Discovery of an Abundant Viral Genus in Polar Regions through the Isolation and Genomic Characterization of a New Virus against Oceanospirillaceae.

The marine bacterial family Oceanospirillaceae, is well-known for its ability to degrade hydrocarbons and for its close association with algal blooms. However, only a few Oceanospirillaceae-infecting phages have been reported thus far. Here, we report on a novel Oceanospirillum phage, namely, vB_OsaM_PD0307, which has a 44,421 bp linear dsDNA genome and is the first myovirus infecting Oceanospirillaceae. A genomic analysis demonstrated that vB_OsaM_PD0307 is a variant of current phage isolates from the NCBI data set but that it has similar genomic features to two high-quality, uncultured viral genomes identified from marine metagenomes. Hence, we propose that vB_OsaM_PD0307 can be classified as the type phage of a new genus, designated Oceanospimyovirus. Additionally, metagenomic read mapping results have further shown that Oceanospimyovirus species are widespread in the global ocean, display distinct biogeographic distributions, and are abundant in polar regions. In summary, our findings expand the current understanding of the genomic characteristics, phylogenetic diversity, and distribution of Oceanospimyovirus phages. IMPORTANCE Oceanospirillum phage vB_OsaM_PD0307 is the first myovirus found to infect Oceanospirillaceae, and it represents a novel abundant viral genus in polar regions. This study provides insights into the genomic, phylogenetic, and ecological characteristics of the new viral genus, namely Oceanospimyovirus.

Proposal of Allopseudospirillum gen. nov. as a replacement name for the illegitimate prokaryotic generic name Pseudospirillum Satomi et al. 2002.

The prokaryotic generic name Pseudospirillum Satomi et al. 2002 is illegitimate because it is a later homonym of Pseudospirillum Alexeieff 1917, a genus of uncertain affiliation, possibly belonging to the Mesomycetozoa (Principle 2 and Rule 51b(5) of the International Code of Nomenclature of Prokaryotes). We therefore propose the replacement generic name Allopseudospirillum, with type species Allopseudospirillum japonicum.

Parathalassolituus penaei gen. nov., sp. nov., a novel member of the family Oceanospirillaceae isolated from a coastal shrimp pond in Guangxi, PR China.

A Gram-stain-negative, strictly aerobic, rod-shaped and motile bacterium with bipolar flagella, designated G-43T, was isolated from a surface seawater sample collected from an aquaculture in Guangxi, PR China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain G-43T was most closely related to the family Oceanospirillaceae and distantly to the most closely related genera Venatorbacter and Thalassolituus (95.52 % and 94.45-94.76 % 16S rRNA gene sequence similarity, respectively), while similarity values to other Oceanospirillaceae type strains were lower than 94.0 %. Strain G-43T was found to grow at 4-30 °C (optimum, 25-28 °C), pH 6-9.0 (optimum, pH 7.0) and with 0-4.0 % NaCl (w/v; optimum at 2 % NaCl). Chemotaxonomic analysis of strain G-43T indicated that the sole respiratory quinone was ubiquinone-8, the predominant cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), and the major polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, aminolipid, diphosphatidylglycerol, phospholipids and an unidentified lipid. The G+C content of the genomic DNA was 55.4 mol%. The phylogenetic, genotypic, phenotypic and chemotaxonomic data demonstrate that strain G-43T represents a novel species in a novel genus within the family Oceanospirillaceae, for which the name Parathalassolituus penaei gen. nov., sp. nov. is proposed. Strain G-43T (=KCTC 72750T= CCTCC AB 2022321T) is the type and only strain of Parathalassolituus penaei.

Analysis of allosteric communication in a multienzyme complex by ancestral sequence reconstruction.

Tryptophan synthase (TS) is a heterotetrameric αßßα complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the α-subunit TrpA and the ß-subunit TrpB via a complex allosteric network. We have analyzed this allosteric network by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect extinct ancestors of modern proteins. Previously, the sequences of TrpA and TrpB from the last bacterial common ancestor (LBCA) have been computed by means of ASR and characterized. LBCA-TS is similar to modern TS by forming a αßßα complex with indole channeling taking place. However, LBCA-TrpA allosterically decreases the activity of LBCA-TrpB, whereas, for example, the modern ncTrpA from Neptuniibacter caesariensis allosterically increases the activity of ncTrpB. To identify amino acid residues that are responsible for this inversion of the allosteric effect, all 6 evolutionary TrpA and TrpB intermediates that stepwise link LBCA-TS with ncTS were characterized. Remarkably, the switching from TrpB inhibition to TrpB activation by TrpA occurred between 2 successive TS intermediates. Sequence comparison of these 2 intermediates and iterative rounds of site-directed mutagenesis allowed us to identify 4 of 413 residues from TrpB that are crucial for its allosteric activation by TrpA. The effect of our mutational studies was rationalized by a community analysis based on molecular dynamics simulations. Our findings demonstrate that ancestral sequence reconstruction can efficiently identify residues contributing to allosteric signal propagation in multienzyme complexes.

Oceanospirillum sediminis sp. nov., Isolated From Coastal Sediment in the Yellow Sea.

A novel Gram-negative, motile, aerobic, spiral-shaped bacterium designated D5T, was isolated from a coastal sediment collected in the Yellow Sea. Optimal growth occurred at 30 °C, pH 7.0-8.0 and in the presence of 1-3% (w/v) NaCl. Strain D5T contained ubiquinone 8 (Q-8) as the predominant respiratory quinone. The major fatty acids (> 10%) were C16:0, C16:1 ω7c/C16:1 ω6c and C18:1w7c/C18:1w6c. The main polar lipids were phosphatidylglycerol and phosphatidylethanolamine. The draft genome is 5.6 Mb in length, and DNA G + C content is 47.2 mol%. 16S rRNA gene sequences showed that strain D5T is most closely related to Oceanospirillum beijerinckii NBRC 15445T (97.8%, sequence similarity). However, the digital DNA-DNA hybridization (dDDH) value and average nucleotide identity (ANI) between strain D5T and O. beijerinckii is only 27.8% and 77.1%. Phylogenetic trees based on 16S rRNA gene sequences and whole genomes all indicated that strain D5T formed a separate branch in the genus Oceanospirillum. Combined results of the polyphasic analyses suggested that strain D5T represents a novel species in the genus Oceanospirillum, for which the name Oceanospirillum sediminis sp. nov. is proposed. The type strain is D5T (= MCCC 1K06061T = KCTC 62987T).

Maribrevibacterium harenarium gen. nov., sp. nov., represented by a marine strain of the family Oceanospirillaceae.

A Gram-stain-negative, non-motile, facultatively anaerobic, short rod-shaped bacterium, designated HB171799T, was isolated from seacoast sandy soil collected at Qishui Bay, Hainan, PR China. The chemotaxonomic analysis revealed that the respiratory quinones were Q-8 and Q-7, and the major cellular fatty acids were summed feature 8 (comprising C18 : 1 ω7c and/or C18 : 1 ω6c), C16 : 0 and C18 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid and an unidentified lipid. The size of the draft genome was 3.68 Mb with a DNA G+C content of 48.0 mol%. Results of phylogenetic analyses based on 16S rRNA gene and genome sequences showed that the novel isolate belonged to the family Oceanospirillaceae and formed a distinct subcluster at the base of the radiation of the genus Marinomonas. The highest sequence similarity (96.0 %) of the novel isolate was found to the type strains of Marinomonas fungiae JCM 18476T and Marinomonas ostreistagni DSM23425T. The whole genome-based phylogeny and differences in cellular fatty acids and polar lipids readily distinguished strain HB171799T from all the closely related validly published type strains. Strain HB171799T is therefore suggested to represent a novel species of a new genus in the family Oceanospirillaceae, for which the name Maribrevibacterium harenarium gen. nov., sp. nov. is proposed. The type strain is HB171799T (=CGMCC 1.16727T=JCM 33332T).

Marinobacterium alkalitolerans sp. nov., with nitrate reductase and urease activity isolated from green algal mat collected from a solar saltern.

A novel Gram-staining-negative, rod-shaped, 0.6-0.8 µm wide and 2.0-3.0 µm in length, motile bacterium designated strain AK62T, was isolated from the green algal mat collected from saltpan, Kakinada, Andhra Pradesh, India. Colonies on ZMA were circular, off-white, shiny, moist, translucent, 1-2 mm in diameter, flat, with an entire margin. The major fatty acids include C16:0, C18:1 ω7c, and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c and/or iso-C14:0 3-OH). Polar lipids include diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminophospholipid, three unidentified phospholipids, and one unidentified lipid. Polyamine includes Spermidine. The DNA G + C content of the strain AK62T was 58.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain AK62T was closely related to the type strains Marinobacterium sediminicola, Marinobacterium coralli and Marinobacterium stanieri with a pair-wise sequence similarity of 96.9, 96.6 and 96.6%, respectively, forming a distinct branch within the genus Marinobacterium and clustered with M. stanieri, M. sediminicola, M. coralli and M. maritimum cluster. Strain AK62T shares average nucleotide identity (ANIb, based on BLAST) of 78.44, 76.69, and 76.95% with M. sediminicola CGMCC 1.7287T, M. stanieri DSM 7027T, and Marinobacterium halophilum Mano11T respectively. Based on the observed phenotypic, chemotaxonomic characteristics, and phylogenetic analysis, strain AK62T is described in this study as a novel species in the genus Marinobacterium, for which the name Marinobacterium alkalitolerans sp. nov. is proposed. The type strain of M. alkalitolerans is AK62T (= MTCC 12102T = JCM 31159T = KCTC 52667T).

Protein expression in the obligate hydrocarbon-degrading psychrophile Oleispira antarctica RB-8 during alkane degradation and cold tolerance.

In cold marine environments, the obligate hydrocarbon-degrading psychrophile Oleispira antarctica RB-8, which utilizes aliphatic alkanes almost exclusively as substrates, dominates microbial communities following oil spills. In this study, LC-MS/MS shotgun proteomics was used to identify changes in the proteome induced during growth on n-alkanes and in cold temperatures. Specifically, proteins with significantly higher relative abundance during growth on tetradecane (n-C14 ) at 16°C and 4°C have been quantified. During growth on n-C14 , O. antarctica expressed a complete pathway for the terminal oxidation of n-alkanes including two alkane monooxygenases, two alcohol dehydrogenases, two aldehyde dehydrogenases, a fatty-acid-CoA ligase, a fatty acid desaturase and associated oxidoreductases. Increased biosynthesis of these proteins ranged from 3- to 21-fold compared with growth on a non-hydrocarbon control. This study also highlights mechanisms O. antarctica may utilize to provide it with ecological competitiveness at low temperatures. This was evidenced by an increase in spectral counts for proteins involved in flagella structure/output to overcome higher viscosity, flagella rotation to accumulate cells and proline metabolism to counteract oxidative stress, during growth at 4°C compared with 16°C. Such species-specific understanding of the physiology during hydrocarbon degradation can be important for parameterizing models that predict the fate of marine oil spills.

Nitrincola iocasae sp. nov., a bacterium isolated from sediment collected at a cold seep field in the South China Sea.

A novel bacterium, designated strain KXZD1103T, was isolated from sediment collected at a cold seep field of the Formosa Ridge in the South China Sea. Cells were Gram-stain-negative, facultatively anaerobic, motile, oxidase- and catalase-positive, and grew optimally at 28 °C, pH 6.0-pH 7.0 and in the presence of 1-3 % (w/v) NaCl. The major cellular fatty acids were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c) and C16 : 0. The major respiratory ubiquinone was Q-8. The predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Analysis of 16S rRNA gene sequences revealed that strain KXZD1103T grouped with members of the genus Nitrincola, with Nitrincola lacisaponensis 4CAT (98.1 % sequence similarity) and Nitrincola schmidtii R4-8T (97.7 %) as its closest neighbours. Genome sequencing revealed a genome size of 4.17 Mb and a DNA G+C content of 50.1 %. Genomic average nucleotide identity values for strain KXZD1103T with the type strains within the genus Nitrincola ranged from 71.0 to 75.7 %, while the in silico DNA-DNA hybridization values for strain KXZD1103T with these strains ranged from 16.1 to 21.6 %. On the basis of the results of phylogenetic, phenotypic and chemotaxonomic analyses, strain KXZD1103T is considered to represent a novel species of the genus Nitrincola, for which the name Nitrincola iocasae sp. nov. is proposed. The type strain is KXZD1103T (=KCTC 72678T=MCCC 1K04283T).

Nitrincola tapanii sp. nov., a novel alkaliphilic bacterium from An Indian Soda Lake.

A novel Gram-stain-negative bacterial strain designated as MEB193T was isolated from a sediment sample collected from Lonar Lake, India. The cells were motile, non-spore-forming and rod-shaped. The strain was oxidase- and catalase-positive. It grew optimally at pH 9.0 and at 1 % (w/v) NaCl concentration at 30 °C. Based on 16S rRNA gene sequence similarity, MEB193T belongs to genus Nitrincola, with Nitrincola alkalilacustris ZV-19T (95.89 %) and Nitrincola lacisaponensis 4CAT (95.87 %) as its closest neighbours. The major fatty acid was summed feature 8 comprising C18:1ω7c/C18:1ω6c (52 %) followed by C16 : 0 (25 %). Phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) were present as the major polar lipids. The draft genome obtained in this study was 2 793 747 bp and the G+C content was 50.79 mol%. Average nucleotide identity (71.76 %) and DNA-DNA hybridization (<20 %) values between strain MEB193T and Nitrincola lacisaponensis 4CAT confirmed the novelty of this new species. Based on phenotypic including chemotaxonomic and genotypic characterization data, strain MEB193T represents a new species of the genus Nitrincola for which the name Nitrincola tapanii sp. nov. is proposed. The type strain is MEB193T (=MCC 2863T=JCM 31570 T=KCTC 52390 T).

Marinospirillum perlucidum sp. nov., a novel helical bacterium isolated from a sea cucumber culture pond.

Strain F3212T, Gram-stain-negative, aerobic, helical and motile bacterium, was isolated from the marine sediment collected in a sea cucumber culture pond located in Rongcheng, China. Strain F3212T grew optimally at pH 8.5, at 30 °C and in the presence of 3.0 % (w/v) NaCl. Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that strain F3212T belongs to the genus Marinospirillum, clustering with M. celere, M. alkaliphilum, M. minutulum, M. megaterium and M. insulare (with 96.4, 94.6, 93.1, 92.4 and 92.1 % 16S rRNA gene sequence similarities, respectively). The chemotaxonomic properties of strain F3212T were similar to those of members of the genus Marinospirillum. Q-8 was the sole respiratory ubiquinone and the genomic DNA G+C content was 53.3 mol%. The major fatty acids were C18 : 1 ω9c, C16 : 0 and C18 : 0. The polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentifed lipid and an unidentified aminophospholipid. The average nucleotide identity scores for strains M. celere DSM 18438T and M. minutulum DSM 6287T were 74.5 and 69.4 %, respectively. The DNA-DNA homologies with M. celere DSM 18438T and M. minutulum DSM 6287T were less than 20 %. It's concluded that strain F3212T represents a new species of the genus Marinospirillum, for which the name Marinospirillum perlucidum sp. nov. is proposed. The type strain is F3212T (=KCTC 52892T=MCCC 1H00198T).

Phenotypic Variations of Oleispira antarctica RB-8(T) in Different Growth Conditions.

The peculiar biotechnological applications of Oleispira spp. in the natural cleansing of oil-polluted marine systems stimulated the study of the phenotypic characteristics of the Oleispira antarctica RB-8(T) strain and modifications of these characteristics in relation to different growth conditions. Bacterial abundance, cell size and morphology variations (by image analysis) and hydrocarbon degradation (by gas chromatography with flame ionization detection, GC-FID) were analysed in different cultures of O. antarctica RB-8(T). The effects of six different hydrocarbon mixtures (diesel, engine oil, naval oil waste, bilge water, jet fuel and oil) used as a single carbon source combined with two different growth temperatures (4° and 15 °C) were analysed (for 22 days). The data obtained showed that the mean cell volume decreased with increasing experimental temperature. Three morphological bacterial shapes were identified: spirals, rods and cocci. Morphological transition from spiral to rod and coccoid shapes in relation to the different substrates (oil mixtures) and/or growth temperatures was observed, except for one experimental condition (naval oil waste) in which spiral bacteria were mostly dominant. Phenotypic traits and physiological status of hydrocarbon-degrading bacteria showed important modifications in relation to culture conditions. These findings suggest interesting potential for strain RB-8(T) for ecological and applicative purposes.

Metagenomic and metatranscriptomic responses of natural oil degrading bacteria in the presence of dispersants.

Oil biodegradation has been extensively studied in the wake of the deepwater horizon spill, but the application of dispersant to oil spills in marine environments remains controversial. Here, we report metagenomic (MG) and metatranscriptomic (MT) data mining from microcosm experiments investigating the oil degrading potential of Canadian west and east coasts to estimate the gene abundance and activity of oil degrading bacteria in the presence of dispersant. We found that the addition of dispersant to crude oil mainly favours the abundance of Thalassolituus in the summer and Oleispira in the winter, two key natural oil degrading bacteria. We found a high abundance of genes related not only to n-alkane and aromatics degradation but also associated with transporters, two-component systems, bacterial motility, secretion systems and bacterial chemotaxis.

Following the Evolutionary Track of a Highly Specific l-Arginine Oxidase by Reconstruction and Biochemical Analysis of Ancestral and Native Enzymes.

Following the evolutionary track of enzymes can help elucidate how enzymes attain their characteristic functions, such as thermal adaptation and substrate selectivity, during the evolutionary process. Ancestral sequence reconstruction (ASR) is effective for following evolutionary processes if sufficient sequence data are available. Selecting sequences from the data to generate a curated sequence library is necessary for the successful design of artificial proteins by ASR. In this study, we tried to follow the evolutionary track of l-arginine oxidase (AROD), a flavin adenine dinucleotide (FAD)-dependent amino acid oxidase (LAAO) that exhibits high specificity for l-arginine. The library was generated by selecting sequences in which the 15th, 50th, 332nd, and 580th residues are Gly, Ser, Trp, and Thr, respectively. We excluded sequences that are either extremely short or long and those with a low degree of sequence identity. Three ancestral ARODs (AncARODn0, AncARODn1, and AncARODn2) were designed using the library. Subsequently, we expressed the ancestral ARODs as well as native Oceanobacter kriegii AROD (OkAROD) in bacteria. AncARODn0 is phylogenetically most remote from OkAROD, whereas AncARODn2 is most similar to OkAROD. Thermal stability was gradually increased by extending AROD sequences back to the progenitor, while the temperature at which the residual activity is half of the maximum measured activity (T1/2) of AncARODn0 was >20°C higher than that of OkAROD. Remarkably, only AncARODn0 exhibited broad substrate selectivity similar to that of conventional promiscuous LAAO. Taken together, our findings led us to infer that AROD may have evolved from a highly thermostable and promiscuous LAAO.IMPORTANCE In this study, we attempted to infer the molecular evolution of a recently isolated FAD-dependent l-arginine oxidase (AROD) that oxidizes l-arginine to 2-ketoarginine. Utilizing 10 candidate AROD sequences, we obtained a total of three ancestral ARODs. In addition, one native AROD was obtained by cloning one of the candidate ARODs. The candidate sequences were selected utilizing a curation method defined in this study. All the ARODs were successfully expressed in Escherichia coli for analysis of their biochemical functions. The catalytic activity of our bacterially expressed ancestral ARODs suggests that our ASR was successful. The ancestral AROD that is phylogenetically most remote from a native AROD has the highest thermal stability and substrate promiscuity. Our findings led us to infer that AROD evolved from a highly thermostable and promiscuous LAAO. As an application, we can design artificial ARODs with improved functions compared with those of native ones.

Neptunomonas marina sp. nov., isolated from seawater.

Strain HPM-16T, isolated from seawater, was characterized using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of an up-to-date bacterial core gene set (92 protein clusters) indicated that strain HPM-16T formed a phylogenetic lineage in the genus Neptunomonas. Strain HPM-16T was most closely related to Neptunomonas concharum LHW37T with 16S rRNA gene sequence similarity of 96.7%. Cells were Gram-stain negative, facultatively anaerobic, motile by means of a single polar flagellum, rod-shaped and formed white colonies. Optimal growth occurred at 30-35 °C, pH 6.5-8, and in the presence of 2-5% NaCl. C18:1ω7c and summed feature 3 (C16:1ω7c and/or C16:1ω6c) were the predominant fatty acids. The only isoprenoid quinone was Q-8. The polar lipid profile revealed the presence of phosphatidylethanolamine, phosphatidylglycerol and several uncharacterized lipids. The major polyamines were putrescine and spermidine. The draft genome was approximately 3.68 Mb in size with a G + C content of 50.5 mol%. Differential phenotypic properties, together with the phylogenetic inference, demonstrate that strain HPM-16T should be classified as a novel species of the genus Neptunomonas, for which the name Neptunomonas marina sp. nov. is presented. The type strain is HPM-16T (= BCRC 80980T = LMG 29560T = KCTC 52235T).

Amphritea opalescens sp. nov., isolated from sediment adjacent to Fildes Peninsula, Antarctica.

A taxonomic study was carried out on a Gram-stain-negative bacterium, namely strain ANRC-JH13T, isolated from a sediment sample collected at Jasper beach, adjacent to Fildes Peninsula, Antarctica. Cells of strain ANRC-JH13T were non-spore-forming rods and motile by the way of flagellum. Strain ANRC-JH13T was facultatively anaerobic, oxidase-positive, and catalase-positive. Growth of strain ANRC-JH13T occurred at 10-42 °C (optimum, 28 °C), pH 4.0-11.0 (pH 7.0) and 0-12.0 % (w/v) NaCl (1.0-2.0 %). Its predominant fatty acids were C16 : 0 (21.7 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 38.3 %), and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 20.1 %). Isoprenoid quinone Q-8 was the major respiratory quinone. Its major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids, and four unknown polar lipids. The DNA G+C content was 48 mol%. Strain ANRC-JH13T showed the highest 16S rRNA gene sequence similarity to Amphritea balenae JAMM 1525T (97.9 %), followed by Amphritea atlantica M41T (97.8 %) and Amphritea japonica JAMM 1866T (97.3 %), and formed a lineage within the genus Amphritea on the phylogenetic trees. However, the in silico average nucleotide identity values between strain ANRC-JH13T and A. balenae JAMM 1525T, A. atlantica M41T, and A. japonica JAMM 1866T were 74.0, 76.7, and 74.9 %, respectively. The in silico DNA-DNA hybridization values between them were 19.8, 20.6, and 19.4 %, respectively. Based on the results from phenotypic, chemotaxonomic, and phylogenetic analyses, strain ANRC-JH13T is considered to represent a novel species of the genus Amphritea, for which the name Amphriteaopalescens sp. nov. is proposed. The type strain is ANRC-JH13T (=MCCC 1K03512T=KCTC 62532T).

Nitrincola tibetensis sp. nov., isolated from Lake XuguoCo on the Tibetan Plateau.

A novel Gram-stain-negative, motile and rod-shaped bacterium, designated xg18T, was isolated from Lake XuguoCo on the Tibetan Plateau. The strain was able to grow optimally at 0-2 % NaCl and tolerate up to 6 % NaCl. Growth occurred at pH 7.0-11.0 (optimum, pH 9.0-10.0) and 15-40 °C (optimum, 37 °C). Vitamins were not required for growth. The major polar lipids of strain xg18T were phosphatidyl ethanolamine and phosphatidylglycerol. The predominant respiratory quinone was Q-8. The major fatty acids were summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The G+C content of genomic DNA was 46.1 mol%. Analysis of 16S rRNA sequences showed that strain xg18T belongs to the genus Nitrincola, with Nitrincola alkalisediminis MEB087T (KC822363, 98.6 %) as its closest neighbour. The DNA-DNA relatedness value of strain xg18T with its closest phylogenetic neighbour, N. alkalisediminis JCM 19317T, was 43.1±3.2 %. Strain xg18T was clearly distinguishable from the type strain of the genus Nitrincola through phylogenetic analysis, fatty acid composition data and a range of physiological and biochemical characteristics comparisons. Based on its phenotypic and chemotaxonomic characteristics, strain xg18T could be classified as a representative of a novel species of the genus for which the name Nitrincola tibetensis sp. nov. is proposed. The type strain is xg18T (=CICC 24457T=KCTC 62401T).

Marinobacterium boryeongense sp. nov., isolated from seawater.

A Gram-stain-negative and strictly aerobic bacterium, designated DMHB-2T, was isolated from a sample of seawater collected off the Yellow Sea coast of the Republic of Korea. Cells were short rods and motile by means of a single polar flagellum. Catalase and oxidase activities were positive. Growth occurred at pH 5.5-10.0 (optimum, pH 6.0), 15-45 °C (optimum, 25 °C) and with 1-9 % NaCl (optimum, 3 %). The respiratory quinone was ubiquinone-8 and the major fatty acids were C16 : 0 (17.9 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 26.1 %) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 37.4 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain DMHB-2T belong to the genus Marinobacterium, with the highest 16S rRNA gene sequence similarity of 95.2 % to Marinobacterium zhoushanense KCTC 42782T. The genomic DNA G+C content of strain DMHB-2T was 60.8 mol%. On the basis of the phenotypic, chemotaxonomic and genotypic characteristics presented in this study, strain DMHB-2T is suggested to represent a novel species of the genus Marinobacterium, for which the name Marinobacteriumboryeongense sp. nov. is proposed. The type strain is DMHB-2T (=KACC 19225T=JCM 31902T).

Characterization of a thermostable flavin-containing monooxygenase from Nitrincola lacisaponensis (NiFMO).

The flavin-containing monooxygenases (FMOs) play an important role in drug metabolism but they also have a high potential in industrial biotransformations. Among the hitherto characterized FMOs, there was no thermostable representative, while such biocatalyst would be valuable for FMO-based applications. Through a targeted genome mining approach, we have identified a gene encoding for a putative FMO from Nitrincola lacisaponensis, an alkaliphilic extremophile bacterium. Herein, we report the biochemical and structural characterization of this newly discovered bacterial FMO (NiFMO). NiFMO can be expressed as active and soluble enzyme at high level in Escherichia coli (90-100 mg/L of culture). NiFMO is relatively thermostable (melting temperature (Tm) of 51 °C), displays high organic solvent tolerance, and accepts a broad range of substrates. The crystal structure of NiFMO was solved at 1.8 Å resolution, which allows future structure-based enzyme engineering. Altogether, NiFMO represents an interesting newly discovered enzyme with the appropriate features to develop into an industrially applied biocatalyst.

Marinobacterium iners (Iizuka and Komagata 1964) comb. nov. arising from the Synonymy of Marinobacterium georgiense González et al. 1997 and Pseudomonas iners Iizuka and Komagata 1964 (Approved Lists 1980).

Although there is documented evidence in the literature that Marinobacterium georgiense González et al. 1997 and Pseudomonas iners Iizuka and Komagata 1964 (Approved Lists 1980) should be treated as heterotypic synonyms, the nomenclatural consequences have not been implemented. Based on the rules of the International Code of Nomenclature of Prokaryotes when Marinobacterium georgiense González et al. 1997 and Pseudomonas iners Iizuka and Komagata 1964 (Approved Lists 1980) are considered to belong to the genus Marinobacterium González et al. 1997, the earliest epithet (from the competing heterotypic synonyms) is to be used for the resulting taxon, i.e., the combination Marinobacterium iners (Iizuka and Komagata 1964) must be created.