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mura1,3
1 Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan
2 Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan
3 The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan
Correspondence
Yoko Takahashi
ytakaha{at}lisci.kitasato-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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A phylogenetic tree based on 16S rRNA gene sequences of all species of the genus Microbacterium and the composition of the medium ‘H0.3’ are available as supplementary material with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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with the type species Microbacterium lacticum, and was emended by Takeuchi & Hatano (1998). Members of the genus Microbacterium are Gram-positive, obligately aerobic and catalase-positive. The peptide subunit of peptidoglycan consists of alanine, D-glutamic acid (plus hydroxyglutamic acid), and either L-lysine, L-ornithine or L-homoserine. Menaquinones are MK-10, MK-11, MK-12, MK-13 and MK-14. Muramic acid occurs in the N-glycolyl form. The G+C content of the genomic DNA ranges from 66 to 72 mol%. Members of the genus Microbacterium are widespread and can be isolated from various environmental habitats (Collins & Bradbury, 1992). The genus Microbacterium is a member of the family Microbacteriaceae of the order Actinomycetales.
Strain YM10-847T was isolated from a sediment sample collected from the mouth of the Samambula River, Fiji (depth: 1 m; GPS location: 1 ° 7' 14'' S, 17 ° 28' 18'' E), in December 2003. Strain YM11-607T was isolated from an unidentified hydroid collected from the Sano Marine Lake, Republic of Palau (depth: 1 m; GPS location: 0 ° 17' 74'' N, 13 ° 26' 92'' E), in March 2004. The samples (0.5–1.0 cm3) were homogenized with a glass rod in 5 ml sterile seawater. Bacteria were isolated from the homogenate (50 µl) by culturing at 25 °C for 30 days on P (YM10-847T) or H0.3 (YM11-607T) media. Compositions of medium P were described in Yoon et al. (2007) and compositions of medium H0.3 are listed in Supplementary material available in IJSEM Online.
Biomass for biochemical and chemotaxonomic characteristics was prepared by culturing in TSB broth at 27 °C.
Morphological observation under a scanning electron microscope (model JSM-5600; JEOL) was performed using cultures grown on glucose–peptone meat extract agar medium (1 % D-glucose, 0.5 % peptone, 0.5 % meat extract, 0.3 % NaCl and 1.2 % agar; pH 7.0) at 27 °C for 3 days. The carbon-assimilation properties of the two strains and three related species were determined by using a yeast nitrogen-based agar medium without amino acids (Nihon Pharmaceutical) (Pridham & Gottlieb, 1948). NaCl tolerance, pH and temperature ranges for growth were determined on 1/5 nutrient agar or marine agar (Difco). The two isolated strains and type strains of the three related species were characterized biochemically using API ZYM (bioMérieux) in accordance with the manufacturer's instructions.
The N-acyl types of muramic acid were determined by using the method of Uchida & Aida (1977). Cell walls were purified by using the method of Kawamoto et al. (1981). One milligram of purified cell wall was hydrolysed at 100 °C with 1 ml 6 M HCl for 16 h. The residue was dissolved in 100 µl water and was used for amino acid analysis. The amino acid composition was determined by HPLC using the Pico Tag method (Waters). Samples were derivatized with phenylisothyocyanate and UV (254 nm) detection was used. The presence of mycolic acid was determined by using the TLC method described in Tomiyasu (1982). Menaquinones were extracted and purified by using the method described in Collins et al. (1977), and were then analysed by HPLC (model 802-SC; Jasco) using a chromatograph equipped with a Capcell Pak C18 column (Shiseido) (Tamaoka et al., 1983). Methyl esters of cellular fatty acids were prepared and analysed by GLC (model HP6890; Hewlett Packard).
DNA was isolated as described by Saito & Miura (1963). DNA base composition was estimated by HPLC (Tamaoka & Komagata, 1984). Levels of DNA–DNA relatedness were determined by the method of Ezaki et al. (1989) using photobiotin and a microplate format.
DNA was prepared using InstaGene matrix (Bio-Rad). The 16S rRNA gene was amplified by PCR using a forward primer corresponding to positions 8–27 and a reverse primer corresponding to positions 1492–1510 (Escherichia coli numbering system; Weisburg et al., 1991) and sequenced with an automated sequence analyser (3730 DNA Analyser; Applied Biosystems) using BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems). Species related to the novel strains were identified by performing sequence database searches using the BLAST program (Altschul et al., 1990). Sequence data for related species were retrieved from GenBank. The CLUSTAL_X software (Thompson et al., 1997) was used for a multiple alignment of nucleotide sequences. Neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony analyses were conducted using MEGA version 3.1 (Kumar et al., 2004). Nucleotide substitution rates (Knuc values) were calculated (Kimura & Ohta, 1972) for the neighbour-joining method. Min-mini algorithm (Nei & Kumar, 2000) was used for the maximum-parsimony method. The maximum-likelihood tree was inferred by using PHYML (Guindon & Gascuel, 2003; Guindon et al., 2005). The distance-based tree was used as a starting tree to be refined by the maximum-likelihood algorithm based on the HKY model (Hasegawa et al., 1985), with a transition/transversion ratio and a gamma shape parameter, which were estimated by maximizing the likelihood of the phylogeny.
The 1465 and 1472 bp of 16S rRNA gene sequences were determined for YM11-607T and YM10-847T, respectively. Subsequent 16S rRNA-based phylogenetic analysis demonstrated that the strains affiliated with the genus Microbacterium as shown in Supplementary Fig. S1 (available in IJSEM Online). YM11-607T was closely related to Microbacterium paludicola in three kinds of trees (Figs 1, 2 and 3). The level of sequence similarity between YM11-607T and M. paludicola was 98.3 %. YM10-847T was closely related to Microbacterium arborescens, Microbacterium imperiale and Microbacterium ulmi (Figs 1, 2 and 3). The level of sequence similarity between YM10-847T and related Microbacterium species: M. arborescens, M. imperiale and M. ulmi was 98.3, 98.1 and 97.2 %, respectively.
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). These characteristics were well matched to the genus description of Microbacterium.
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, thus confirming that the isolated strains are two independent novel species of the genus Microbacterium.
The chemotaxonomic and morphological characteristics (Tables 1
and 2) of these two isolated strains are consistent with their assignment to the genus Microbacterium (Takeuchi & Hatano, 1998). The phenotypes and characteristics that distinguish the isolated strains from one another and from their phylogenetic neighbours are listed in Table 2.
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Description of Microbacterium sediminicola sp. nov.
Microbacterium sediminicola (sedi.mi.ni.co'la. L. n. sedimen -inis, sediment; L. suff. -cola, inhabitant dweller; N.L. n. sediminicola, sediment-dweller).
Cells are rod-shaped, vary in cell size from 0.4 to 0.7 by 0.8 to 1.5 µm. Gram-positive, catalase-positive, aerobic. Colonies are pale yellow. Growth occurs between pH 6 and 11, and 19 and 38 °C. In 1/5 nutrient agar medium, NaCl is tolerated up to 7 %. L-Arabinose, D-galactose, D-glucose, maltose, D-mannitol, D-mannose, L-rhamnose, trehalose and D-xylose are assimilated, but D-fructose, raffinose and sucrose are not. Esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, 
-galactosidase, 
-galactosidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase are detected by the API ZYM enzyme assay; alkaline phosphatase, chymotrypsin, naphthol-AS-BI-phosphohydrolase, -glucuronidase, -mannosidase and -fucosidase are negative. Weak reaction for lipase (C14) and trypsin is detected. The acyl type of the peptidoglycan was N-glycolyl. The major menaquinones are MK-10 and MK-11. The major cellular fatty acids are anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The DNA G+C content is 67.8 mol%.
The type strain, YM10-847T (=MBIC08264T=DSM 18905T), was isolated from sediment collected from the mouth of the Samambula River, Fiji.
Description of Microbacterium marinilacus sp. nov.
Microbacterium marinilacus (mari.ni.la'cus. L. adj. marinus, marine; L. n. lacus -us, lake; N.L. gen. n. marinilacus, of a marine lake).
Cells are rod-shaped, vary in cell size from 0.4 to 0.6 by 0.9 to 1.4 µm. Gram-positive, catalase-positive, aerobic. Colonies are lemon yellow. Growth occurs between pH 6 and 11, and 12 and 38 °C. In 1/5 nutrient agar medium, NaCl is tolerated up to 8 %. L-Arabinose, D-fructose, D-glucose, maltose, D-mannitol, D-mannose, L-rhamnose, trehalose and D-xylose are assimilated, but D-galactose, raffinose and sucrose are not. Esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, naphthol-AS-BI-phosphohydrolase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase and -fucosidase are detected by the API ZYM enzyme assay; alkaline phosphatase, -galactosidase, -galactosidase, -glucuronidase and -mannosidase are negative. Weak reaction for lipase (C14), chymotrypsin, acid phosphatase and trypsin is detected. The acyl type of the peptidoglycan was N-glycolyl. The major menaquinones are MK-11 and MK-12. The major cellular fatty acids are anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The DNA G+C content is 71.6 mol%.
The type strain, YM11-607T (=MBIC07778T=DSM 18904T), was isolated from an unidentified hydroid collected from the Sano Marine Lake, Republic of Palau.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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