Lutimonas zeaxanthinifaciens sp. nov., a zeaxanthin-producing marine bacterium isolated from coastal sediment.

A Gram-stain-negative, yellow-pigmented, strictly aerobic, non-flagellated, motile by gliding, rod-shaped bacterium, designated strain YSD2104T, was isolated from a coastal sediment sample collected from the southeastern part of the Yellow Sea. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain YSD2104T was closely related to three type strains, Lutimonas vermicola IMCC1616T (97.4 %), Lutimonas saemankumensis SMK-142T (96.9 %), and Lutimonas halocynthiae RSS3-C1T (96.8 %). Strain YSD2104T has a single circular chromosome of 3.54 Mbp with a DNA G+C content of 38.3 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain YSD2104T and the three type strains (L. vermicola IMCC1616 T, L. saemankumensis SMK-142T, and L. halocynthiae RSS3-C1T) were 74.0, 86.2 and 73.6 %, and 17.9, 30.3 and 17.8 %, respectively. Growth was observed at 20-30 °C (optimum, 30 °C), at pH 6.5-8.5 (optimum, pH 7.0), and with NaCl concentrations of 1.5-3.5 % (optimum, 2.5 %). The major carotenoid was zeaxanthin, and flexirubin-type pigment was not produced. The major respiratory quinone was menaquinone-6. The major fatty acids (>10 %) were iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), and summed feature 9 (iso-C17 : 1 ω9c and/or 10-methyl C16 : 0). The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, two unidentified aminolipids, and eight unidentified lipids. Conclusively, based on this polyphasic approach, we classified strain YSD2104T (=KCTC 102008T=JCM 36287T) as representing a novel species of the genus Lutimonas and proposed the name Lutimonas zeaxanthinifaciens sp. nov.

Falsirhodobacter algicola sp. nov., a member of the Rhodobacteraceae isolated from the marine red algae Grateloupia sp.

A Gram-negative, pale yellow-pigmented, non-flagellated, motile, rod-shaped and aerobic bacterium, designated strain PG104T, was isolated from red algae Grateloupia sp. collected from the coastal area of Pohang, Republic of Korea. Growth of strain PG104T was observed at 15-35 °C (optimum, 30 °C), pH 6.0-10.0 (optimum, pH 7.5-8.0) and in the presence of 0-8.0 % (w/v) NaCl (optimum, 5.0 %). The predominant fatty acids included C17 : 0, C18 : 0, 11-methyl C18 : 1 ω7c and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and the major respiratory quinone was Q-10. Polar lipids included phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified lipid and one unidentified aminolipid. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain PG104T formed a phylogenetic lineage with members of the genus Falsirhodobacter and exhibited 16S rRNA gene sequence similarities of 97.1 and 96.6 % to Falsirhodobacter deserti W402T and Falsirhodobacter halotolerans JA744T, respectively. The complete genome of strain PG104T consisted of a single circular chromosome of approximately 2.8 Mbp with five plasmids. Based on polyphasic taxonomic data, strain PG104T represents a novel species in the genus Falsirhodobacter, for which the name Falsirhodobacter algicola sp. nov. is proposed. The type strain of Falsirhodobacter algicola is PG104T (=KCTC 82230T=JCM 34380T).

Ferrimonas lipolytica sp. nov., a facultatively anaerobic bacterium isolated from seawater.

A Gram-stain-negative, motile, facultatively anaerobic rod-shaped bacterium with a polar flagellum, designated strain S7T was isolated from seawater sample collected at Uljin marina, in the East Sea of the Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain S7T was affiliated with members of genus Ferrimonas, showing the highest sequence similarities to the type strains Ferrimonas senticii P2S11T (95.7 %), Ferrimonas balearica PATT (95.7 %) and Ferrimonas pelagia CBA4601T (95.1 %). The genome was 4.13 Mbp with a DNA G+C content of 49.4 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between S7T and F. senticii P2S11T and F. balearica PATT yielded ANI values of 71.9 and 70.7 %, and dDDH values of 15.1 and 13.9 %, respectively. The genome of S7T was predicted to encode triacylglycerol lipase, phospholipase A1/A2 and lysophospholipase as well as esterase involved in lipolytic processes. Growth was observed at 8-31 °C (optimum 27 °C), at pH 7-9 (optimum pH 7), and with 1-6 % NaCl (optimum 2 %). The respiratory quinones were MK-7 and Q-7 and the major fatty acids (>10 %) were C16 : 0, C16 : 1ω9c, C17 : 1ω8c, and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The major polar lipids were identified as phosphatidylethanolamine, phosphatidylglycerol, two unidentified phospholipids, and three unidentified lipids. On the basis of the results of this polyphasic analysis, it was determined that the strain represents a novel species of the genus Ferrimonas, for which the name Ferrimonas lipolytica sp. nov. is proposed. The type strain is S7T (=KCTC 72490T=JCM 33793T).

Tamlana carrageenivorans sp. nov., a carrageenan-degrading bacterium isolated from seawater.

A Gram-stain-negative, aerobic, non-motile, rod-shaped, agarolytic and carrageenolytic bacterial strain, designated UJ94T, was isolated from seawater of Uljin in the Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain UJ94T shared sequence similarities of 98.4, 96.1 and 95.4 % with Tamlana agarivorans JW-26T, Tamlana sedimentorum KMM 9545T and Tamlana crocina HST1-43T, respectively. Growth of strain UJ94T was observed at 4-37 °C and pH 6.5-8.0 in the presence of 2-9 % (w/v) NaCl. The major fatty acids of strain UJ94T were iso-C15 : 0, summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and iso-C17 : 0 3-OH; MK-6 was the predominant menaquinone. Phosphatidylethanolamine, two unidentified aminolipids and five unidentified lipids were detected as major polar lipids. The whole circular genome comprised 4 116 543 bp and had a G+C content of 35.2 mol%. The ranges of average nucleotide identity and in silico DNA-DNA hybridization estimated by genome-to-genome distance were 90.6-74.2 % and 47.6-14.6 %, respectively, with the type strains of T. agarivorans and T. sedimentorum. The present polyphasic study, including phylogenetic, chemotaxonomic, biochemical and genomic data, suggested that strain UJ94T represents a novel species of the genus Tamlana, for which the name Tamlana carrageenivorans sp. nov. is proposed. The type strain is UJ94T (=KCTC 62451T=NBRC 113234T).

Complete Genome Sequence of Marinobacterium aestuarii ST58-10T, a Benzene-Degrading Bacterium Isolated from Estuarine Sediment.

Marinobacterium aestuarii ST58-10T was identified as a benzene-degrading aerobic bacterium isolated from estuarine sediment in the Republic of Korea. The genome of strain ST58-10T was found to be composed of a single circular chromosome (5,191,608 bp) with a G+C content of 58.78% and harboring 4,473 protein-coding genes. The assembled sequence data will help elucidate potential metabolic pathways and mechanisms responsible for the hydrocarbon-degrading ability of M. aestuarii ST58-10T.

Marinobacterium aestuarii sp. nov., a benzene-degrading marine bacterium isolated from estuary sediment.

A Gram-stain-negative, aerobic, motile, flagellated rod-shaped bacterium, designated ST58-10T, was isolated from an estuarine sediment in the Republic of Korea. The strain was able to degrade benzene. Growth of strain ST58-10T was observed at 4-35 °C (optimum, 20-25 °C), pH 5-9 (optimum, pH 7-8) and 1-8 % NaCl (optimum, 3 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ST58-10T formed a phyletic lineage within the genus Marinobacterium of the family Oceanospirillaceae. Strain ST58-10T was most closely related to Marinobacterium profundum PAMC 27536T (99.6 %) and Marinobacterium rhizophilum CL-YJ9T (98.3 %), and to other members of the genus Marinobacterium(94.5-91.5 %). However, the mean value estimated by using the Genome-to-Genome Distance Calculator was 50.6±7.4 % with M. profundum PAMC 27536T and 30.9±2.8 with M. rhizophilum CL-YJ9T, respectively. An average nucleotide identity value was 89.0 % with M. profundum PAMC 27536T and 85.6 % with M. rhizophilum CL-YJ9T, respectively. The major fatty acids of strain ST58-10T were summed feature 3 (comprising C16 : 1ω7c/C16 : 1ω6c), summed feature 8 (comprising C18 : 1 ω7c/C18 : 1ω6c), C16 : 0 and C10 : 0 3-OH, and contained ubiquinone (Q-8) as the sole isoprenoid quinone. Phosphatidylethanolamine, phosphatidylglycerol, three unidentified aminolipids, an unidentified glycolipid and an unidentified lipid were detected as polar lipids. The DNA G+C content of strain ST58-10T was 58.78 mol%. On the basis of the phenotypic, chemotaxonomic and molecular properties, strain ST58-10T represents a novel species of the genus Marinobacterium, for which the name Marinobacterium aestuarii sp. nov. is proposed. The type strain is ST58-10T (=KCTC 52193T=NBRC 112103T).

Echinicola strongylocentroti sp. nov., isolated from a sea urchin Strongylocentrotus intermedius.

A yellowish-orange-pigmented marine bacterium, designated MEBiC08714T was isolated from a sea urchin Strongylocentrotus intermedius collected at the west edge of the East Sea of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that MEBiC08714T was affiliated with the genus Echinicola and that the strain was most closely related to Echinicola vietnamensis KCTC 12713T (96.9 %), followed by 'Echinicola shivajiensis' JCM 17847 (96.3 %), Echinicola jeungdonensis KCTC 23122T (96.1 %), and Echinicola pacifica KCTC 12368T (95.0 %). Cells were strictly aerobic, Gram-stain-negative, rod shaped, flexirubin-type pigments-negative and motile by gliding. Growth was observed at 20-35 °C (optimum 25 °C), at pH 6-11 (optimum pH 7.0), and with 0-13 % NaCl (optimum 2 %). This strain was able to hydrolyze agar and starch. The polar lipids of MEBiC08714T contained phosphatidylethanolamine, an unidentified phospholipid, and four unidentified lipids. The major fatty acids were iso-C15 : 0 (27.5 %), iso-C17 : 0 3-OH (11.5 %), anteiso-C15 : 0 (5.2 %), summed feature 3 (C16:1ω7c and/or C16:1ω6c, 20.3 %) and summed feature 9 (iso-C17 : 1ω9c and/or10-methyl C16 : 06, 6.3 %). The DNA G+C content was 44.2 mol% and the major respiratory quinone was MK-7. On the basis of these polyphasic taxonomic data, MEBiC08714T represents a novel species of the genus Echinicola, for which the name Echinicola strongylocentroti sp. nov. is proposed. The type strain is MEBiC08714T (=KCTC 52052T=JCM 31307T).

A biological process effective for the conversion of CO-containing industrial waste gas to acetate.

Acetogens have often been observed to be inhibited by CO above an inhibition threshold concentration. In this study, a two-stage culture consisting of carboxydotrophic archaea and homoacetogenic bacteria is found to be effective in converting industrial waste gas derived from a steel mill process. In the first stage, Thermococcus onnurineus could grow on the Linz-Donawitz converter gas (LDG) containing ca. 56% CO as a sole energy source, converting the CO into H2 and CO2. Then, in the second stage, Thermoanaerobacter kivui could grow on the off-gas from the first stage culture, consuming the H2 and CO in the off-gas completely and producing acetate as a main product. T. kivui alone could not grow on the LDG gas. This work represents the first demonstration of acetate production using steel mill waste gas by a two-stage culture of carboxydotrophic hydrogenogenic microbes and homoacetogenic bacteria.

Formate-driven growth coupled with H(2) production.

Although a common reaction in anaerobic environments, the conversion of formate and water to bicarbonate and H(2) (with a change in Gibbs free energy of ΔG° = +1.3 kJ mol(-1)) has not been considered energetic enough to support growth of microorganisms. Recently, experimental evidence for growth on formate was reported for syntrophic communities of Moorella sp. strain AMP and a hydrogen-consuming Methanothermobacter species and of Desulfovibrio sp. strain G11 and Methanobrevibacter arboriphilus strain AZ. The basis of the sustainable growth of the formate-users is explained by H(2) consumption by the methanogens, which lowers the H(2) partial pressure, thus making the pathway exergonic. However, it has not been shown that a single strain can grow on formate by catalysing its conversion to bicarbonate and H(2). Here we report that several hyperthermophilic archaea belonging to the Thermococcus genus are capable of formate-oxidizing, H(2)-producing growth. The actual ΔG values for the formate metabolism are calculated to range between -8 and -20 kJ mol(-1) under the physiological conditions where Thermococcus onnurineus strain NA1 are grown. Furthermore, we detected ATP synthesis in the presence of formate as a sole energy source. Gene expression profiling and disruption identified the gene cluster encoding formate hydrogen lyase, cation/proton antiporter and formate transporter, which were responsible for the growth of T. onnurineus NA1 on formate. This work shows formate-driven growth by a single microorganism with protons as the electron acceptor, and reports the biochemical basis of this ability.

Marinoscillum gen. nov., a member of the family 'Flexibacteraceae', with Marinoscillum pacificum sp. nov. from a marine sponge and Marinoscillum furvescens nom. rev., comb. nov.

A novel strain, designated MRN461(T), was isolated from a marine sponge in Micronesia. The 16S rRNA gene sequence of the isolate showed 95.6 % similarity with that of 'Microscilla furvescens' IFO (now NBRC) 15994. Phylogenetic analysis revealed that the isolate and 'Microscilla furvescens' IFO 15994 formed a distinct phyletic line within the family 'Flexibacteraceae'. Cells of strain MRN461(T) were Gram-negative, long filamentous rods, motile by gliding. Growth was observed at 15-40 degrees C (optimum, 33 degrees C), at pH 5.0-9.5 (optimum, pH 7.5) and in the presence of 0.5-7.0 % sea salts (optimum, 2.5 %). The major isoprenoid quinone was MK-7. The dominant fatty acids were summed feature 3 (comprising iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c; 34.8 %), C(16 : 1)omega5c (21.6 %) and iso-C(16 : 1) (19.8 %). The DNA G+C content was 41.5 mol%. On the basis of evidence from our polyphasic taxonomic study, strain MRN461(T) is classified within a novel genus and species in the family 'Flexibacteraceae', for which the name Marinoscillum pacificum gen. nov., sp. nov. is proposed. The type strain of Marinoscillum pacificum is strain MRN461(T) (=KCCM 42325(T) =JCM 14064(T)). The misclassified species '[Microscilla] furvescens' is transferred to the new genus as Marinoscillum furvescens (ex Lewin 1969) nom. rev., comb. nov., with LMG 13023(T) (=DSM 4134(T) =ATCC 23129(T) =NBRC 15994(T)) as the type strain.

Monitoring nutrient impact on bacterial community composition during bioremediation of anoxic PAH-contaminated sediment.

Marine harbor sediments are frequently polluted with significant amount of polycyclic aromatic hydrocarbons (PAHs) some of which are naturally toxic, recalcitrant, mutagenic, and carcinogenic. To stimulate biodegradation of PAHs in PAH-contaminated sediments collected from near Gwangyang Bay, Korea, lactate was chosen as a supplementary carbonaceous substrate. Sediment packed into 600 ml air-tight jar was either under no treatment condition or lactate amended condition (1%, w/v). Microbial community composition was monitored by bacteria-specific and archaea-specific PCR-terminal restriction fragment length polymorphism (T-RFLP), in addition to measuring the residual PAH concentration. Results showed that lactate amendment enhanced biodegradation rate of PAHs in the sediment by 4 to 8 times, and caused a significant shift in archaebacterial community in terms of structure and diversity with time. Phylogenetic analysis of 23 archaeal clones with distinctive RFLP patterns among 288 archaeal clones indicated that majority of the archaeal members were closest to unculturable environmental rDNA clones from hydrocarbon-contaminated and/or methanogenesis-bearing sediments. Lactate amendment led to the enrichment of some clones that were most closely related to PAH-degrading Methanosarcina species. These results suggest a possible contribution of methanogenic community to PAH degradation and give us more insights on how to effectively remediate PAH-contaminated sediments.

Description of Croceitalea gen. nov. in the family Flavobacteriaceae with two species, Croceitalea eckloniae sp. nov. and Croceitalea dokdonensis sp. nov., isolated from the rhizosphere of the marine alga Ecklonia kurome.

Two novel bacterial strains, designated DOKDO 025(T) and DOKDO 023(T), were isolated on Dokdo Island, Korea, from the rhizosphere of the brown alga Ecklonia kurome. The strains were subjected to a polyphasic taxonomy study and were found to be Gram-negative, aerobic, rod-shaped, non-motile and orange-coloured. The isolates shared 96.3 % 16S rRNA gene sequence similarity. They showed 93.8-95.6 % 16S rRNA gene sequence similarity with respect to members of the genus Muricauda in the family Flavobacteriaceae, but formed a distinct phyletic line. Moreover, the cellular appendages reported for all Muricauda species were absent from strains DOKDO 025(T) and DOKDO 023(T). The predominant cellular fatty acids of strain DOKDO 025(T) were iso-C(15 : 0), iso-C(15 : 1) and one with an equivalent chain-length of 13.565 and those of strain DOKDO 023(T) were iso-C(15 : 0), iso-C(15 : 1) and iso-C(17 : 0) 3-OH. The DNA G+C content of strains DOKDO 025(T) and DOKDO 023(T) were 59.5 and 66.5 mol%, respectively, higher than any values found in recognized members of the family Flavobacteriaceae. The major respiratory quinone was MK-6. On the basis of evidence from the polyphasic study, strains DOKDO 025(T) and DOKDO 023(T) represent two novel species in a new genus, Croceitalea gen. nov., for which the names Croceitalea eckloniae sp. nov. (the type species) and Croceitalea dokdonensis sp. nov. are proposed. The type strain of Croceitalea eckloniae sp. nov. is DOKDO 025(T) (=KCCM 42309(T) =JCM 13827(T)) and that of Croceitalea dokdonensis sp. nov. is DOKDO 023(T) (=KCCM 42308(T) =JCM 13826(T)).

Critical factors to high thermostability of an alpha-amylase from hyperthermophilic archaeon Thermococcus onnurineus NA1.

Genomic analysis of a hyperthermophilic archaeon, Thermococcus onnurineus NA1 [1], revealed the presence of an open reading frame consisting of 1,377 bp similar to alpha-amylases from Thermococcales, encoding a 458-residue polypeptide containing a putative 25-residue signal peptide. The mature form of the alpha-amylase was cloned and the recombinant enzyme was characterized. The optimum activity of the enzyme occurred at 80 degrees C and pH 5.5. The enzyme showed a liquefying activity, hydrolyzing maltooligosaccharides, amylopectin, and starch to produce mainly maltose (G2) to maltoheptaose (G7), but not pullulan and cyclodextrin. Surprisingly, the enzyme was not highly thermostable, with half-life (t(1/2)) values of 10 min at 90 degrees C, despite the high similarity to alpha-amylases from Pyrococcus. Factors affecting the thermostability were considered to enhance the thermostability. The presence of Ca2+ seemed to be critical, significantly changing t(1/2) at 90 degrees C to 153 min by the addition of 0.5 mM Ca2+. On the other hand, the thermostability was not enhanced by the addition of Zn2+ or other divalent metals, irrespective of the concentration. The mutagenetic study showed that the recovery of zinc-binding residues (His175 and Cys189) enhanced the thermostability, indicating that the residues involved in metal binding is very critical for the thermostability.

Flagellimonas eckloniae gen. nov., sp. nov., a mesophilic marine bacterium of the family Flavobacteriaceae, isolated from the rhizosphere of Ecklonia kurome.

A marine bacterium, DOKDO 007(T), was isolated from the rhizosphere of the marine alga Ecklonia kurome collected from Dokdo Island, Korea, in October 2004. The strain produced orange-coloured colonies on marine agar 2216. 16S rRNA gene sequence analysis indicated that the novel isolate belonged to the family Flavobacteriaceae and showed relatively high sequence similarities with members of the genus Muricauda (92.0-94.0 %). Phylogenetic analysis based on nearly complete 16S rRNA gene sequences revealed that the novel isolate shared a lineage with members of the genera Muricauda and Costertonia. Cells were aerobic, Gram-negative rods producing non-diffusible carotenoid pigments. In contrast to all other members of the family Flavobacteriaceae, cells of DOKDO 007(T) were motile by means of a polar flagellum. Optimal growth occurred in the presence of 3.5-4 % (w/v) sea salts (corresponding to 2.7-3.1 % NaCl), at pH 8 and at temperatures of 26-29 degrees C. The novel strain required Ca(2+) ions in addition to NaCl for growth. The dominant fatty acids were iso-15 : 0, iso-15 : 1omega10c and 10-methyl-16 : 0. The major respiratory quinone was MK-6. The DNA G+C content was 56.3 mol%, an unusually high value for members of the family Flavobacteriaceae. On the basis of these polyphasic taxonomic data, strain DOKDO 007(T) should be classified as representing a new genus and novel species in the family Flavobacteriaceae, for which the name Flagellimonas eckloniae gen. nov., sp. nov. is proposed. The type strain is DOKDO 007(T) (=KCCM 42307(T)=JCM 13831(T)).

Cloning, expression, and characterization of a DNA ligase from a hyperthermophilic archaeon Thermococcus sp.

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed an ORF of 1689 bases encoding 562 amino acids that showed a high similarity to DNA ligases from other hyperthermophilic archaea. The ligase, which was designated TNA1_lig (Thermococcus sp. NA1 ligase), was cloned and expressed in Escherichia coli. The recombinant TNA1_lig was purified by metal affinity chromatography. The optimum ligase activity of the recombinant TNA1_lig occurred at 80 degrees C and pH 7.5. The enzyme was activated by MgCl2 and ZnCl2 but was inhibited by MnCl2 and NiCl2. Additionally, the enzyme was activated by either ATP or NAD+.