Characterization of a Novel Moderately Thermophilic Solvent-Tolerant Esterase Isolated From a Compost Metagenome Library.

A novel esterase, EstCS1, was isolated from a compost metagenomics library. The EstCS1 protein, which consists of 309 amino acid residues with an anticipated molecular mass of 34 kDa, showed high amino acid sequence identities to predicted esterases and alpha/beta hydrolases (59%) from some cultured bacteria and to predicted lipases/esterases from uncultured bacteria. The phylogenetic analysis suggested that the EstCS1 belongs to the hormone-sensitive lipase family of lipolytic enzyme classification and contains a catalytic triad including Ser155-Asp255-His285. The Ser155 residue of the catalytic triad in the EstCS1 was located in the consensus active-site motif, GXSXG. Besides, a conserved HGGG motif placed in an oxyanion hole of the hormone-sensitive lipase family was discovered, too. The EstCS1 demonstrated the highest activity toward p-nitrophenyl propionate (C3) and caproate (C6) and was normally stable up to 60°C with optimal activity at 50°C. In addition, an optimal activity was observed at pH 8, and the EstCS1 possessed its stability within the pH range between 5 and 10. Interestingly, EstCS1 had an outstanding stability in up to 30% (v/v) organic solvents and activity over 50% in the presence of 50% (v/v) acetone, ethanol, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide. The EstCS1 hydrolyzed sterically hindered tertiary alcohol esters of t-butyl acetate and linalyl acetate. Considering the properties, such as the moderate thermostability, stability against organic solvents, and activity toward esters of tertiary alcohols, the EstCS1 will be worthwhile to be used for organic synthesis and related industrial applications.

Enzymatic characterization of a soluble aggregate induced by N-terminal extension to a lipolytic enzyme.

A self-assembling peptide (27PEP) was isolated from an open reading frame (ORF). The ORF consisted of an unknown functional domain and a catalytic (lipolytic and phospholipolytic) domain (MPlaG) on metagenomic fosmid clone. This extension of 27 amino acids prior to the N-terminus of the catalytic domain (27PEP-MPlaG), starting at Met261, produced an aggregate of high molecular weight (> 700 kDa). Compared with MPlaG, 27PEP-MPlaG showed the same temperature and pH effect for maximum activity but was stable in the presence of inhibitors such as EDTA and PMSF. The 27PEP-MPlaG exhibited lower specific activity than that of MPlaG, but when pre-incubated for 30 min at temperatures between 4 and 100 °C, its activity increased at temperatures greater than 40 °C under alkaline conditions and eventually reached the specific activity level of MPlaG at 60 °C. We experimentally determined that the aggregate caused by 27PEP was dissociated at elevated temperatures resulting in an active catalytic monomer. The 27PEP-indued aggregation may be attractive as application tool for improving or engineering of biocatalysts and biomaterials.

Engineered biosynthesis of milbemycins in the avermectin high-producing strain Streptomyces avermitilis.

BACKGROUND: Milbemycins, produced from Streptomyces hygroscopicus subsp. aureolacrimosus and Streptomyces bingchenggensis, are 16-membered macrolides that share structural similarity with avermectin produced from Streptomyces avermitilis. Milbemycins possess strong acaricidal, insecticidal, and anthelmintic activities but low toxicity. Due to the high commercial value of the milbemycins and increasing resistance to the avermectins and their derivatives, it is imperative to develop an efficient combinatorial biosynthesis system exploiting an overproduction host strain to produce the milbemycins and novel analogs in large quantities. RESULTS: The respective replacement of AveA1 and AveA3 (or module 7 in AveA3) of the avermectin polyketide synthase (PKS) in the avermectin high-producing strain S. avermitilis SA-01 with MilA1 and MilA3 (or module 7 in MilA3) of the milbemycin PKS resulted in the production of milbemycins A3, A4, and D in small amounts and their respective C5-O-methylated congener milbemycins B2, B3, and G as major products with total titers of approximately 292 mg/l. Subsequent inactivation of the C5-O-methyltransferase AveD led to a production of milbemycins A3/A4 (the main components of the commercial product milbemectin) in approximately 225 and 377 mg/l in the flask and 5 l fermenter culture, respectively, along with trace amounts of milbemycin D. CONCLUSIONS: We demonstrated that milbemycin biosynthesis can be engineered in the avermectin-producing S. avermitilis by combinatorial biosynthesis with only a slight decrease in its production level. Application of a similar strategy utilizing higher producing industrial strains will provide a more efficient combinatorial biosynthesis system based on S. avermitilis for further enhanced production of the milbemycins and their novel analogs with improved insecticidal potential.

Quantitative analyses of individual sugars in mixture using FRET-based biosensors.

Molecular biosensors were developed and applied to measure individual sugars in biological mixtures such as bacterial culture broths. As the sensing units, four sugar-binding proteins (SBPs for allose, arabinose, ribose, and glucose) were selected from the Escherichia coli genome and connected to a cyan fluorescent protein and yellow fluorescent protein via dipeptide linkers (CFP-L-SBP-YFP). The putative sensors were randomized in the linker region (L) and then investigated with regard to the intensity of fluorescence resonance energy transfer on the binding of the respective sugars. As a result, four representatives were selected from each library and examined for their specificity using 16 available sugars. The apparent dissociation constants of the allose, arabinose, ribose, and glucose sensors were estimated to be 0.35, 0.36, 0.17, and 0.18 µM. Finally, the sugar sensors were applied to monitor the consumption rate of individual sugars in an E. coli culture broth. The individual sugar profiles exhibited a good correlation with those obtained using an HPLC method, confirming that the biosensors offer a rapid and easy-to-use method for monitoring individual sugars in mixed compositions.

Novel metagenome-derived, cold-adapted alkaline phospholipase with superior lipase activity as an intermediate between phospholipase and lipase.

A novel lipolytic enzyme was isolated from a metagenomic library obtained from tidal flat sediments on the Korean west coast. Its putative functional domain, designated MPlaG, showed the highest similarity to phospholipase A from Grimontia hollisae CIP 101886, though it was screened from an emulsified tricaprylin plate. Phylogenetic analysis showed that MPlaG is far from family I.6 lipases, including Staphylococcus hyicus lipase, a unique lipase which can hydrolyze phospholipids, and is more evolutionarily related to the bacterial phospholipase A(1) family. The specific activities of MPlaG against olive oil and phosphatidylcholine were determined to be 2,957 ± 144 and 1,735 ± 147 U mg(-1), respectively, which means that MPlaG is a lipid-preferred phospholipase. Among different synthetic esters, triglycerides, and phosphatidylcholine, purified MPlaG exhibited the highest activity toward p-nitrophenyl palmitate (C(16)), tributyrin (C(4)), and 1,2-dihexanoyl-phosphatidylcholine (C(8)). Finally, MPlaG was identified as a phospholipase A(1) with lipase activity by cleavage of the sn-1 position of OPPC, interfacial activity, and triolein hydrolysis. These findings suggest that MPlaG is the first experimentally characterized phospholipase A(1) with lipase activity obtained from a metagenomic library. Our study provides an opportunity to improve our insight into the evolution of lipases and phospholipases.

Algoriphagus namhaensis sp. nov., isolated from seawater.

A Gram-staining-negative, non-motile, non-spore-forming bacterial strain, DPG-3(T), was isolated from seawater from the South Sea in Korea, and its taxonomic position was investigated using a polyphasic approach. Strain DPG-3(T) grew optimally at 30 °C and in the presence of 2% (w/v) NaCl. In a neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain DPG-3(T) fell within a clade comprising Algoriphagus species and appeared most closely related to Algoriphagus halophilus JC 2051(T) (96.1%16S rRNA gene sequence similarity) and Algoriphagus lutimaris S1-3(T) (96.4%). The type strains of other Algoriphagus species showed 16S rRNA gene sequence similarities of 92.9-96.0% with strain DPG-3(T). The predominant menaquinone of strain DPG-3(T) was MK-7. The major fatty acids were iso-C(15:0) and iso-C(15:0) 2-OH and/or C(16:1)ω7c (summed feature 3). The major polar lipids detected in strain DPG-3(T) were phosphatidylcholine, phosphatidylethanolamine and an unidentified lipid. The genomic DNA G+C content was 44.8 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain DPG-3(T) is considered to represent a novel species of the genus Algoriphagus, for which the name Algoriphagus namhaensis sp. nov. is proposed. The type strain is DPG-3(T) (=KCTC 23419(T)=CCUG 60523(T)).

Tropicimonas aquimaris sp. nov., isolated from seawater, and emended description of the genus Tropicimonas Harwati et al. 2009.

A Gram-staining-negative, aerobic, non-motile and rod-shaped bacterial strain, designated DPG-21(T), was isolated from seawater from the South Sea in Korea, and investigated using a polyphasic taxonomic approach. Strain DPG-21(T) grew optimally at pH 7.0-8.0, at 30 °C and in the presence of 2% (w/v) NaCl. In a neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain DPG-21(T) clustered with Tropicimonas isoalkanivorans B51(T) (with a sequence similarity of 97.1%); the novel strain showed lower 16S rRNA gene sequence similarities (<95.4%) with the other species included in the tree. The mean DNA-DNA relatedness value between strain DPG-21(T) and T. isoalkanivorans DSM 19548(T) was 12%. The predominant ubiquinones of strain DPG-21(T) were Q-10 and Q-9 while C(18:1)ω7c was the strain's major fatty acid. The polar lipid profile of strain DPG-21(T) was similar to that of T. isoalkanivorans DSM 19548(T). The genomic DNA G+C content of the novel strain was 69.6 mol%. Some phenotypic properties and the phylogenetic and genetic data indicated that strain DPG-21(T) was distinct from T. isoalkanivorans and represents a novel species of the genus Tropicimonas, for which the name Tropicimonas aquimaris sp. nov. is proposed. The type strain is DPG-21(T) (=KCTC 23424(T)=CCUG 60524(T)).

Cellulophaga geojensis sp. nov., a member of the family Flavobacteriaceae isolated from marine sand.

A Gram-stain-negative, aerobic, non-flagellated, non-spore-forming, motile (by gliding) bacterial strain, designated M-M6(T), was isolated from marine sand of Geoje island, Korea. Strain M-M6(T) grew optimally at 25 °C, at pH 7.0-8.0 and in the presence of 2 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain M-M6(T) fell within the clade comprising Cellulophaga species, forming a coherent cluster with Cellulophaga lytica ATCC 23178(T) and Cellulophaga fucicola NN015860(T), with which it shared 16S rRNA gene sequence similarities of 98.1 and 98.2 %, respectively. Sequence similarities between strain M-M6(T) and the type strains of other recognized Cellulophaga species were in the range 92.4-93.8 %. Strain M-M6(T) contained MK-6 as the predominant menaquinone and iso-C(15:0), iso-C(15:1) G, iso-C(17:0) 3-OH, and C(16:1)ω7c and/or iso-C(15:0) 2-OH as the major fatty acids. The major polar lipids detected in strain M-M6(T) and the type strains of C. lytica and C. fucicola were two unidentified lipids, one unidentified aminolipid and one unidentified aminophospholipid. The DNA G+C content of strain M-M6(T) was 35.4 mol%. Levels of DNA-DNA relatedness between strain M-M6(T) and C. lytica JCM 8516(T) and C. fucicola JCM 21778(T) were 33 and 35 %, respectively. Differential phenotypic properties and phylogenetic and genetic distinctiveness distinguished strain M-M6(T) from all recognized Cellulophaga species. On the basis of the data presented, strain M-M6(T) is considered to represent a novel species of the genus Cellulophaga, for which the name Cellulophaga geojensis sp. nov. is proposed. The type strain is M-M6(T) ( = KCTC 23498(T) = CCUG 60801(T)).

Pseudorhodobacter aquimaris sp. nov., isolated from seawater, and emended description of the genus Pseudorhodobacter Uchino et al. 2002.

A Gram-stain-negative, aerobic, motile, ovoid bacterial strain, designated HDW-19(T), was isolated from seawater of the west coast of Korea and subjected to a polyphasic taxonomic study. Strain HDW-19(T) grew optimally at pH 7.0-8.0, at 30 °C and in the presence of 2-3% (w/v) NaCl. Bacteriochlorophyll a was not produced by strain HDW-19(T). Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences showed that strain HDW-19(T) clustered with Pseudorhodobacter ferrugineus IAM 12616(T), with which it shared 96.4% similarity. A neighbour-joining phylogenetic tree based on gyrB gene sequences showed that strain HDW-19(T) also clustered with the type strain of P. ferrugineus, sharing 83.0% similarity. Strain HDW-19(T) contained Q-10 as the predominant ubiquinone and C(18:1)ω7c as the major fatty acid. The major polar lipids were phosphatidylcholine, phosphatidylglycerol, three unidentified aminophospholipids and two unidentified aminolipids. The DNA G+C content of strain HDW-19(T) was 60.9 mol%. Differential phenotypic properties, together with phylogenetic distinctiveness, showed that strain HDW-19(T) can be differentiated from P. ferrugineus. On this basis, strain HDW-19(T) is considered to represent a novel species of the genus Pseudorhodobacter, for which the name Pseudorhodobacter aquimaris sp. nov. is proposed. The type strain is HDW-19(T) (=KCTC 23043(T) =CCUG 58879(T)).

A novel family VII esterase with industrial potential from compost metagenomic library.

BACKGROUND: Among the vast microbial genomic resources now available, most microbes are unculturable in the laboratory. A culture-independent metagenomic approach is a novel technique that circumvents this culture limitation. For the screening of novel lipolytic enzymes, a metagenomic library was constructed from compost, and the clone of estCS2 was selected for lipolytic properties on a tributyrin-containing medium. RESULTS: The estCS2 sequence encodes a protein of 570 amino acid residues, with a predicted molecular mass of 63 kDa, and based on amino acid identity it most closely matches (45%) the carboxylesterase from Haliangium ochraceum DSM 14365. EstCS2 belong to family VII, according to the lipolytic enzyme classification proposed by Arpigny and Jaeger, and it retains the catalytic triad Ser245-Glu363-His466 that is typical of an α/ß hydrolase. The Ser245 residue in the catalytic triad of EstCS2 is located in the consensus active site motif GXSXG. The EstCS2 exhibits strong activity toward p-nitrophenyl caproate (C6), and it is stable up to 60°C with an optimal enzymatic activity at 55°C. The maximal activity is observed at pH 9, and it remains active between pH 6-10. EstCS2 shows remarkable stability in up to 50% (v/v) dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). The enzyme has the ability to cleave sterically hindered esters of tertiary alcohol, as well as to degrade polyurethanes, which are widely used in various industries. CONCLUSIONS: The high stability of EstCS2 in organic solvents and its activity towards esters of ketoprofen and tertiary alcohols, and in polyurethane suggests that it has potential uses for many applications in biotransformation and bioremediation.

Roseovarius marinus sp. nov., isolated from seawater.

A Gram-stain-negative, non-motile, ovoid- to rod-shaped bacterium, designated HDW-9(T), belonging to the class Alphaproteobacteria, was isolated from seawater of the Yellow Sea, Korea. Strain HDW-9(T) grew optimally at pH 7.0-8.0, at 30 °C and with 2-3 % (w/v) NaCl. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences showed that strain HDW-9(T) clustered with Roseovarius crassostreae CV919-312(T), with which it exhibited 95.5 % 16S rRNA gene sequence similarity. Strain HDW-9(T) exhibited 92.5-94.7 % 16S rRNA gene sequence similarity with the other type strains of species of the genus Roseovarius. Strain HDW-9(T) contained Q-10 as the predominant ubiquinone and C(18 : 1)ω7c as the major fatty acid. The DNA G+C content was 58.3 mol%. Differential phenotypic properties distinguished strain HDW-9(T) from the other members of the genus Roseovarius. Strain HDW-9(T) is considered to represent a novel species of the genus Roseovarius, for which the name Roseovarius marinus sp. nov. is proposed. The type strain is HDW-9(T) (=KCTC 22805(T) =CCUG 58403(T)).

Ruegeria faecimaris sp. nov., isolated from a tidal flat sediment.

A Gram-negative-staining, non-motile and rod-shaped bacterial strain, HD-28(T), was isolated from a tidal flat of the Yellow Sea, Korea. Strain HD-28(T) grew optimally at pH 7.0-8.0 and 30 °C in the presence of 2-3 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain HD-28(T) was most closely related to species of the genus Ruegeria and exhibited 95.5-96.9 % 16S rRNA gene sequence similarity to the type strains of Ruegeria species. A neighbour-joining phylogenetic tree based on gyrB gene sequences also showed that strain HD-28(T) fell within the cluster comprising recognized species of the genus Ruegeria, showing 77.5-83.9 % sequence similarity. Strain HD-28(T) contained Q-10 as the predominant ubiquinone and C(18 : 1)ω7c as the major fatty acid. The major polar lipids detected in strain HD-28(T) were phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid and two unidentified lipids. The DNA G+C content was 57.9 mol%. Differential phenotypic properties, together with phylogenetic distinctiveness, demonstrated that strain HD-28(T) could be distinguished from recognized species of the genus Ruegeria. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain HD-28(T) is considered to represent a novel species of the genus Ruegeria, for which the name Ruegeria faecimaris sp. nov. is proposed. The type strain is HD-28(T) ( = KCTC 23044(T) = CCUG 58878(T)).

Lysobacter dokdonensis sp. nov., isolated from soil.

A Gram-negative, non-motile, rod-shaped bacterial strain, DS-58(T), was isolated from a soil sample from Dokdo, an island of Korea, and its taxonomic position was investigated. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DS-58(T) fell within the family Xanthomonadaceae. The isolate showed 96.9 % 16S rRNA gene sequence similarity with its closest phylogenetic neighbour, Lysobacter niastensis GH41-7(T), and 93.4-95.7 % 16S rRNA gene sequence similarity with other members of the genus Lysobacter. Strain DS-58(T) contained Q-8 as the predominant ubiquinone and iso-C(16 : 0), iso-C(15 : 0) and iso-C(17 : 1)ω9c as the major fatty acids. The DNA G+C content was 68.1 mol%. Strain DS-58(T) could be distinguished phenotypically from type strains of closely related species of the genus Lysobacter and phylogenetically from all members of the genus Lysobacter. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain DS-58(T) is considered to represent a novel species of the genus Lysobacter, for which the name Lysobacter dokdonensis sp. nov. is proposed. The type strain is DS-58(T) ( = KCTC 12822(T)  = DSM 17958(T)).

Mucilaginibacter boryungensis sp. nov., isolated from soil.

A Gram-stain-negative, non-motile, non-spore-forming bacterial strain, BDR-9(T), was isolated from soil collected from Boryung on the west coast of the Korean peninsula, and its taxonomic position was investigated by using a polyphasic study. Strain BDR-9(T) grew optimally at 25 °C, at pH 6.0-7.5 and in the absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain BDR-9(T) fell within the clade comprising species of the genus Mucilaginibacter within the phylum Bacteroidetes. 16S rRNA gene sequence similarity values between strain BDR-9(T) and the type strains of species of the genus Mucilaginibacter were in the range 94.0-95.6 %. Strain BDR-9(T) contained MK-7 as the predominant menaquinone and iso-C(15 : 0) and C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH as the major fatty acids. The DNA G+C content was 44.3 mol%. Differential phenotypic properties and phylogenetic distinctiveness of strain BDR-9(T) demonstrated that this strain is distinguishable from species of the genus Mucilaginibacter. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BDR-9(T) is considered to represent a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter boryungensis sp. nov. is proposed. The type strain is BDR-9(T) ( = KCTC 23157(T)  = CCUG 59599(T)).

Paraperlucidibaca baekdonensis gen. nov., sp. nov., isolated from seawater.

A Gram-negative, non-spore-forming, rod-shaped bacterial strain, RL-2(T), was isolated from seawater of the East Sea in Korea and was subjected to a polyphasic taxonomic study. Strain RL-2(T) grew optimally at pH 7.5-8.0, at 20 °C and in the absence of NaCl. Phylogenetic trees based on 16S rRNA gene sequence analysis showed that strain RL-2(T) forms a cluster with Perlucidibaca piscinae IMCC1704(T) and various uncultured and unidentified gammaproteobacteria. Strain RL-2(T) exhibited 16S rRNA gene sequence similarity values of 96.1 % to Perlucidibaca piscinae IMCC1704(T) and 93.7-99.7 % to the uncultured bacterial clones belonging to the cluster and an unidentified gammaproteobacterium. The fatty acid profile of strain RL-2(T) was similar to that of Perlucidibaca piscinae IMCC1704(T), but the predominant ubiquinone type (Q-11) of strain RL-2(T) was different from that (Q-8) of Perlucidibaca piscinae IMCC1704(T). The DNA G+C content of strain RL-2(T) was 61.3 mol%. On the basis of phylogenetic, chemotaxonomic and phenotypic data, strain RL-2(T) is considered to represent a novel species of a new genus in the family Moraxellaceae, for which the name Paraperlucidibaca baekdonensis gen. nov., sp. nov. is proposed. The type strain of Paraperlucidibaca baekdonensis is RL-2(T) ( = KCTC 23145(T)  = CCUG 59307(T)).

Planococcus salinarum sp. nov., isolated from a marine solar saltern, and emended description of the genus Planococcus.

A Gram-positive, non-motile and coccoid-, short rod- or rod-shaped bacterial strain, ISL-16(T), was isolated from a marine solar saltern in Korea and its taxonomic position was investigated using a polyphasic taxonomic approach. Strain ISL-16(T) grew optimally at pH 7.0-8.0, at 30 degrees C and in the presence of 2 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ISL-16(T) joined the cluster comprising species of the genus Planococcus. Its 16S rRNA gene sequence contained the same signature nucleotides as those defined for the genus Planococcus. Strain ISL-16(T) exhibited 16S rRNA gene sequence similarity values of 96.9-98.2 % to the type strains of species of the genus Planococcus. Strain ISL-16(T) contained MK-8 and MK-7 as the predominant menaquinones and anteiso-C(15 : 0), C(16 : 1)omega7c alcohol and anteiso-C(17 : 0) as the major fatty acids. The DNA G+C content was 48.3 mol%. DNA-DNA relatedness values between strain ISL-16(T) and the type strains of species of the genus Planococcus were 15-28 %. Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness, enabled strain ISL-16(T) to be differentiated from recognized species of the genus Planococcus. On the basis of the data presented, strain ISL-16(T) is considered to represent a novel species of the genus Planococcus, for which the name Planococcus salinarum sp. nov. is proposed. The type strain is ISL-16(T) (=KCTC 13584(T)=CCUG 57753(T)). An emended description of the genus Planococcus is also given.

Jannaschia seohaensis sp. nov., isolated from a tidal flat sediment.

A Gram-negative, motile and pleomorphic bacterial strain, SMK-146(T), was isolated from a tidal flat sediment of the Yellow Sea, Korea, and its taxonomic position was investigated. Strain SMK-146(T) grew optimally at pH 7.0-8.0 and 30 degrees C. It contained Q-10 as the predominant ubiquinone and C(18 : 1)omega7c and 11-methyl C(18 : 1)omega7c as the major fatty acids. The major polar lipids were phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 68.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SMK-146(T) belongs to the genus Jannaschia. Strain SMK-146(T) exhibited 16S rRNA gene sequence similarity values of 95.3-97.0 % to the type strains of the five recognized Jannaschia species. The mean DNA-DNA relatedness value between strain SMK-146(T) and Jannaschia seosinensis KCCM 42114(T), the closest phylogenetic neighbour, was 17 %. Differential phenotypic properties also revealed that strain SMK-146(T) differs from the recognized Jannaschia species. On the basis of phenotypic, phylogenetic and genetic data, strain SMK-146(T) represents a novel species of the genus Jannaschia, for which the name Jannaschia seohaensis sp. nov. is proposed. The type strain is SMK-146(T) (=KCTC 22172(T) =CCUG 55326(T)).

Algoriphagus lutimaris sp. nov., isolated from a tidal flat sediment.

A Gram-negative, non-motile, non-spore-forming bacterial strain, S1-3(T), was isolated from a tidal flat sediment on the west coast of Korea and its taxonomic position was investigated. Strain S1-3(T) grew optimally at 30 degrees C and in the presence of 2 % (w/v) NaCl. Strain S1-3(T) contained MK-7 as the predominant menaquinone and C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH and iso-C(15 : 0) as the major fatty acids. The DNA G+C content was 41.4 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain S1-3(T) fell within the clade comprising Algoriphagus species, clustering with Algoriphagus halophilus IMSNU 14013(T), with which it exhibited 99.6 % 16S rRNA gene sequence similarity. The 16S rRNA gene sequence similarity between strain S1-3(T) and the type strains of other Algoriphagus species was 94.0-97.1 %. Differential phenotypic properties and phylogenetic and genetic distinctiveness of strain S1-3(T) demonstrated that this strain is distinguishable from the other Algoriphagus species as well as A. halophilus. On the basis of phenotypic, chemotaxonomic, phylogenetic and genetic data, strain S1-3(T) is considered to represent a novel species of the genus Algoriphagus, for which the name Algoriphagus lutimaris sp. nov. is proposed. The type strain is S1-3(T) (=KCTC 22630(T) =CCUG 57608(T)).

Salimicrobium flavidum sp. nov., isolated from a marine solar saltern.

A Gram-variable-staining, motile and coccoid-, ovoid- or rod-shaped bacterium, strain ISL-25(T), was isolated from a marine solar saltern of the Yellow Sea, Korea, and its taxonomic position was investigated by means of a polyphasic study. Strain ISL-25(T) grew optimally at pH 7.0-8.0 and 30-37 degrees C. Strain ISL-25(T) contained meso-diaminopimelic acid as the cell-wall peptidoglycan, MK-7 as the predominant menaquinone and anteiso-C(15 : 0), anteiso-C(17 : 0) and iso-C(16 : 0) as the major fatty acids. The DNA G+C content was 49.3 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ISL-25(T) belongs to the genus Salimicrobium. The similarity values between the 16S rRNA gene sequence of strain ISL-25(T) and those of the type strains of the three currently recognized Salimicrobium species were 97.6-98.3 %. Mean DNA-DNA relatedness values between strain ISL-25(T) and the type strains of the genus Salimicrobium were 9-15 %. Differential phenotypic properties of strain ISL-25(T), together with the phylogenetic and genetic distinctiveness, revealed that this strain could be differentiated from other Salimicrobium species. Therefore, strain ISL-25(T) represents a novel species within the genus Salimicrobium, for which the name Salimicrobium flavidum sp. nov. is proposed. The type strain is ISL-25(T) (=KCTC 13260(T)=CCUG 56755(T)).

Seohaeicola saemankumensis gen. nov., sp. nov., isolated from a tidal flat.

A Gram-negative, non-motile and rod-, oval- or coccoid-shaped bacterial strain, SD-15(T), was isolated from a tidal flat of the Yellow Sea, Korea. The novel strain, which was phylogenetically closely related to the genera Phaeobacter, Leisingera and Marinovum, was studied using a polyphasic taxonomic approach. Strain SD-15(T) grew optimally at pH 7.0-8.0 and 30 degrees C in the presence of 2 % (w/v) NaCl. It contained Q-10 as the predominant ubiquinone and C(18 : 1)omega7c and 11-methyl C(18 : 1)omega7c as the major fatty acids. The major polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and an unidentified lipid. The DNA G+C content was 63.4 mol%. Strain SD-15(T) exhibited the highest 16S rRNA gene sequence similarity values (95.1-96.4 %) to the type strains of species of the genus Phaeobacter, Leisingera methylohalidivorans MB2(T) and Marinovum algicola ATCC 51440(T). Strain SD-15(T) could be differentiated from members of the genera Phaeobacter, Leisingera and Marinovum by differences in the contents of some fatty acids, by the absence of aminolipid and by differences in some phenotypic properties. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain SD-15(T) represents a new genus and novel species, for which the name Seohaeicola saemankumensis gen. nov., sp. nov. is proposed. The type strain of the type species is Seohaeicola saemankumensis SD-15(T) (=KCTC 22175(T)=CCUG 55328(T)).