Parvicella tangerina gen. nov., sp. nov. (Parvicellaceae fam. nov., Flavobacteriales), first cultured representative of the marine clade UBA10066, and Lysobacter luteus sp. nov., from activated sludge of a seawater-processing wastewater treatment plant.

Two strains isolated from a sample of activated sludge that was obtained from a seawater-based wastewater treatment plant on the southeastern Mediterranean coast of Spain have been characterized to achieve their taxonomic classification, since preliminary data suggested they could represent novel taxa. Given the uniqueness of this habitat, as this sort of plants are rare in the world and this one used seawater to process an influent containing intermediate products from amoxicillin synthesis, we also explored their ecology and the annotations of their genomic sequences. Analysis of their 16S rRNA gene sequences revealed that one of them, which was orange-pigmented, was distantly related to Vicingus serpentipes (family Vicingaceae) and to other representatives of neighbouring families in the order Flavobacteriales (class Flavobacteriia) by 88-89 % similarities; while the other strain, which was yellow-pigmented, was a putative new species of Lysobacter (family Xanthomonadaceae, order Xanthomonadales, class Gammaproteobacteria) with Lysobacter arseniciresistens as closest relative (97.3 % 16S rRNA sequence similarity to its type strain). Following a polyphasic taxonomic approach, including a genome-based phylogenetic analysis and a thorough phenotypic characterization, we propose the following novel taxa: Parvicella tangerina gen. nov., sp. nov. (whose type strain is AS29M-1T=CECT 30217T=LMG 32344T), Parvicellaceae fam. nov. (whose type genus is Parvicella), and Lysobacter luteus sp. nov. (whose type strain is AS29MT=CECT 30171T=LMG 32343T).

Pseudidiomarina piscicola sp. nov., isolated from cultured European seabass, Dicenthrarchus labrax.

Strain CECT 9734 T, a Gram-negative, aerobic, chemoorganotrophic bacterium, motile by polar flagella, was isolated from cultured European seabass, Dicenthrarchus labrax, in Spain. It grows from 5 to 42 ºC, 6-9 pH and 1-12% total salinity. Major cellular fatty acids are C15:0 iso, summed feature 9 (C17:1 iso w9c/C16:0 10-methyl) and C17:0 iso. The genome size is 2.5 Mbp and G + C content is 49.5 mol%. Comparative analysis of the 16S rRNA gene sequence shows that the strain is a member of Pseudidiomarina, with highest similarities with Pseudidiomarina halophila (97.0%) and Pseudidiomarina salinarum (96.9%). Phylogenomic tree based on UBCG program shows P. halophila as its closest relative. ANI and in-silico DDH with other Pseudidiomarina spp. are lower than 87 and 20%, respectively, suggesting that strain CECT 9734 T represents a new species, for which we propose the name Pseudidiomarina piscicola sp. nov. and CECT 9734 T (= LUBLD50 7aT = LMG 31044 T) as type strain.

Chryseobacterium potabilaquae sp. nov., Chryseobacterium aquaeductus sp. nov. and Chryseobacterium fistulae sp. nov., from drinking water systems.

A polyphasic taxonomic study was conducted on three strains isolated from drinking water systems that had previously been deposited as Chryseobacterium species at the Spanish Type Culture Collection in order to complete their classification. Strains CECT 9293T, CECT 9390T and CECT 9393T were isolated from sites in Barcelona, Spain, in the framework of a project aimed at generating the first MALDI-TOF database specific for bacteria present in water for human consumption. Their partial 16S rRNA sequences showed that their closest relatives among the type strains of Chryseobacterium exhibited 98 % similarity or less, supporting their taxonomic novelty. At the same time, comparison between them revealed that strains CECT 9293T and CECT 9393T could perhaps be related at the species level as they shared 99.5 % similarity. However, whole genome sequencing was performed and the subsequent calculation of relatedness indices, average nucleotide identity and estimated DNA-DNA hybridization, ruled out that possibility and confirmed instead that each of the strains should be considered a separate species in the genus Chryseobacterium. Having clarified their status, we also performed phylogenomic analyses and searched for possible environmental or non-type material sequences that could be related to any of them at the species level. In parallel, the strains were characterized phenotypically and compared to their closest relatives to determine diagnostic traits to support their formal proposal. The proposed species are Chryseobacterium potabilaquae sp. nov. with the type strain CECT 9293T (=LMG 32084T), Chryseobacterium aquaeductus sp. nov. with the type strain CECT 9390T (=LMG 32085T) and Chryseobacterium fistulae sp. nov. with the type strain CECT 9393T (=LMG 32086T).

Diversity and distribution of marine heterotrophic bacteria from a large culture collection.

BACKGROUND: Isolation of marine microorganisms is fundamental to gather information about their physiology, ecology and genomic content. To date, most of the bacterial isolation efforts have focused on the photic ocean leaving the deep ocean less explored. We have created a marine culture collection of heterotrophic bacteria (MARINHET) using a standard marine medium comprising a total of 1561 bacterial strains, and covering a variety of oceanographic regions from different seasons and years, from 2009 to 2015. Specifically, our marine collection contains isolates from both photic (817) and aphotic layers (744), including the mesopelagic (362) and the bathypelagic (382), from the North Western Mediterranean Sea, the North and South Atlantic Ocean, the Indian, the Pacific, and the Arctic Oceans. We described the taxonomy, the phylogenetic diversity and the biogeography of a fraction of the marine culturable microorganisms to enhance our knowledge about which heterotrophic marine isolates are recurrently retrieved across oceans and along different depths. RESULTS: The partial sequencing of the 16S rRNA gene of all isolates revealed that they mainly affiliate with the classes Alphaproteobacteria (35.9%), Gammaproteobacteria (38.6%), and phylum Bacteroidetes (16.5%). In addition, Alteromonas and Erythrobacter genera were found the most common heterotrophic bacteria in the ocean growing in solid agar medium. When comparing all photic, mesopelagic, and bathypelagic isolates sequences retrieved from different stations, 37% of them were 100% identical. This percentage increased up to 59% when mesopelagic and bathypelagic strains were grouped as the aphotic dataset and compared to the photic dataset of isolates, indicating the ubiquity of some bacterial isolates along different ocean depths. Finally, we isolated three strains that represent a new species, and the genome comparison and phenotypic characterization of two of these strains (ISS653 and ISS1889) concluded that they belong to a new species within the genus Mesonia. CONCLUSIONS: Overall, this study highlights the relevance of culture-dependent studies, with focus on marine isolated bacteria from different oceanographic regions and depths, to provide a more comprehensive view of the culturable marine bacteria as part of the total marine microbial diversity.

Thalassocella blandensis gen. nov., sp. nov., a novel member of the family Cellvibrionaceae.

Strain ISS155T, isolated from surface Mediterranean seawater, has cells that are Gram-reaction-negative, motile, strictly aerobic chemoorganotrophic, oxidase-positive, unable to reduce nitrate to nitrite, and able to grow with cellulose as the sole carbon and energy source. It is mesophilic, neutrophilic, slightly halophilic and has a requirement for sodium and magnesium ions. Its 16S rRNA gene sequence places the strain among members of Cellvibrionaceae, in the Gammaproteobacteria, with Agarilytica rhodophyticola 017T as closest relative (94.3 % similarity). Its major cellular fatty acids are C18 : 1, C16 : 0 and C16 : 1; major phospholipids are phosphatidyl glycerol, phosphatidyl ethanolamine and an unidentified lipid, and the major respiratory quinone is Q8. The genome size is 6.09 Mbp and G+C content is 45.2 mol%. A phylogenomic analysis using UBCG merges strain ISS155T in a clade with A. rhodophyticola, Teredinibacter turnerae, Saccharophagus degradans and Agaribacterium haliotis type strain genomes, all of them possessing a varied array of carbohydrate-active enzymes and the potential for polysaccharide degradation. Average amino acid identity indexes determined against available Cellvibrionaceae type strain genomes show that strain ISS155T is related to them by values lower than 60 %, with a maximum of 58 % to A. rhodophyticola 017T and 57 % to T. turnerae T7902T and S. degradans 2-40T. These results, together with the low 16S rRNA gene sequence similarities and differences in phenotypic profiles, indicate that strain ISS155T represents a new genus and species in Cellvibrionaceae, for which we propose the name Thalassocella blandensis gen. nov., sp. nov., and strain ISS155T (=CECT 9533T=LMG 31237T) as the type strain.

Mesonia oceanica sp. nov., isolated from oceans during the Tara oceans expedition, with a preference for mesopelagic waters.

Strain ISS653T, isolated from Atlantic seawater, is a yellow pigmented, non-motile, Gram-reaction-negative rod-shaped bacterium, strictly aerobic and chemoorganotrophic, slightly halophilic (1-15 % NaCl) and mesophilic (4-37 °C), oxidase- and catalase-positive and proteolytic. Its major cellular fatty acids are iso-C15 : 0, iso-C15 : 0 2-OH, and iso-C17 : 0 3-OH; the major identified phospholipid is phosphatidylethanolamine and the major respiratory quinone is MK6. Genome size is 4.28 Mbp and DNA G+C content is 34.9 mol%. 16S rRNA gene sequence similarity places the strain among members of the family Flavobacteriaceae, with the type strains of Mesonia phycicola (93.2 %), Salegentibacter mishustinae (93.1 %) and Mesonia mobilis (92.9 %) as closest relatives. Average amino acid identity (AAI) and average nucleotide identity (ANI) indices show highest values with M. mobilis (81 % AAI; 78.9 % ANI), M. phycicola (76 % AAI; 76.3 % ANI), Mesonia maritima (72 % AAI, 74.9 % ANI), Mesonia hippocampi (64 % AAI, 70.8 % ANI) and Mesonia algae (68 % AAI; 72.2 % ANI). Phylogenomic analysis using the Up-to-date-Bacterial Core Gene set (UBCG) merges strain ISS653T in a clade with species of the genus Mesonia. We conclude that strain ISS653T represents a novel species of the genus Mesonia for which we propose the name Mesonia oceanica sp. nov., and strain ISS653T (=CECT 9532T=LMG 31236T) as the type strain. A second strain of the species, ISS1889 (=CECT 30008) was isolated from Pacific Ocean seawater. Data obtained throughout the Tara oceans expedition indicate that the species is more abundant in the mesopelagic dark ocean than in the photic layer and it is more frequent in the South Pacific, Indian and North Atlantic oceans.

Shimia thalassica sp. nov., and reclassification of Pseudopelagicola gijangensis as Shimia gijangensis comb. nov., and Thalassobius activus as Cognatishimia activa comb. nov.

Strain CECT 7735T, a marine Gram-reaction negative, aerobic, non-motile bacterium, was isolated from coastal seawater in Valencia, Spain. Strain CECT 7735T is chemoorganotrophic, mesophilic, slightly halophilic, grows at 15-28 °C but not at 4 or 37 °C, requires seawater for growth and grows up to 6 % salinity. The major cellular fatty acid is summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The G+C content of the genome is 55.7 mol%. Comparative analysis of the 16S rRNA gene sequence shows the strain is affiliated to the family Rhodobacteraceae, in the class Alphaproteobacteria, with highest similarities to Phaeobacter species (97.0-97.5 %), Shimia species (96.5-97.3 %) and Pseudopelagicola gijangensis (96.5 %). Further phylogenomic analysis through the up-to-date-bacterial core gene (UBCG) set showed P. gijangensis to be its closest relative. Average nucleotide identity and in silico DNA-DNA hybridization values are lower than 85 and 21 %, respectively, with its phylogenetic relatives, suggesting that strain CECT 7735T represents a new species. The average amino acid identity value was over 70 % with the genome of the type strain of P. gijangensis and with all those of Shimia species. These values, together with UBCG set trees, suggest that the new species and P. gijangensisbelong to the same genus and that Pseudopelagicola should be reclassified as a Shimia species. We conclude that strain CECT 7735T represents a new species in the genus Shimia, for which we propose the name Shimiathalassica sp. nov. In addition, Pseudopelagicola gijangensis is reclassified as Shimiagijangensis comb. nov. From the same phylogenomic study, it can be concluded that Thalassobius activus should be reclassified in the genus Cognatishimia as Cognatishimiaactiva comb. nov.

Ruegeria denitrificans sp. nov., a marine bacterium in the family Rhodobacteraceae with the potential ability for cyanophycin synthesis.

Strain CECT 5091T, an aerobic, marine, Gram-reaction- and Gram-stain-negative, chemoheterotrophic bacterium was isolated from oysters harvested off the Spanish Mediterranean coast. Analysis of the 16S rRNA gene sequence placed the strain within the genus Ruegeria, in the family Rhodobacteraceae, with 16S rRNA gene similarities of 98.7, 98.7 and 98.4 % to Ruegeria conchae, Ruegeria atlanticaand Ruegeria arenilitoris, respectively. Average nucleotide identities (ANI) and in silico DNA-DNA hybridization (DDH) were determined, comparing the genome sequence of CECT 5091T with those of the type strains of 12 species of the genus Ruegeria: the values obtained were always below the thresholds (95-96 % ANI, 70 % in silico DDH) used to define genomic species, proving that CECT 5091T represents a novel species of the genus Ruegeria. The strain was slightly halophilic and mesophilic, with optimum growth at 26 °C, pH 7.0 and 3 % salinity, it required sodium and magnesium ions for growth and was able to reduce nitrate to dinitrogen. Carbon sources for growth include some carbohydrates (d-ribose, d-glucose, l-rhamnose, N-acetyl-d-glucosamine) and multiple organic acids and amino acids. The major cellular fatty acid was summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), representing 70 % of the total fatty acids. Carbon monoxide oxidation, cyanophycin synthetic ability and phosphatidylglycerol, diphosphatidylglycerol and phosphatidylcholine production are predicted from genome annotation, while bacteriochlorophyll a production was absent. The DNA G+C content of the genome was 56.7 mol%. We propose the name Ruegeriadenitrificans sp. nov. and strain CECT 5091T (=5OM10T=LMG 29896T) as the type strain for the novel species.

Roseivivax atlanticus (Li, Lai, Liu, Sun and Shao, 2015) is a later heterotypic synonym of Roseivivax marinus (Dai, Shi, Gao, Liu and Zhang, 2014).

The genomes of the type strains of Roseivivax atlanticusand Roseivivax marinus(Rhodobacteraceae, Alphaproteobacteria), were analysed to determine their respective Average Nucleotide Identity (ANI) and in silico DNA-DNA hybridization (DDH) values. These species were proposed and effectively published relatively closely in time (February and August 2014, respectively) and so not taking account of the other. The intergenomic relatedness between both type strains, 97.0-97.4 % ANI and 82.8 % in silico DDH, confirm that they represent members of the same genomic species. This conclusion is also supported at the phenotypic level. Since the nameRoseovarius marinuswas validly published earlier, R. atlanticus (Validation List 161, IJSEM 65, 1-4. 2015) should be considered a later heterotypic synonym of R. marinus(Dai, Shi, Gao, Liu and Zhang, 2014), in application of the priority rule.

Whole genome sequences reveal Vibrio hemicentroti Kim et al. 2013 as a later heterotypic synonym of Vibrio splendidus (Beijerinck 1900) Baumann et al. 1981.

The synonymy between Vibrio hemicentrotiKim et al. 2013 and Vibrio splendidus(Beijerinck 1900) Baumann et al. 1981 was suggested after a recent multilocus sequence analysis of the Splendidus clade, which included the type strains of both species. To clarify their status, we have determined genomic indexes from whole genome sequences of strains V. hemicentroti CECT 8714T and V. splendidus NCCB 53037T. Average Nucleotide Identities of 96.0-96.7 % and an in silico DNA-DNA hybridization value of 70.2 %, as well as similarity levels of selected housekeeping gene sequences support the consideration of V.hemicentroti as a later heterotypic synonym of V. splendidus.

Vibrio palustris sp. nov. and Vibrio spartinae sp. nov., two novel members of the Gazogenes clade, isolated from salt-marsh plants (Arthrocnemum macrostachyum and Spartina maritima).

Two bacterial strains, EAod9T and SMJ21T, isolated from salt-marsh plants, were determined to be related to species of the genus Vibriofrom from 16S rRNA sequence comparisons. Their closest phylogenetic relatives are members of the Gazogenes clade, Vibrio mangrovi and Vibrio rhizosphaerae , which show the greatest similarity to the SMJ21TrRNA sequence (97.3 and 97.1 %, respectively), while EAod9T had less than 97.0 % similarity to any other species of the genus Vibrio. Both strains share the basic characteristics of the genus Vibrio, as they are Gram-stain negative, motile, slightly halophilic, facultatively anaerobic bacteria. In addition, they are oxidase-negative and unable to grow on TCBS Agar; they grow between 15 to 26 °C, pH 6 to 8 and in up to 10 % (w/v) total salinity. They produce indol, are positive in the Voges-Proskauer test and are negative for arginine dihydrolase, lysine and ornithine decarboxylases. Strain SMJ21T is aerogenic and red-pigmented, due to prodigiosin production, while strain EAod9T ferments glucose without gas and is not pigmented. The major cellular fatty acids of both novel strains were C16 : 1ω7c/C16 : 1ω6c and C16 : 0. WGSobtained for both strains, along with the other five members of the clade, allowed the determination of ANI indexes and in silico estimations of DDH values, which confirmed that the two strains represent two novel species of the genus Vibrio: Vibriopalustris sp. nov. (with EAod9T=CECT 9027T=LMG 29724T as the proposed type strain) and Vibrio spartinae sp. nov. (with SMJ21T=CECT 9026T=LMG 29723T as the proposed type strain).

Grimontia celer sp. nov., from sea water.

Strain 96-237T, a Gram-reaction-negative, curved- to spiral-shaped motile bacterium, isolated from coastal marine water, was found to be related to species of the genus Grimontia by 16S rRNA gene sequence comparison, sharing 98.3 % similarity to Grimontia marina CECT 8713Tand 98.8 % to 'Grimontiaindica' AK16. Phenotypic analysis revealed that strain 96-237T is slightly halophilic, mesophilic and facultatively anaerobic, fermenting d-glucose, d-ribose, d-mannose, d-mannitol, maltose and sucrose. It was positive for oxidase and indole production and negative for arginine dihydrolase and lysine and ornithine decarboxylases. Its major fatty acids were C16 : 1ω7c/C16 : 1ω6c (SF3), C18 : 1ω7c and C16 : 0. Its DNA G+C content was 48.4 mol%. The strain was different at the species level from all other species of the genusGrimontia, with average nucleotide identity indices of 79.6 % to Grimontia. hollisae CECT 8713T, 87.8 % to G. marina CECT 5069T and 89.1 % to 'G. indica' AK16 genomes. Thus, the strain represents a novel species for which we propose the name Grimontia celer and 96-237T (=CECT 9029T =KCTC 42960T =LMG 29238T) as the type strain.

Marinomonas spartinae sp. nov., a novel species with plant-beneficial properties.

Two strains of Gram-stain-negative, chemo-organotrophic, aerobic and halophilic gammaproteobacteria, isolated from within the stem and roots of Spartina maritima in salt marshes from the south Atlantic Spanish coast, were found to represent a novel species in the genus Marinomonas through phylogenetic analysis of their 16S rRNA genes and phenotypic characterization. 16S rRNA gene sequences of the two strains shared < 96.2% similarity with other Marinomonas species, with Marimonas alcarazii being the most similar in sequence. They required sodium ions for growth, were able to thrive at low (4 °C) temperatures and at salinities of 12-15%, were unable to hydrolyse any tested macromolecule except casein, and grew with different monosaccharides, disaccharides, sugar alcohols, organic acids and amino acids. The novel species differed from other Marinomonas species in the use of several sole carbon sources, its temperature and salinity ranges for growth, ion requirements and cellular fatty acid composition, which included C16:0, C16:1 and C18:1 as major components and C10:0 3-OH, C12:0 and C12:0 3-OH as minor components. The name Marinomonas spartinae sp. nov. is proposed, with SMJ19T (=CECT 8886T=KCTC 42958T) as the type strain.

Marinomonas blandensis sp. nov., a novel marine gammaproteobacterium.

A novel Gram-staining-negative, chemoorganotrophic, moderately halophilic, strictly aerobic bacterium, strain MED121T, was isolated from a seawater sample collected at the Blanes Bay Microbial Observatory in the north-western Mediterranean Sea. Analysis of its 16S rRNA gene sequence, retrieved from the whole-genome sequence, showed that this bacterium was most closely related to Marinomonas dokdonensis and other Marinomonas species (96.3 and 93.3-95.7 % sequence similarities, respectively), within the family Oceanospirillaceae. Strain MED121T was included into a whole-genome sequencing study and, subsequently, it was characterized using a polyphasic taxonomic approach. It was found to be oxidase and catalase positive, its cells are cocci to short rods, it does not ferment carbohydrates and does not reduce nitrate to nitrite or gas and it requires at least 2.5 % (w/v) marine salts and tolerates up to 7 % (w/v) salts. Its major cellular fatty acids in order of abundance are C16 : 1ω7c/C16 : 1ω6c, C18 : 1ω7c, C16 : 0 and C10 : 0 3-OH. Its genome had an approximate length of 5.1 million bases and a DNA G+C content equal to 40.9 mol%. Analysis of the annotated genes reveals the capacity for the synthesis of ubiquinone 8 (Q8) and the polar lipids phosphatidylglycerol and phosphatidylethanolamine, in agreement with other members of the genus. All the data collected supported the creation of a novel species to accommodate this bacterium, for which the name Marinomonas blandensis sp. nov. is proposed. The type strain is MED121T (=CECT 7076T=LMG 29722T).

Vibrio renipiscarius sp. nov., isolated from cultured gilthead sea bream (Sparus aurata).

Two strains of Gram-negative, facultatively anaerobic, slightly halophilic bacteria, isolated from healthy gilthead sea bream (Sparus aurata) cultured in Spanish Mediterranean fish farms, were different from their closest relatives, Vibrio scophthalmi and V. ichthyoenteri, by phenotypic, phylogenetic and genomic standards. The strains were negative for decarboxylase tests and lacked extracellular hydrolytic activities, but were able to ferment d-mannitol, sucrose, cellobiose and d-gluconate, among other carbohydrates. The major cellular fatty acids were C16:1 and C16:0, in agreement with other species of the genus Vibrio. Their 16S rRNA gene sequences were 98.4 and 97.2 % similar to those of the type strains of V. scophthalmi and V. ichthyoenteri, and the similarities using other housekeeping genes (ftsZ, rpoD, recA, mreB and gyrB) and indices of genomic resemblance (average nucleotide identity and estimated DNA-DNA hybridization) between the isolates and those type strains were clearly below intraspecific levels, supporting the recognition of the strains as members of a separate novel species. Thus, we propose the name Vibrio renipiscarius sp. nov., with DCR 1-4-2T ( = CECT 8603T = KCTC 42287T) as the type strain.

Roseovarius albus sp. nov., a new Alphaproteobacterium isolated from the Mediterranean Sea.

Strain 4SM10(T), an aerobic marine, Gram-negative, heterotrophic and non pigmented bacterium isolated from seawater from Vinaroz in Castellón, Spain, was characterized using a polyphasic approach. Analysis of the 16S rRNA gene sequence placed the strain within the Roseobacter clade in the family Rhodobacteraceae. Phylogenetic analyses also showed that strain 4SM10(T) forms a stable clade with species of the genus Roseovarius, being related to Roseovarius nubinhibens ISM(T) and Roseovarius aestuarii SMK-122(T) at 97.5 and 97.4 % 16S rRNA sequence similarity, respectively. Average Nucleotide Identity (ANI) values, determined as a measure of overall genomic resemblance, confirmed that strain 4SM10(T) does not belong to the same species as R. aestuarii CECT 7745(T) and Roseovarius nubinhibens CECT 7750(T) displaying ANI values well below the 95 % boundary for genomic species. Strain 4SM10(T) requires Na(+) plus a divalent cation (either Mg(2+) or Ca(2+)) to grow, reduces nitrate to nitrite and uses a large number of amino acids and organic acids (but no carbohydrates) as sole carbon sources. Enzymatic activities displayed in API ZYM tests are alkaline phosphatase, leucine arylamidase and acid phosphatase. The major cellular fatty acids were identified as C18:1 ω7c and/or C18:1 ω6c (67.1 %). The DNA G+C content was determined to be 54.27 mol%. Based on the genotypic and phenotypic data obtained, the name Roseovarius albus sp. nov. is proposed for this novel taxon, with the type strain 4SM10(T) (=CECT 7450(T) = KCTC 22653(T)).

Genomic and phenotypic characterization of 26 novel marine bacterial strains with relevant biogeochemical roles and widespread presence across the global ocean.

Prokaryotes dominate global oceans and shape biogeochemical cycles, yet most taxa remain uncultured and uncharacterized as of today. Here we present the characterization of 26 novel marine bacterial strains from a large isolate collection obtained from Blanes Bay (NW Mediterranean) microcosm experiments made in the four seasons. Morphological, cultural, biochemical, physiological, nutritional, genomic, and phylogenomic analyses were used to characterize and phylogenetically place the novel isolates. The strains represent 23 novel bacterial species and six novel genera: three novel species pertaining to class Alphaproteobacteria (families Rhodobacteraceae and Sphingomonadaceae), six novel species and three new genera from class Gammaproteobacteria (families Algiphilaceae, Salinispheraceae, and Alteromonadaceae), 13 novel species and three novel genera from class Bacteroidia (family Flavobacteriaceae), and one new species from class Rhodothermia (family Rubricoccaceae). The bacteria described here have potentially relevant roles in the cycles of carbon (e.g., carbon fixation or energy production via proteorhodopsin), nitrogen (e.g., denitrification or use of urea), sulfur (oxidation of sulfur compounds), phosphorus (acquisition and use of different forms of phosphorus and remodeling of membrane phospholipids), and hydrogen (oxidation of hydrogen to obtain energy). We mapped the genomes of the presented strains to the Tara Oceans metagenomes to reveal that these strains were globally distributed, with those of the family Flavobacteriaceae being the most widespread and abundant, while Rhodothermia being the rarest and most localized. While molecular-only approaches are also important, our study stresses the importance of culturing as a powerful tool to further understand the functioning of marine bacterial communities.

Actibacterium mucosum gen. nov., sp. nov., a marine alphaproteobacterium from Mediterranean seawater.

Strain R46(T), a marine alphaproteobacterium, was isolated from Mediterranean seawater at Malvarrosa beach, Valencia, Spain. It is an aerobic chemo-organotrophic, mesophilic and slightly halophilic organism, with complex ionic requirements. Phylogenetic analyses based on the 16S rRNA and gyrB gene sequences showed that strain R46(T) formed a separate branch within the family Rhodobacteraceae, bearing similarities below 94.7 and 80.3%, respectively, to any other recognized species. It contained Q10 as the predominant isoprenoid quinone and C(18:1)ω7c/C(18:1)ω6c as the major cellular fatty acid. Phosphatidylglycerol was the only identified polar lipid, although other lipids were also detected. The DNA G+C content of the genomic DNA was 61.3 mol%. On the basis of extensive phenotypic and phylogenetic comparative analysis, it is concluded that the strain represents a novel genus and species, for which the name Actibacterium mucosum gen. nov., sp. nov. is proposed. The type strain of the type species is Actibacterium mucosum R46(T) ( = CECT 7668(T) = KCTC 23349(T)).

Tropicibacter multivorans sp. nov., an aerobic alphaproteobacterium isolated from surface seawater.

Strain MD5T, an aerobic marine alphaproteobacterium, was isolated from Mediterranean seawater at Malvarrosa beach, Valencia, Spain. The strain was characterized in a polyphasic study and was placed phylogenetically within the Roseobacter clade in the family Rhodobacteraceae. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MD5T is related to Tropicibacter naphthalenivorans C02T, Phaeobacter inhibens T5T, P. gallaeciensis BS107T and P. daeponensis TF-218T, with 96.9, 96.2, 96.1 and 96.1 % sequence similarity, respectively. Phylogenetic analyses also showed that strain MD5T forms a stable clade only with T. naphthalenivorans C02T. Strain MD5T requires Na+ plus a divalent cation (either Mg2+ or Ca2+) to grow, does not reduce nitrate to nitrite and uses a large number of carbohydrates as sole carbon sources. It is positive for ß-galactosidase and urease activities and aesculin hydrolysis. Enzyme activities displayed in the API ZYM strip were alkaline phosphatase, leucine arylamidase, acid phosphatase and α-glucosidase. Major cellular fatty acids were summed feature 8 (C18:1ω7c and/or C18:1ω6c; 70.9 %) and C16:0 (8.2 %). The results of physiological and biochemical tests allowed clear phenotypic differentiation of this isolate from the only described species of the genus Tropicibacter. It is evident from the genotypic and phenotypic data obtained that the strain should be classified in a novel species in the genus Tropicibacter. The name Tropicibacter multivorans sp. nov. is proposed, with the type strain MD5T (=CECT 7557T=KCTC 23350T).

Pseudomonas litoralis sp. nov., isolated from Mediterranean seawater.

Strains 2SM5(T) and 2SM6, two strictly aerobic chemo-organotrophic gammaproteobacteria, were isolated from Mediterranean seawater off the coast of Vinaroz, Castellón, Spain, in February, 1990. They were extensively characterized by a polyphasic study that placed them in the genus Pseudomonas. Phylogenetic analysis of 16S rRNA gene sequences showed that both strains shared 100 % sequence similarity and were closely related to members of the Pseudomonas pertucinogena clade, with less than 97.3 % similarity to strains of established species; Pseudomonas xiamenensis was the closest relative. Analysis of sequences of three housekeeping genes, rpoB, rpoD and gyrB, further confirmed the phylogenetic assignment of the Mediterranean isolates. Chemotaxonomic traits such as quinone and polar lipid composition also corroborated the placement of strains 2SM5(T) and 2SM6 in the gammaproteobacteria. Other phenotypic traits, including fatty acid composition, enabled clear differentiation of both isolates from other species of Pseudomonas. We therefore conclude that strains 2SM5(T) and 2SM6 represent a novel species of Pseudomonas, for which the name Pseudomonas litoralis is proposed; the type strain is 2SM5(T) ( = CECT 7670(T) = KCTC 23093(T)).