IMCC35002T were considered to represent each novel species in the genus Ferrimonas, for which the names Ferrimonas sediminicola sp. nov. (IMCC35001T=KACC 21161T=NBRC 113699T) and Ferrimonas aestuarii (IMCC35002T=KACC 21162T=NBRC 113700T) sp. nov. are proposed.

Aequoribacter fuscus gen. nov., sp. nov., a new member of the family Halieaceae, isolated from coastal seawater.

A Gram-stain-negative, rod-shaped, obligately aerobic, nonflagellated, and chemoheterotrophic bacterium, designated IMCC3088T, was isolated from coastal seawater of the Yellow Sea. The 16S rRNA gene sequence analysis indicated that this strain belonged to the family Halieaceae which shared the highest sequence similarities with Luminiphilus syltensis NOR5-1BT (94.5%) and Halioglobus pacificus S1-72T (94.5%), followed by 92.3-94.3% sequence similarities with other species within the aforementioned family. Phylogenetic analyses demonstrated that strain IMCC3088T was robustly clustered with Luminiphilus syltensis NOR5-1BT within the family Halieaceae. However, average amino acid identity (AAI), percentages of conserved proteins (POCP), average nucleotide identity (ANI), and alignment fraction (AF) between strain IMCC3088T and Luminiphilus syltensis NOR5-1BT were 54.5%, 47.7%, 68.0%, and 16.5%, respectively, suggesting that they belonged to different genera. Whole-genome sequencing of strain IMCC3088T revealed a 3.1 Mbp genome size with a DNA G + C content of 51.7 mol%. The genome encoded diverse metabolic pathways including sulfur oxidation, phenol degradation, and proteorhodopsin phototrophy. Mono-unsaturated fatty acids were found to be the predominant cellular fatty acid components in the strain. Phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol were the primarily identified polar lipids, and ubiquinone-8 was identified as a major respiratory quinone. The taxonomic data collected herein suggested that strain IMCC3088T represented a novel genus and species of the family Halieaceae, for which the name Aequoribacter fuscus gen. nov., sp. nov. is proposed with the type strain (= KACC 15529T = NBRC 108213T).

Sphingorhabdus lacus sp. nov. and Sphingorhabdus profundilacus sp. nov., isolated from freshwater environments.

Two Gram-stain-negative, aerobic, non-motile bacteria, designated IMCC1753T and IMCC26285T, were isolated from a shallow eutrophic pond and a deep oligotrophic lake, respectively. Results of 16S rRNA gene sequence analysis indicated that the two strains shared 99.8 % sequence similarity and were most closely related to Sphingorhabdus contaminans JC216T(98.7-98.8 %). The whole genome sequences of strains IMCC1753T and IMCC26285T were 3.5 and 2.9 Mbp in size with 56.6 and 55.5 mol% DNA G+C content, respectively. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the two strains were 82.2 and 25.8 %, respectively, indicating that they are separate species. The two strains showed ≤98.8 % 16S rRNA gene sequence similarities and ≤82.2 % ANI and ≤28.7 % dDDH values to closely related species of the genus Sphingorhabdus, indicating that the two strains each represent novel species. Major fatty acid constituents of strain IMCC1753T were C17 : 1 ω6c, C17 : 1 ω8c and summed features 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) and 8 (C18 : 1 ω6c and/or C18 : 1 ω7c); those of strain IMCC26285T were summed features 3 and 8. The predominant isoprenoid quinone detected in both strains was ubiquinone-10 and the most abundant polyamine was spermidine. Both strains contained phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol and sphingoglycolipid as major polar lipids. On the basis of the phylogenetic and phenotypic characteristics, strains IMCC1753T and IMCC26285T were considered to represent two distinct novel species in the genus Sphingorhabdus, for which the names Sphingorhabdus lacus (IMCC1753T=KCTC 52480T=KACC 18985T=NBRC 112442T) and Sphingorhabdus profundilacus (IMCC26285T=KCTC 52479T=KACC 18986T=NBRC 112454T) are proposed, respectively.

Halioglobus maricola sp. nov., isolated from coastal seawater.

A Gram-stain-negative, rod-shaped, aerobic, non-flagellated, chemoheterotrophic bacterium, designated IMCC14385T, was isolated from surface seawater of the East Sea, Republic of Korea. The 16S rRNA gene sequence analysis indicated that IMCC14385T represented a member of the genus Halioglobus sharing 94.6-97.8 % similarities with species of the genus. Whole-genome sequencing of IMCC14385T revealed a genome size of 4.3 Mbp and DNA G+C content of 56.7 mol%. The genome of IMCC14385T shared an average nucleotide identity of 76.6 % and digital DNA-DNA hybridization value of 21.6 % with the genome of Halioglobus japonicus KCTC 23429T. The genome encoded the complete poly-ß-hydroxybutyrate biosynthesis pathway. The strain contained summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and C17 : 1 ω8c as the predominant cellular fatty acids as well as ubiquinone-8 (Q-8) as the respiratory quinone. The polar lipids detected in the strain were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, five unidentified phospholipids, an unidentified aminolipid, an unidentified aminophospholipid and four unidentified lipids. On the basis of taxonomic data obtained in this study, it is suggested that IMCC14385T represents a novel species of the genus Halioglobus, for which the name Halioglobus maricola sp. nov. is proposed. The type strain is IMCC14385T (=KCTC 72520T=NBRC 114072T).

Photochemical characterization of flavobacterial rhodopsin: The importance of the helix E region for heat stability.

Although many microbial rhodopsins have been discovered many of organisms in a variety of habitats, little is known about the property and diversity of rhodopsin in flavobacteria. Recent studies discovered that many proteorhodopsin (PR)-like proteins exist in genomes of flavobacteria. Following the isolation of a flavobacterial rhodopsins (FR) from the flavobacteria IMCC1997 from the East Sea of Korea, we characterized its photochemical features. We confirmed that the FR expression is induced by light in the IMCC1997 cell. Upon receiving light energy in vitro, the proton acceptor (D83) and donor (E94) of the FR translocate protons from intracellular to extracellular regions. Compared with proteorhodopsin (PR), the FR from IMCC 1997 cells is very unstable, which may be explained by their primary sequence differences. The ratio of all trans/13-cis retinal conformation does not influence this stability. To measure the stability of FR, we tested heat endurance at 70 °C and found that the heat endurance time of some FR mutants increased. Based upon these results, we found the helix E of this protein to be critical for the unstability of FR.

Isolation and genome analysis of Winogradskyella algicola sp. nov., the dominant bacterial species associated with the green alga Dunaliella tertiolecta.

Microalgae and bacteria are known to be closely associated in diverse environments. To isolate dominant bacterial species associated with a green alga, Dunaliella tertiolecta, a photoreactor culture of the microalga was investigated using culture-based and culture-independent approaches. The bacterial community structure of the algal culture showed that the most abundant bacterial species under the culture conditions was related to the genus Winogradskyella. The closely related amplicon sequences, showing ≥ 99.5% 16S rRNA gene sequence similarity to one of the isolates, designated IMCC-33238T, constituted > 49% of the bacterial community and was therefore regarded as the most dominant species in the algal culture. Strain IMCC33238T was characterized by Gramstaining-negative and orange-colored rods. Phylogenetic analyses of the 16S rRNA genes as well as whole genome sequences revealed that strain IMCC33238T belonged to Winogradskyella and shared more than 97.2% 16S rRNA gene sequence similarity with Winogradskyella species. The strain contained iso-C15:1 G, iso-C15:0, iso-C15:0 3-OH, and summed feature 3 (C16:1ω6c and/or C16:1ω7c) as major fatty acids and MK-6 as the predominant quinone. The polar lipids found in strain IMCC33238T were phosphatidylethanolamine, two unidentified aminolipids, and three unidentified lipids. The genome of strain IMCC33238T was 3.37 Mbp in size with 33.9 mol% G + C content and proteorhodopsin. Many genes encoding folate and vitamin production are considered to play an important role in the bacteria-algae interaction. On the basis of phylogenetic and phenotypic characteristics, strain IMCC33238T represents a novel species in the genus Winogradskyella, for which the name Winogradskyella algicola sp. nov. is proposed. The type strain is IMCC33238T (= KACC 21192T = NBRC 113704T).

Rhodoferax lacus sp. nov., isolated from a large freshwater lake.

A Gram-staining-negative, aerobic, motile with a single polar flagellum and rod-shaped bacterium as a bacterial host of podovirus P26218, designated IMCC26218T, was isolated from Lake Soyang, South Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain IMCC26218T belonged to the genus Rhodoferax of the family Comamonadaceae and shared 97.7-99.0 % sequence similarities with Rhodoferax species. The draft whole genome sequence of strain IMCC26218T was ca. 4.9 Mbp in size with the DNA G+C content of 62.3 mol%. Average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) values between strain IMCC26218T and other Rhodoferax were 74.0-77.3 % and 19.5-21.0 %, respectively, showing that the strain represents a new Rhodoferax species. The strain contained summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) and C16 : 0 as the major fatty acids and phosphatidylethanolamine, three unidentified phospholipids, two unidentified aminolipids and two unidentified lipids as major polar lipids. The predominant isoprenoid quinone of the strain was ubiquinone-8 (Q-8). On the basis of the phylogenetic and phenotypic characteristics, strain IMCC26218T is considered to represent a novel species of the genus Rhodoferax, for which the name Rhodoferax lacus sp. nov. is proposed. The type strain is IMCC26218T (=KACC 18983T=NBRC 112709T).

Pelagibacterium sediminicola sp. nov., isolated from tidal flat sediment.

A Gram-stain-negative, obligately aerobic, cream-coloured, non-gliding, motile with a single polar flagellum and rod-shaped bacterium, designated IMCC34151T, was isolated from tidal flat sediment of the Yellow Sea, Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain IMCC34151T belonged to the genus Pelagibacterium of the family Hyphomicrobiaceae and shared 94.7-96.8 % sequence similarities to Pelagibacterium species. Whole-genome sequencing of strain IMCC34151T revealed a genome size of 3.2 Mbp and a DNA G+C content of 62.6 mol%. The strain contained summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C19 : 0cyclo ω8c and C16 : 0 as the major fatty acids and ubiquinone-10 (Q-10) as the major respiratory quinone. The polar lipids detected in the strain were phosphatidylglycerol, diphosphatidylglycerol, two unidentified glycolipids and 12 unidentified lipids. On the basis of its phylogenetic and phenotypic characteristics, strain IMCC34151T is considered to represent a novel species of the genus Pelagibacterium, for which the name Pelagibacteriumsediminicola sp. nov. (type strain IMCC34151T =KACC 19595T=NBRC 113420T) is proposed.

Isolation, cultivation, and genome analysis of proteorhodopsin-containing SAR116-clade strain Candidatus Puniceispirillum marinum IMCC1322.

Strain IMCC1322 was isolated from a surface water sample from the East Sea of Korea. Based on 16S rRNA analysis, IMCC1322 was found to belong to the OCS28 sub-clade of SAR116. The cells appeared as short vibrioids in logarithmic-phase culture, and elongated spirals during incubation with mitomycin or in aged culture. Growth characteristics of strain IMCC1322 were further evaluated based on genomic information; proteorhodopsin (PR), carbon monoxide dehydrogenase, and dimethylsulfoniopropionate (DMSP)-utilizing enzymes. IMCC1322 PR was characterized as a functional retinylidene protein that acts as a light-driven proton pump in the cytoplasmic membrane. However, the PR-dependent phototrophic potential of strain IMCC1322 was only observed under CO-inhibited and nutrient-limited culture conditions. A DMSP-enhanced growth response was observed in addition to cultures grown on C1 compounds like methanol, formate, and methane sulfonate. Strain IMCC1322 cultivation analysis revealed biogeochemical processes characteristic of the SAR116 group, a dominant member of the microbial community in euphotic regions of the ocean. The polyphasic taxonomy of strain IMCC1322 is given as Candidatus Puniceispirillum marinum, and was confirmed by chemotaxonomic tests, in addition to 16S rRNA phylogeny and cultivation analyses.

Sulfitobacter profundi sp. nov., isolated from deep seawater.

A Gram-stain-negative, rod-shaped, obligately aerobic, chemoheterotrophic bacterium which is motile by means of a single polar flagellum, designated SAORIC-263T, was isolated from deep seawater of the Pacific Ocean. Phylogenetic analyses based on 16S rRNA gene sequences and genomebased phylogeny revealed that strain SAORIC-263T belonged to the genus Sulfitobacter and shared 96.1-99.9% 16S rRNA gene sequence similarities with Sulfitobacter species. Wholegenome sequencing of strain SAORIC-263T revealed a genome size of 3.9Mbp and DNA G+C content of 61.3 mol%. The SAORIC-263T genome shared an average nucleotide identity and digital DNA-DNA hybridization of 79.1-88.5% and 18.9-35.0%, respectively, with other Sulfitobacter genomes. The SAORIC-263T genome contained the genes related to benzoate degradation, which are frequently found in deep-sea metagenome. The strain contained summed feature 8 (C18:1ω7c), C18:1ω7c 11-methyl, and C16:0 as the predominant cellular fatty acids as well as ubiquinone-10 (Q-10) as the major respiratory quinone. The major polar lipids of the strain were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, and aminolipid. On the basis of taxonomic data obtained in this study, it is suggested that strain SAORIC-263T represents a novel species of the genus Sulfitobacter, for which the name Sulfitobacter profundi sp. nov. is proposed. The type strain is SAORIC-263T (= KACC 21183T = NBRC 113428T).

Genome analysis of Rubritalea profundi SAORIC-165T, the first deep-sea verrucomicrobial isolate, from the northwestern Pacific Ocean.

Although culture-independent studies have shown the presence of Verrucomicrobia in the deep sea, verrucomicrobial strains from deep-sea environments have been rarely cultured and characterized. Recently, Rubritalea profundi SAORIC-165T, a psychrophilic bacterium of the phylum Verrucomicrobia, was isolated from a depth of 2,000 m in the northwestern Pacific Ocean. In this study, the genome sequence of R. profundi SAORIC-165T, the first deep-sea verrucomicrobial isolate, is reported with description of the genome properties and comparison to surface-borne Rubritalea genomes. The draft genome consisted of four contigs with an entire size of 4,167,407 bp and G+C content of 47.5%. The SAORIC-165T genome was predicted to have 3,844 proteincoding genes and 45 non-coding RNA genes. The genome contained a repertoire of metabolic pathways, including the Embden-Meyerhof-Parnas pathway, pentose phosphate pathway, tricarboxylic acid cycle, assimilatory sulfate reduction, and biosynthesis of nicotinate/nicotinamide, pantothenate/coenzyme A, folate, and lycopene. The comparative genomic analyses with two surface-derived Rubritalea genomes showed that the SAORIC-165T genome was enriched in genes involved in transposition of mobile elements, signal transduction, and carbohydrate metabolism, some of which might be related to bacterial enhancement of ecological fitness in the deep-sea environment. Amplicon sequencing of 16S rRNA genes from the water column revealed that R. profundi-related phylotypes were relatively abundant at 2,000 m and preferred a particle-associated life style in the deep sea. These findings suggest that R. profundi represents a genetically unique and ecologically relevant verrucomicrobial group well adapted to the deep-sea environment.

Flavobacterium aquariorum sp. nov., isolated from freshwater of the North Han River.

A non-motile, yellow-pigmented bacterial strain, designated IMCC34762T, was isolated from a freshwater sample collected from Lake Cheongpyeong in Korea. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain IMCC-34762T formed a lineage within the genus Flavobacterium and was most closely related to F. pectinovorum DSM 6368T (98.3% sequence similarity), followed by F. piscis CCUG 60099T (98.3%), F. branchiicola 59B-3-09T (98.2%), and F. saccharophilum DSM 1811T (98.2%). The average nucleotide identity and the genome-to-genome distance between strain IMCC34762T and the closely related strains were 61-62% and 26-27%, respectively, indicating that IMCC34762T is a novel species of the genus Flavobacterium. The major fatty acids (> 5%) of strain IMCC34762T were summed feature 3 (C16:1ω6c and/or C16:1ω7c, 17.3%), iso-C15:0 (15.0%), iso-C15:0 G (9.0%), C15:0ω6c (7.4%), iso-C15:0 (7.4%), and iso-C16:0 (5.3%). The major respiratory quinone and polyamine were MK-6 and sym-homospermidine, respectively. The major polar lipids were phosphatidylethanolamine, an unidentified aminophospholipid, and an unidentified lipid. The DNA G + C content of strain IMCC34762T was 34.4 mol%. Based on the taxonomic data presented in this study, strain IMCC-34762T represents a novel species within the genus Flavobacterium, for which the name Flavobacterium aquariorum, sp. nov. is proposed. The type strain is IMCC34762T (= KACC 19725T = NBRC 113425T).