Search

Two Gram-positive, round-spore-forming, rod-shaped, halophilic bacterial strains, 5B73CT and 5B133E, were isolated from field soil in Kakegawa, Shizuoka, Japan, and were characterized taxonomically using a polyphasic approach. These two strains were found to comprise strictly aerobic, motile rods that formed subterminal endospores. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains 5B73CT and 5B133E are phylogenetically affiliated to the genus Virgibacillus, exhibiting sequence similarities of 94.1–96.4 % with respect to the type strains of Virgibacillus species. The DNA G+C contents of strains 5B73CT and 5B133E were 42.6 and 42.3 mol%, respectively. The cell-wall peptidoglycan type (meso-diaminopimelic acid), the major cellular fatty acids (anteiso-C15 : 0, iso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0) and the quinone type (MK-7) of the isolates support their affiliation to the genus Virgibacillus. On the basis of their genotypic and phenotypic characteristics, the isolates represent a novel species of the genus Virgibacillus, for which the name Virgibacillus halophilus sp. nov. is proposed. The type strain is 5B73CT (=IAM 15308T=KCTC 13935T).

The taxonomic positions of two bacterial strains, MBIC01146(T) and MBIC01099, isolated from marine environments of Japan and Palau, respectively, were determined by using a polyphasic approach. The bacteria were aerobic, motile, Gram-negative rods and formed star-shaped aggregations. The G+C content of the genomic DNA ranged from 58.5 to 58.7 mol%. The major respiratory quinone was ubiquinone-10 and the predominant cellular fatty acids were C(16 : 0), C(18 : 1)omega6c and C(18 : 1)omega7c. 16S rRNA gene sequence analyses based on both neighbour-joining and maximum-parsimony methods revealed that strains MBIC01146(T) and MBIC01099 were positioned within the cluster comprising Ruegeria atlantica and Silicibacter lacuscaerulensis within subgroup alpha-3 of the Proteobacteria. The phenotypic and chemotaxonomic characteristics of the novel strains were similar to those of Ruegeria atlantica; however, DNA-DNA hybridization tests showed that the isolates represented an independent species. The isolates could be differentiated from Ruegeria atlantica based on several characteristics. Therefore, strains MBIC01146(T) and MBIC01099 are considered to represent a novel species of the genus Ruegeria, for which the name Ruegeria mobilis sp. nov. is proposed. The type strain is MBIC01146(T) (=NBRC 101030(T)=CIP 109181(T)). An emended description of Ruegeria atlantica Uchino et al. 1999 is also given.

We isolated three orange or yellow pigment-producing marine bacteria, strains 04OKA-13-27 (MBIC08261), 04OKA-17-12 (MBIC08260), and YM6-073 (MBIC06409), off the coast of Okinawa Prefecture in Japan. These strains were classified as novel species of the family Flavobacteriaceae based on their 16S rRNA gene sequence. They were cultured, and the major carotenoids produced were purified by chromatographic methods. Their structures were determined by spectral data to be (3R)-saproxanthin (strain 04OKA-13-27), (3R,2'S)-myxol (strain YM6-073), and (3R,3'R)-zeaxanthin (strains YM6-073 and 04OKA-17-12). Saproxanthin and myxol, which are monocyclic carotenoids rarely found in nature, demonstrated significant antioxidative activities against lipid peroxidation in the rat brain homogenate model and a neuro-protective effect from L-glutamate toxicity.

Urukthapelstatin A, a novel cyclic peptide, was isolated from the cultured mycelia of marine-derived Thermoactinomycetaceae bacterium Mechercharimyces asporophorigenens YM11-542. The peptide was purified by solvent extraction, silica gel chromatography, ODS flash chromatography, and finally by preparative HPLC. Urukthapelstatin A dose-dependently inhibited the growth of human lung cancer A549 cells with an IC(50) value of 12 nM. Urukthapelstatin A also showed potent cytotoxic activity against a human cancer cell line panel.

A polyphasic taxonomic approach was applied to determine the taxonomic position of a hydrocarbon-degrading actinomycete, strain Hou_blueT, which was isolated from soil samples collected from an oil spring in Niigata, Japan. The results of 16S rRNA and gyrB gene sequence comparisons indicated that strain Hou_blueT represented a novel lineage in the suborder Corynebacterineae. Colonies were malachite green-like in colour on 1/10 trypticase soy agar and the cell morphology was coccoid in all growth phases. The cell-wall diamino acid and sugar indicated chemotype IV and variation A1γ. The sugars of the peptidoglycan were glycolated. The polar lipids were composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and some unspecified glycolipids. The organism contained two novel cyclic forms of menaquinone, smaragdiquinone A-8(H4, ω-cycl) and smaragdiquinone B-8(H4, dicycl). The major fatty acids were cis-9-18 : 1 (34.46 %) and 16 : 0 (25.1 %). Small amounts of 10-methyl-branched fatty acids were also present (10-methyl-17 : 0, 0.17 %), but not tuberculostearic acid (10-methyl-18 : 0), which has been shown to be present in all nocardiae. Gas-chromatographic analysis of the mycolic acid revealed a carbon-chain length of C43–C49. The DNA G+C content was 63.7 mol%. On the basis of phenotypic and phylogenetic distinctness, the organism is proposed to represent a novel genus and species, Smaragdicoccus niigatensis gen. nov., sp. nov., with the type strain Hou_blueT (=MBIC 06267T=DSM 44881T).

Three strains, 002-048T, RB589 and 002-051T, isolated from field soil in Japan, were characterized using a polyphasic approach. The isolates were Gram-positive, strictly aerobic, non-motile rods that formed ellipsoidal, subterminal endospores. The chemotaxonomic characteristics of these isolates included the presence of meso-diaminopimelic acid as the cell-wall peptidoglycan, anteiso-C15 : 0 and anteiso-C17 : 0 as the major cellular fatty acids and MK-7 as the predominant menaquinone. The DNA G+C content was 44–46 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the isolates represented an independent lineage that is distinct from related taxa and exhibited less than 94.3 % sequence similarity with respect to those taxa. Moreover, a DNA–DNA hybridization analysis showed that the three isolates represented two species. On the basis of their phenotypic and phylogenetic distinctiveness, the isolates represent two species within a novel genus, for which the names Terribacillus saccharophilus gen. nov., sp. nov. and Terribacillus halophilus sp. nov. are proposed. The type strain of T. saccharophilus is 002-048T (=IAM 15309T=KCTC 13936T) and the type strain of T. halophilus is 002-051T (=IAM 15310T=KCTC 13937T).

Strains YM18-098T and A5E-52T were both Gram-positive, aerobic, irregular rod-shaped bacteria, with lysine and ornithine as the diagnostic diamino acids of their peptidoglycans, respectively. The acyl type of the peptidoglycan in both cases was N-glycolyl. The major menaquinones were MK-11, -12 and -13. Mycolic acids were not detected. The G+C content of the DNA was 69–70 mol%. Comparative 16S rRNA studies revealed that the isolates belonged to the genus Microbacterium, that YM18-098T was closely related to the species Microbacterium lacticum and Microbacterium schleiferi, and that A5E-52T was closely related to the species Microbacterium aurum, Microbacterium aoyamense, Microbacterium deminutum and Microbacterium pumilum. DNA-DNA relatedness analysis showed that the isolated strains represented two separate genomic species. Based on both phenotypic and genotypic data, the following new species of the genus Microbacterium are proposed: Microbacterium flavum sp. nov. and Microbacterium lacus sp. nov., with the type strains YM18-098T (= MBIC08278T, DSM 18909T) and A5E-52T (= MBIC08279T, DSM 18910T), respectively.

A Gram stain-negative, strictly aerobic, brown-pigmented, non-motile, rod-shaped, chemoheterotrophic bacterial strain-designated A6F-119(T) was isolated from a marine sponge (Rhabdastrella sp.). Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the new strain represented a member of the family Flavobacteriaceae of the phylum Bacteroidetes and that it showed highest sequence similarity (93 %) to Tenacibaculum maritimum NBRC 15946(T). The strain could be differentiated phenotypically from the recognized members of the family Flavobacteriaceae. The DNA G + C content of strain A6F-119(T) was determined to be 30.8 mol%; MK-6 was identified as the major menaquinone; and the presence of iso-C15:0, iso-C17:0 3-OH, and C16:1 ω7c and/or C16:1 ω6c as the major (>10 %) cellular fatty acids. A polar lipid profile was present consisting of phosphatidylethanolamine, an unidentified aminolipid, and three unidentified lipids. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel genus for which the name Spongiiferula fulva gen. nov., sp. nov. is proposed. The type strain of S. fulva is A6F-119(T) (= KCTC 42752(T) = NBRC 111402(T)).

A Gram-stain negative, strictly aerobic, chemoheterotrophic, tangerine orange pigmented, curved-rod shaped bacterium, designated N5DA8-2C(T), was isolated from a sea squirt by use of a bait-streaked agar technique. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the novel marine strain is affiliated with the family Flavobacteriaceae of the phylum Bacteroidetes and that it shared high (92.6 %) sequence similarity with Frondibacter aureus A5Q-67(T). The strain could be differentiated phenotypically from the related members of the family Flavobacteriaceae. The major fatty acids of strain N5DA8-2C(T) were iso-C15:1 G, iso-C17:0 3-OH, iso-C15:0 and C16:1 ω7c and/or C16:1 ω6c. A polar lipid profile was present consisting of phosphatidylethanolamine and three unidentified amino lipids. The DNA G+C content of the strain was determined to be 41 mol% and the major respiratory quinone was identified as menaquinone 6 (MK-6). From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel genus in the family Flavobacteriaceae, for which the name Ascidiimonas aurantiaca gen. nov., sp. nov. is proposed. The type strain of A. aurantiaca gen. nov., sp. nov. is N5DA8-2C(T) (= KCTC 32992(T) = NBRC 110020(T)).

A polyphasic taxonomic investigation was performed on a bacterial strain, 38-Ka-2(T), which was isolated from the green alga Ulva pertusa Kjellman (Chlorophyta) in Hokkaido, Japan. The bacterial cells were observed to be golden-yellow coloured, Gram-negative, strictly aerobic, non-spore-forming, non-motile and rod-shaped. Phylogenetic analyses based on the 16S rRNA gene sequence indicated that the new strain is a member of the family Flavobacteriaceae within the phylum Bacteroidetes and that it shows high sequence similarity (94.8 %) to Aquimarina addita JC2680(T). The genomic DNA G+C content of strain 38-Ka-2(T) was determined to be 36 mol%; MK-6 was identified as the major menaquinone; and the presence of iso-C15:0, C18:0 and iso-C17:0 3-OH as the major (10 %) cellular fatty acids. The polar lipid profile was found to consist of phosphatidylethanolamine, an unidentified glycolipid and two unidentified lipids. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, strain 38-Ka-2(T) is considered to represent a novel genus for which the name Algitalea ulvae gen. nov., sp. nov. is proposed. The type strain of A. ulvae is 38-Ka-2(T) (=KCTC 32994(T) = NBRC 110017(T)).

A study was carried out to clarify the taxonomy of four Gram-positive, heterotrophic mesophiles isolated from marine lakes in the Republic of Palau. The strains, designated YM3-251T, YM3-653, YM3-671 and YM11-542T, formed aerial and substrate mycelia. The cell-wall peptidoglycan contained meso-diaminopimelic acid, glutamic acid and alanine. The G+C content of their genomic DNA was approximately 45 mol%. The major fatty acid was iso-C15 : 0 and the major isoprenoid quinone was MK-9. The strains formed a distinct group in the 16S rRNA gene tree and shared a range of phenotypic properties that distinguished them from members of related genera in Thermoactinomycetaceae fam. nov. The name proposed to accommodate the new isolates is Mechercharimyces gen. nov., comprising two species based on genotypic and phenotypic criteria, including comparative gyrB and DNA–DNA relatedness data. The names proposed for these taxa are Mechercharimyces mesophilus sp. nov., the type species, and Mechercharimyces asporophorigenens sp. nov., with the type strains YM3-251T (=MBIC06230T=DSM 44894T) and YM11-542T (=MBIC06487T=DSM 44955T), respectively.

Chemically synthesized 4-hydroxybenzoate (4HBA) is widely used in the chemical and electrical industries as a material for producing polymers such as those of the liquid crystal type. Its alkyl esters, called parabens, have been the most widely used preservatives by the food and cosmetic industries. We report here for the first time a microorganism, a marine bacterium, which biosynthesizes these petrochemical products. The marine bacterial strain, A4B-17, which was found to belong to the genus Microbulbifer on the basis of its rRNA and gyrB sequences, was isolated from an ascidian in the coastal waters of Palau. Strain A4B-17 was, surprisingly, found to produce 10 mg/liter of 4HBA, together with its butyl (24 mg/liter), heptyl (0.4 mg/liter), and nonyl (6 mg/liter) esters. We therefore characterized 23 other marine bacteria belonging to the genus Microbulbifer , which our institute had previously isolated from various marine environments, and found that these bacteria also produced 4HBA, although with low production levels (less than one-fifth of that produced by A4B-17). We also show that the alkyl esters of 4HBA produced by strain A4B-17 were effective in preventing the growth of yeasts, molds, and gram-positive bacteria.

3株のA. acidoterrestrisのgyrB遺伝子塩基配列を決定し, その他のAlicyclobacillus属細菌由来gyrB遺伝子塩基配列と相同性解析を実施したところ, gyrB遺伝子はA. acidoterrestris内で高度に保存されていることがわかった (97.6%以上).その結果をもとに, A.acidoterrestrisに特異的な領域を含むプライマー領域の検索を行ったところ, 5種類のプライマー (フォワード・プライマー: 3種類, リバース・プライマー: 2種類) を設計することができた.このプライマーの総当たりの組み合わせで, A. acidoterrestris由来DNAを鋳型にPCR反応を実施したところ, いずれの組み合わせにおいても目的の長さの断片が増幅された.他方, A.acidoterrestris以外の菌種については, 一部の菌株で目的の断片長ではない増幅産物が見られたが, ほとんどの場合で増幅産物は見られなかった.さらに, それらのプライマー領域をLAMP法へ応用したところ, A.acidoterrestrisのみに遺伝子増幅が起こることが確認された.以上のことから, 設計したプライマーを用いてPCR-電気泳動法あるいはLAMP法を行うことにより, A. acidoterrestrisを正確かっ短時間で鑑別可能であることが示された.

A novel Ferrimonas species is described on the basis of phenotypic, chemotaxonomic and phylogenetic studies. Four halophilic organisms were isolated from marine sand and marine macroalgae samples by using high-pH marine agar 2216. An analysis of the nearly complete 16S rRNA gene sequences of these new isolates indicated that they were phylogenetically close (16S rRNA gene sequence similarity >99·5 %, gyrB gene sequence similarity >97·8 %), and were most closely related to Ferrimonas balearica (16S rRNA gene sequence similarity 97·1–97·3 %, gyrB gene sequence similarity 84·4–85·0 %). Chemotaxonomic data (major menaquinone MK7; major fatty acids C16 : 0 and C18 : 1 ω9c) supported the affiliation of the new isolates to the genus Ferrimonas. The results of physiological and biochemical tests allowed phenotypic differentiation of the isolates from F. balearica. It is therefore proposed that the new isolates represent a novel species with the name Ferrimonas marina sp. nov. and type strain A4D-4T (=MBIC06480T=DSM 16917T).

A carotenoid biosynthesis gene cluster mediating the production of 2-hydroxyastaxanthin was isolated from the marine bacterium Brevundimonas sp. strain SD212 by using a common crtI sequence as the probe DNA. A sequence analysis revealed this cluster to contain 12 open reading frames (ORFs), including the 7 known genes, crtW , crtY , crtI , crtB , crtE , idi , and crtZ . The individual ORFs were functionally analyzed by complementation studies using Escherichia coli that accumulated various carotenoid precursors due to the presence of other bacterial crt genes. In addition to functionally identifying the known crt genes, we found that one (ORF11, named crtG ) coded for a novel enzyme, carotenoid 2,2′-β-hydroxylase, which showed intriguingly partial homology with animal sterol-C5-desaturase. When this crtG gene was introduced into E. coli accumulating zeaxanthin and canthaxanthin, the resulting transformants produced their 2-hydroxylated and 2,2′-dihydroxylated products which were structurally novel or rare xanthophylls, as determined by their nuclear magnetic resonance and high-performance liquid chromatography/photodiode array detector/atmospheric pressure chemical ionization mass spectrometry spectral data. The new carotenoid produced was suggested to have a strong inhibitory effect on lipid peroxidation.

A Gram-negative, strictly aerobic, beige-pigmented, non-motile, rod-shaped bacterial strain designated N5DB13-4(T) was isolated from the red alga Gracilaria vermiculophylla (Rhodophyta) collected at Sodegaura Beach, Chiba, Japan. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the novel isolate is affiliated with the family Flavobacteriaceae within the phylum Bacteroidetes and that it showed highest sequence similarity (97.3 %) to Wenyingzhuangia heitensis H-MN17(T). The hybridization values for DNA-DNA relatedness between the strains N5DB13-4(T) and W. heitensis H-MN17(T) were 34.1 ± 3.5 %, which is below the threshold accepted for the phylogenetic definition of a novel prokaryotic species. The DNA G+C content of strain N5DB13-4(T) was determined to be 31.8 mol%; MK-6 was identified as the major menaquinone; and the presence of iso-C15:0, iso-C15:0 3-OH and iso-C17:0 3-OH as the major (10 %) cellular fatty acids. A complex polar lipid profile was present consisting of phosphatidylethanolamine, two unidentified glycolipids and four unidentified lipids. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel species of the genus Wenyingzhuangia for which the name Wenyingzhuangia gracilariae sp. nov. is proposed. The type strain of W. gracilariae sp. nov. is N5DB13-4(T) (=KCTC 42246 (T)=NBRC 110602(T)).

A Gram-negative, strictly aerobic, pink-pigmented, non-motile, spherical bacterium designated YM26-125T was isolated from iron sand. Phylogenetic analyses based on the 16S rRNA gene sequence indicated that the new strain was affiliated with the phylum Planctomycetes and shared 96.1% sequence similarity with Rhodopirellula baltica SH 1T. The strain could be also differentiated phenotypically from the Rhodopirellula baltica SH 1T. The major fatty acids of strain YM26-125T were identified as C16:0 and C18:1 ω9c as defined by the MIDI system. Amino acid analysis of cell wall hydrolysates indicated that the novel isolate did not contain diaminopimelic acid in their cell walls. The DNA G+C content was determined to be 57.5 mol%, the major respiratory quinone was identified as menaquinone 6 (MK-6) and a polar lipid profile was present consisting of a phosphatidylglycerol, a diphosphatidylglycerol and three unidentified phospholipids. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel species for which the name Rhodopirellula caenicola sp. nov. is proposed. The type strain of R. caenicola is YM26-125T (=KCTC 32995T=NBRC 110016T).

A new chlorinated metabolite designated chlokamycin (1), was isolated along with ikarugamycin (2) from the culture broth of the marine-derived Streptomyces sp. MA2–12. The structure of 1 was elucidated based on spectroscopic analyses (1D and 2D NMR data and ROESY correlations). Chlokamycin moderately inhibited the growth of Jurkat cells and HCT116 cells with IC50 values of 24.7 and 33.5 μM, respectively.

A novel Gram-stain negative, spherical, non-motile, strictly aerobic, heterotrophic, yellow pigmented bacterium, designated strain 04OKA003-7(T) was isolated from the hard coral Galaxea fascicularis L. collected at Akajima, Okinawa, Japan. Phylogenetic analysis based on the 16S rRNA gene sequence revealed the novel isolate is affiliated with the family Flavobacteriaceae of the phylum Bacteroidetes and that it showed highest sequence similarity (92.9 %) to Vitellibacter aestuarii JC2436(T) and Aureitalea marina S1-66(T). The strain could be differentiated phenotypically from recognized members of the family Flavobacteriaceae. The major fatty acids of strain 04OKA003-7(T) were identified as iso-C15:0 and iso-C17:0 3-OH as defined by the MIDI system. The DNA G+C content was determined to be 41 mol%, the major respiratory quinone was identified as menaquinone 6 (MK-6) and a polar lipid profile was present consisting of phosphatidylethanolamine, two unidentified aminolipids and an unidentified lipid. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel genus for which the name Aureisphaera galaxeae gen. nov., sp. nov. is proposed. The type strain of A. galaxeae is 04OKA003-7(T) (=KCTC 32993(T) = NBRC 110018(T)).

A Gram-negative, strictly aerobic, lemon-yellow pigmented, non-motile, rod-shaped strain designated A7G-39(T) was isolated from a yellow coloured marine sponge (Tethya sp.). Phylogenetic analyses based on the 16S rRNA gene sequence indicated that the new strain represented a member of the family Flavobacteriaceae of the phylum Bacteroidetes and that it showed highest sequence similarity (94.1 %) to Algibacter wandonensis WS-MY22(T). The strain could be differentiated phenotypically from recognised members of the family Flavobacteriaceae. The major fatty acids of strain A7G-39(T) were identified as iso-C15:0 3-OH and summed feature 1 (iso-C15:1 H and/or C13:0 3-OH) as defined by the MIDI system. The DNA G+C content was determined to be 32.6 mol%, the major respiratory quinone was identified as menaquinone 7 (MK-7) and a polar lipid profile was present consisting of phosphatidylethanolamine, an unidentified aminolipid and an unidentified lipid. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel genus for which the name Spongiivirga citrea gen. nov., sp. nov. is proposed. The type strain of S. citrea is A7G-39(T) (=KCTC 32990(T) = NBRC 110022(T)).

Phylogenetic analysis of the genus Pseudomonas was conducted by using the combined gyrB and rpoD nucleotide sequences of 31 validly described species of Pseudomonas (a total of 125 strains). Pseudomonas strains diverged into two major clusters designated intrageneric cluster I (IGC I) and intrageneric cluster II (IGC II). IGC I was further split into two subclusters, the ‘P. aeruginosa complex’, which included P. aeruginosa, P. alcaligenes, P. citronellolis, P. mendocina, P. oleovorans and P. pseudoalcaligenes, and the ‘P. stutzeri complex’, which included P. balearica and P. stutzeri. IGC II was further split into three subclusters that were designated the ‘P. putida complex’, the ‘P. syringae complex’ and the ‘P. fluorescens complex’. The ‘P. putida complex’ included P. putida and P. fulva. The ‘P. syringae complex’ was the cluster of phytopathogens including P. amygdali, P. caricapapayae, P. cichorii, P. ficuserectae, P. viridiflava and the pathovars of P. savastanoi and P. syringae. The ‘P. fluorescens complex’ was further divided into two subpopulations, the ‘P. fluorescens lineage’ and the ‘P. chlororaphis lineage’. The ‘P. fluorescens lineage’ contained P. fluorescens biotypes A, B and C, P. azotoformans, P. marginalis pathovars, P. mucidolens, P. synxantha and P. tolaasii, while the ‘P. chlororaphis lineage’ included P. chlororaphis, P. agarici, P. asplenii, P. corrugata, P. fluorescens biotypes B and G and P. putida biovar B. The strains of P. fluorescens biotypes formed a polyphyletic group within the ‘P. fluorescens complex’.

The conventional methods for identifying mycobacterial species are based on their phenotypic characterization. Since some problematic species are slow growers, their taxonomy takes several weeks or months to identify. The ribosomal DNA (rDNA) sequence-based identification strategy has been adopted to solve this problem. More recently, the gyrB sequences have been shown to be useful phylogenetic markers for the identification of species. We determined the gyrB sequences of 43 slowly growing strains belonging to 15 species in the genus Mycobacterium . The frequencies of base substitutions in the gyrB sequences were comparable to those in the 16S-23S rDNA internal transcribed spacer (ITS) sequences. The ITS sequences of four species belonging to the M. tuberculosis complex ( M. tuberculosis , M. bovis , M. africanum , and M. microti ) were 100% identical, while four synonymous substitutions were found in the gyrB sequences of these strains. Based on the differences found in the gyrB sequences, we developed PCR and PCR-restriction fragment length polymorphism methods to discriminate these species.

A Gram-negative, strictly aerobic, reddish-pink-pigmented, non-motile, rod-shaped strain designated A6F-52(T) was isolated from a lake sediment sample. Preliminary analysis based on the 16S rRNA gene sequence revealed that the novel isolate could be affiliated with the family Catalimonadaceae of the phylum Bacteroidetes and that it showed highest sequence similarity (97.2%) to Catalinimonas alkaloidigena CNU-914(T). The level of DNA-DNA relatedness between strains A6F-52(T) and Catalinimonas alkaloidigena CNU-914(T) was less than 70%, which is accepted as the phylogenetic definition of a species. The DNA G+C content of strain A6F-52(T) was 44.4 mol%; MK-7 was the major menaquinone; and the presence of iso-C15：0, C16：1 ω5C and iso-C17：0 3-OH as the major cellular fatty acids supported the identification of the novel isolate as a member of the genus Catalinimonas. A complex polar lipid profile was present consisting of phosphatidylethanolamine, an unidentified phospholipid, and an unidentified lipid. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel species for which the name Catalinimonas niigatensis sp. nov. is proposed. The type strain of Catalinimonas niigatensis is A6F-52(T) (= KCTC 32474(T) = NBRC 109829(T)).

A Gram-negative, strictly aerobic, pale-yellow pigmented, rod-shaped bacterium designated A4O-5(T) was isolated from a red coloured marine alga (Chondrus ocellatus Holmes). Phylogenetic analyses based on the 16S rRNA gene sequence indicated that the new strain was affiliated with the family Flavobacteriaceae and shared approximately 90 % sequence similarity with Leptobacterium flavescens YM3-0301(T) and Gangjinia marincola GJ16(T). The strain could be differentiated phenotypically from the related members of the family Flavobacteriaceae. The major fatty acids of strain A4O-5(T) were identified as iso-C15:0, iso-C17:0 3-OH and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). A complex polar lipid profile was present consisting of phosphatidylethanolamine, an unidentified phospholipid, three unidentified amino lipids and an unidentified lipid. The DNA G+C content of the strain was determined to be 41.9 mol% and the major respiratory quinone was identified as menaquinone 6 (MK-6). From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel genus in the family Flavobacteriaceae, for which the name Citreitalea marina gen. nov., sp. nov. is proposed. The type strain of C. marina is A4O-5(T) (=KCTC 32483(T)=NBRC 109918(T)).

Metabolites from thermophilic bacteria I: N- propionylanthranilic acid, a co-metabolite of the bacillamide class antibiotics and tryptophan metabolites with herbicidal activity from Laceyella sacchari

We have characterized genomic structure and transcription of a murine odorant receptor (OR) gene, MOR23. This gene has two distinct transcriptional initiation regions, which are differentially utilized in the olfactory and testicular cells. We have found that the MOR23 gene has an intron within the 5'-untranslated region upstream of the coding sequence. In olfactory cells, transcription of the gene is initiated in the region upstream of this intron, while in testicular cells, transcription starts within the intron. Differential initiation of transcription is discussed in the context of tissue-specific expression of the OR genes.

A novel actinobacterial strain, designated YM16-381(T), was isolated from sediments of Lake Nakaumi in Shimane Prefecture, Japan. The cell of the strain was motile, non-spore-forming and Gram-positive. The colony was gray-pink and circular on marine agar 2216 medium. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain belongs to the family Dermatophilaceae of the suborder Micrococcineae. The highest sequence similarity value of the isolate was 96.2% against Kineosphaera limosa. The diaminopimelic acid in the cell wall was meso-A2pm. The major menaquinone was MK-8(H4). The DNA G+C contents were 70.5 mol%. The major cellular fatty acids were C17:1ω8c, C16:0, C15:0 and C18:1ω9c. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and ninhydrin-positive phosphoglycolipid. On the basis of polyphasic taxonomic studies, strain YM16-381(T) represents a novel species of the genus Kineosphaera within the family Dermatophilaceae, for which the name Kineosphaera nakaumiensis sp. nov. is proposed. The type strain is YM16-381(T) (=KCTC 29138(T)=NBRC 109121(T)).

During the course of systematic nucleotide sequence analysis of the terC region of E.coli K-12 by using the ordered lambda phage clones, we found the presence of a gene, termed hrpA, that showed a high degree of sequence similarity to the PRP2, PRP16 and PRP22 genes of Saccharomyces cerevisiae. The products of these yeast genes are known to play their roles in mRNA splicing, and belong to a group of proteins collectively called the DEAH family. The hrpA gene is the first example of a DEAH family gene in prokaryotes. The N-terminal region of the protein it encodes contains conserved sequence stretches characteristic of an RNA helicase. Its molecular mass is calculated to be 146 kDa. Previously, a 135 kDa protein was identified by Moir et al. [J. Bacteriol. (1992) 174, 2102-2110] in this region which is most likely identical to that encoded by hrpA. The C-terminal region of the hrpA gene product seems to contain an RNA binding motif weakly resembling that of ribosomal protein S1 of E.coli. Disruption of the hrpA gene suggested that it is not essential for the growth of E.coli.

Polyphasic taxonomic investigation was conducted on a strictly aerobic, Gram-negative, non-motile, pale-yellow-pigmented, rod-shaped strain designated HJ24(T) which was isolated from an unidentified orange-colored marine sponge. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the novel isolate could be affiliated with the family Flavobacteriaceae of the phylum Bacteroidetes and that it showed highest sequence similarity (88.6%) with Pseudozobellia thermophila KMM 3531(T). The novel isolate is phenotypically and physiologically different from other related genera. The DNA G+C contents were 41 mol%, MK-6 was the major menaquinone and iso-C15:0, iso-C15:1 and iso-C17:0 3-OH were the predominant fatty acids. On the basis of polyphasic taxonomic studies, it was concluded that the novel strain represents a new genus and a novel species of the family Flavobacteriaceae. We propose the name Spongitalea numazuensis gen. nov., sp. nov. for this strain; its type strain is HJ24(T) (= DSM 21243(T)).

A Gram-negative, strictly aerobic, golden-yellow pigmented, non-motile, rod-shaped strain designated J36A-7(T) was isolated from an unidentified yellow-green coloured marine sponge. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the novel isolate was affiliated with the family Flavobacteriaceae of the phylum Bacteroidetes and that it showed highest sequence similarity (85.6 %) to Nonlabens dokdonensis DSW-6(T). The strain could be differentiated phenotypically from recognized members of the family Flavobacteriaceae. The major fatty acids of strain J36A-7(T) were identified as iso-C16:0, iso-C17:0 3-OH and summed feature three as defined by the MIDI system (C16:1 ω7c and/or iso-C15:0 2-OH). The DNA G+C content was determined to be 31.2 mol%, the major respiratory quinone was identified as menaquinone 6 (MK-6) and a complex polar lipid profile was present consisting of phosphatidylethanolamine, two unidentified glycolipids, one unidentified aminolipid and one unidentified lipid. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel genus for which the name Spongiimonas flava gen. nov., sp. nov. is proposed. The type strain of S. flava is J36A-7(T) (=KCTC 32176(T) = NBRC 109319(T)).

A strictly aerobic, Gram-negative, reddish-orange pigmented, non-motile and rod-shaped bacterium, designated AK17-053(T) was isolated from a marine crustacean (Squillidae) living on tidal flats on the coast of the Ariake Sea, Nagasaki, Japan. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the novel isolate could be affiliated with the family Saprospiraceae of the phylum Bacteroidetes and that it showed highest sequence similarity (84%) with Lewinella marina MKG-38(T). The strain could be differentiated phenotypically from recognized members of the family Saprospiraceae. The G+C content of DNA was 55.3 mol%, MK-7 was the major menaquinone and iso-C(15:0) and C(16:1)ω7c were the major fatty acids. On the basis of polyphasic taxonomic studies, it was concluded that strain AK17-053(T) represents a new genus of the family Saprospiraceae. We propose the name Rubidimonas crustatorum gen. nov., sp. nov. for this strain; its type strain is AK17-053(T) (= MBIC08356(T) = NBRC 107717(T)).

A Gram-negative, strictly aerobic, reddish-pink pigmented, non-motile, rod-shaped strain designated N5DB8-4(T), was isolated from an orange-striped sea anemone Diadumene lineata by a differential growth screening method. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the novel isolate was affiliated with the family Flammeovirgaceae of the phylum Bacteroidetes and that it showed highest sequence similarity (89.1%) to Porifericola rhodea N5EA6-3A2B(T). The strain could be differentiated phenotypically from recognized members of the family Flammeovirgaceae. The G+C content of the DNA is 52.6 mol%, the major respiratory quinone is menaquinone 7 (MK-7) and iso-C15:0, C16:1ω5c and iso-C15:1 G (the double-bond position indicated by capital letter is unknown) were the major fatty acids. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain represents a novel taxon for which the name Tunicatimonas pelagia gen. nov., sp. nov. is proposed. The type strain of Tunicatimonas pelagia is N5DB8-4(T) (=KCTC 23473(T )= NBRC 107804(T)).

A simulation method was studied to calculate vibration response of a bar group installed between restraints in a single row under seismic excitation. Because impact phenomena take place between adjacent bars or a bar and a restraint, nonlinear force caused by the impact should be considered in the response calculation. In this study, a new method for determining the nonlinear force without convergence iteration was proposed. The simulation method is basically a combination of the mode superposition method and the pseudo external force method. The method proposed by Nigam is applied for time integration. An impact point is modeled as a set of a gap, a linear spring and a linear damper. The nonlinear forces at the next time step can be determined with matrix calculation by introducing influence coefficient matrices, which express the influence of impact forces on intersections at impact points. In order to verify the above method, the calculation results were compared with experimental ones, which showed that the method can simulate the experimental results well.

Clinical signs, changes in blood characteristics, plasma estrogen and progesterone levels were investigated during the period of the 7 days before expulsion to the 3 days after expulsion in 5 cows having twins. Values obtained were compared with those of 8 cows with single birth. An average of gestation length of the cows with twins was 8 days shorter than those of the cows with single birth. At expulsion, proper handling at the dystocia was needed in 2/5 twinnings. Retained placenta occurred in all twin calvings, although in 3 cases either of the 2 placentas was discharged within 12 h after the expulsion. In many twinnings, the rectal temperature did not decrease below 38.5C before the expulsion, and increased above 39.6C after expulsion. Ligamentum sacrotuberale latum relaxated at expulsion did not fully resume to the former condition after the expulsion. Maximum values of glucose concentration before and after the expulsion in twinnings were lower than those at the same periods in the single birth. The ratios of segmentcytes to lymphocytes after the expulsion and numbers of eosinophils before and after expulsion were lower in the cows with twins. In some twinnings, a decrease in estrogen levels was slower and a slight increase in progesterone levels was observed after expulsion.

Two aerobic, Gram-reaction-negative, golden-yellow pigmented and rod-shaped bacteria, designated strains A5Q-118T and A5Q-27, were isolated from an unidentified sea squirt that thrives in the coral reefs off the coast of Okinawa, Japan. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the novel isolates were affiliated with the family ‘Flammeovirgaceae’ of the phylum Bacteroidetes. Strains A5Q-118T and A5Q-27 shared 100 % sequence similarity with each other and showed Flammeovirgaceae’. The novel isolates were phenotypically and physiologically different from strains described previously. The DNA G+C content was 35.5–36.2 mol%, MK-7 was the major menaquinone and iso-C15 : 0 and C16 : 1ω5c were the major fatty acids. Based on the results of this polyphasic taxonomic study, it was concluded that strains A5Q-118T and A5Q-27 represent a novel species in a new genus of the family ‘Flammeovirgaceae’, for which the name Aureibacter tunicatorum gen. nov., sp. nov. is proposed. Proposal for designation of the Flammeovirgaceae fam. nov. is also presented. The type strain of Aureibacter tunicatorum is A5Q-118T ( = KCTC 23232T = NBRC 107587T).

A Gram-negative-staining, obligately aerobic, non-motile, rod-shaped and chemoheterotrophic bacterium, designated strain MN1-1006T, was isolated from an ascidian (sea squirt) sample, and was studied using a polyphasic taxonomic approach. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the new isolate shared approximately 93–99% sequence similarity with recognized species of the genus Rubritalea within the phylum ‘Verrucomicrobia’. DNA–DNA hybridization values between strain MN1-1006T and Rubritalea squalenifaciens HOact23T and Rubritalea sabuli YM29-052T were 57% and 14.5%, respectively. Strain MN1-1006T produced carotenoid compounds that rendered the cell biomass a reddish pink colour. The strain also contained squalene. The cell-wall peptidoglycan of the novel strain contained muramic acid and meso-diaminopimelic acid. The DNA G+C content of strain MN1-1006T was 51.4 mol%. The major cellular fatty acids were iso-C14:0, iso-C16:0 and anteiso-C15:0. The major isoprenoid quinone was MK-9. On the basis of these data, it was concluded that strain MN1-1006T represents a novel species of the genus Rubritalea, for which the name Rubritalea halochordaticola sp. nov. is proposed. The type strain is MN1-1006T ( = KCTC 23186T = NBRC 107102T).

Two peptides, tumescenamides A and B, were isolated from the fermentation broth of a marine bacterium, Streptomyces tumescens YM23-260. The structure of tumescenamide A was determined to be a cyclic depsipeptide consisting of α-amino-2-butenoic acid, tyrosine, valine, leucine and threonine, substituted with a 2,4-dimethylheptanoyl residue at the α-NH(2) position. Tumescenamide B possesses a 2,4,6-trimethylnonanoyl residue in place of the 2,4-dimethylheptanoyl substituent in tumescenamide A. Tumescenamide A induced reporter gene expression under the control of the insulin-degrading enzyme promoter.

We previously reported that extra-slow-growing Bradyrhizobium japonicum isolates obtained from three field sites in Japan, designated as HRS (highly reiterated sequence-possessing) strains, have high copy numbers of the insertion sequences RSalpha and RSbeta. When strain collections in the USA, Japan, Korea, Thailand and China were examined by Southern hybridization using RSalpha, RSbeta and IS1631 as probes, HRS strains were found in the Japanese, Chinese, and American collections, but not in the Korean and Thai ones. Copy number analyses of RSalpha and RSbeta, calibrated with the copy number of rrs (16S rRNA gene), indicated that the HRS stains can be divided into two major groups. Group A is comprised of members with a high copy number of RSalpha (mean+/-S.D., 139+/-27), a low number of RSbeta (mean+/-S.D., 30+/-13) sequences, and extremely slow growth rates (mean doubling time+/-S.D., 27+/-9 h). In contrast, group B is comprised of strains with a high copy number of RSbeta (mean+/-S.D., 93+/-6) and a lower number of RSalpha. These groupings of HRS strains were well correlated with phylogenetic clusters based on rrs, gyrB and serogroups (110/122 and 123/135). Growth rate of B. japonicum strains was also correlated exclusively with RSalpha copy number. The ecological, evolutionary and biotechnological implications of the findings are discussed.

Phylogenetic and taxonomic characterization was performed for 14 strains of bacteria that produce anticancer antibiotics (pelagiomicins) (represented by strain Ni-2088(T)) and one strain that produces UV-absorbing substances (strain F-104(T)), isolated from marine algae and seagrass collected from coastal areas of tropical Pacific islands and a subtropical island of Japan. All 15 isolates were Gram-negative, strictly aerobic, non-motile and non-spore-forming. Sequence analysis of the 16S rRNA gene showed that the isolates occupied positions in the phylogenetic radiation of the genus Microbulbifer, with similarities of 93.6-97.6 %. The cells possessed a clearly discernible rod-coccus cell cycle in association with the growth phase; cells were rods during the growth phase and all converted to coccoid-ovoid cells when proliferation ceased. The coccoid-ovoid cells were optically denser than the rod cells and were viable for extended periods. They were considered to constitute a resting form. The type strains of described species of Microbulbifer were also found to possess identical rod-coccus cell cycles. The G+C content of the DNA was 48.1-49.7 mol%. The major respiratory quinone system was ubiquinone-8. The major fatty acids were C(18 : 1)omega7c and C(16 : 0), and the hydroxy acids comprised C(10 : 0) 3-OH, C(12 : 0) 3-OH and iso-C(11 : 0) 3-OH. The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine. The group of 14 pelagiomicin-producing strains and strain F-104(T) each constituted a single genomic species. Based on phylogenetic affiliation, phenotypic characteristics and genomic distinctness, the isolates represent two novel species in the genus Microbulbifer, for which the names Microbulbifer variabilis sp. nov. (type strain Ni-2088(T) =MBIC01082(T) =ATCC 700307(T)) and Microbulbifer epialgicus sp. nov. (type strain F-104(T) =MBIC03330(T) =DSM 18651(T)) are proposed.

Six obligately aerobic, Gram-negative, non-motile, pale-yellow-pigmented, rod-shaped bacterial strains, designated YM3-301(T), HG868, 04PA2 Co4-8B, 04PA2 Co4-99A, 04PA2 018SW-3 and 04PA2 018SW-18, were isolated from a marine sponge and seawater and were subjected to a polyphasic taxonomic investigation. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the novel isolates were affiliated with the family Flavobacteriaceae (phylum Bacteroidetes) and that they showed the highest sequence similarity (90.3-90.9 %) to members of the genus Leeuwenhoekiella and to Zhouia amylotica. The 16S rRNA gene sequence similarities with respect to members of other related genera were only 86.3-89.1 %. In contrast, the six isolates shared high levels of 16S rRNA gene sequence similarity (99.7-100 %) and DNA-DNA relatedness (72-94 %) with each other. The novel isolates were phenotypically and physiologically different from members of related genera. The G+C content of the DNA was 41.8-43.5 mol%, MK-6 was the major menaquinone and i15 : 0, i15 : 1 and i17 : 0 3-OH were the major fatty acids. On the basis of the data from the taxonomic studies, it was concluded that these six novel strains represent a novel genus and species of the family Flavobacteriaceae, for which the name Leptobacterium flavescens gen. nov., sp. nov. is proposed. The type strain of Leptobacterium flavescens is YM3-301(T) (=KCTC 22160(T)=MBIC 06275(T)=NBRC 104141(T)).

Planococcus maritimus strain iso-3 was isolated from an intertidal sediment sample from the Clyde estuary in the UK. A novel red pigment glyco-carotenoic acid ester, methyl glucosyl-3,4-dehydro-apo-8'-lycopenoate has been isolated from this marine bacterium using chromatographic methods. The structure of methyl glucosyl-3,4-dehydro-apo-8'-lycopenoate was determined to be methyl 1-(beta-D-glucopyranosyloxy)-3,4-didehydro-1,2-dihydro-8'-apo-psi-caroten-8'-oate by the degradation experiment and the spectroscopic analyses. The methyl glucosyl-3,4-dehydro-apo-8'-lycopenoate showed potent antioxidative activity in the (1)O(2) suppression model.

The SSP1 gene of Schizosaccharomyces pombe which is homologous to the SSC1 gene of Saccharomyces cerevisiae was cloned and its nucleotide sequence determined. A heat-shock element and three possible TATA boxes were found upstream of the coding region. Dual modes of transcriptional initiation were observed in primer extension analyses using as templates the mRNAs prepared from cells before and after heat-shock. Initiation sites situated 50 to 60 nucleotides downstream of the normal one were found to be additionally used in cells after heat-shock. Thus, the mode of transcription in heat-shocked cells of S. pombe appears to be more similar to the one observed in higher eukaryotes than that in S. cerevisiae. The SSP1 gene contains two methionine codons in the region predicted to encode a mitochondrial targeting signal sequence of its protein (Ssp1) and the stretch between the two methionine codons is capable of forming stem-loop structures. Both of the two methionine codons were confirmed to function as translational initiators in vitro. A possible mechanism is proposed for the response of the SSP1 gene towards heat-shock by the differential initiation of its transcription and translation.

Six Gram-negative, non-motile, rod-shaped, chemoheterotrophic bacteria designated strains YM23-227(T), 06SJR1-1(T), AK18-024(T), 05IJR53-1(T), MN1-1037(T) and MN1-1047 were isolated from various marine environments and subjected to a polyphasic taxonomic study. Preliminary analysis based on 16S rRNA gene sequences revealed that the novel isolates could be affiliated with the family Verrucomicrobiaceae of the phylum 'Verrucomicrobia'. The strains shared approximately 94-100 % sequence similarity with each other and showed less than 94 % similarity with members of the family Verrucomicrobiaceae with validly published names. The DNA-DNA relatedness between strains YM23-227(T) and 06SJR1-1(T) was less than 70 %, a value that is accepted as a phylogenetic definition of a species. The cell wall peptidoglycan of the strains contained muramic acid and meso-diaminopimelic acid. The novel isolates produced carotenoid pigments and squalene. The DNA G+C contents of the six strains were 63-65 mol%. The major menaquinone was MK-9 and iso-C(14 : 0) was the major fatty acid. Based on the evidence from the polyphasic taxonomic study, it was concluded that the six strains should be classified as representing a new genus and five novel species of the family Verrucomicrobiaceae within the phylum 'Verrucomicrobia', for which the names Haloferula rosea gen. nov., sp. nov. (type strain 06SJR1-1(T)=MBIC08340(T)=KCTC 22201(T)), Haloferula harenae sp. nov. (type strain YM23-227(T)=MBIC08299(T)=KCTC 22198(T)), Haloferula phyci sp. nov. (type strain AK18-024(T)=MBIC08341(T)=KCTC 22200(T)), Haloferula helveola sp. nov. (type strain 05IJR53-1(T)=MBIC08342(T)=KCTC 22199(T)) and Haloferula sargassicola sp. nov. (type strain MN1-1037(T)=MBIC08343(T)=KCTC 22202(T)) are proposed.