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1 Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkultren GmbH, Inhoffenstrasse 7b, D-38124 Braunschweig, Germany
3 Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
Correspondence
Hiroaki Kasai
hiroaki.kasai{at}mbio.jp
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ABSTRACT |
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. 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, 
-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).
A scanning electron micrograph of cells of strain Hou_blueT, a phylogenetic tree based on gyrB gene sequences and structures of the novel quinones present in strain Hou_blueT are available as supplementary figures in IJSEM Online.
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MAIN TEXT |
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) malachite green-like, round colonies of 1–3 mm diameter were obtained after incubation for 7 days in a hexadecane-saturated atmosphere at 25 °C. Subcultivation was done on 1/10 trypticase soy agar (TSA; Difco) at 30 °C for 7 days. On this medium, strain Hou_blueT was able to grow at 4–37 °C, but not at 45 °C. Growth at 30 °C was also observed on International Streptomyces Project (ISP) medium 2 (Daigo), medium 6 and TSA.
Gram-staining was performed as described by Gerhardt et al. (1994). Cell morphology was observed under a Nikon phase-contrast microscope at x1000, with cells grown for 14 days at 30 °C on 1/10 TSA. The size of the cells was determined by using a scanning electron microscope with specimens that were fixed with 2.5 % glutaraldehyde in 0.1 M cacodylate buffer containing several drops of 4 % osmium tetroxide for 1 h at room temperature. Suspensions were transferred to the surface of a polylysine-coated glass plate, and dehydrated in ethanol and t-butyl alcohol at room temperature. Preparations were sputter-coated with Pt/Pd on aluminium mounts and observed with a Hitachi S2500 scanning electron microscope (see Supplementary Fig. S1 in IJSEM Online). The 16S rRNA and gyrB genes were analysed as described by Katsuta et al. (2005). Sequences were aligned by using CLUSTAL_X (Thompson et al., 1997) based on aligned sequences supplied by RDP-II release 9 (Cole et al., 2005). A neighbour-joining phylogenetic tree based on genetic distances calculated using the Kimura two-parameter model was constructed with MEGA version 3.1 (Kumar et al., 2004). PHYML (version 2.4.4; Guindon & Gascuel, 2003) was used to construct a maximum-likelihood tree. The phylogenetic relationship based on the GTR substitution model was analysed, and a tree was built up from the neighbour-joining tree as the starter tree. The robustness of the topology was evaluated by using the maximum-likelihood method with bootstrap analysis based on 100 replications (Fig. 1). The 16S rRNA gene sequence of strain Hou_blueT was a continuous stretch of 1478 bp. Sequence similarity calculations after a BLAST search against GenBank indicated that the closest relatives of strain Hou_blueT were Nocardia africana (95.0 %), Nocardia araoensis (94.5 %), Nocardia arthritidis (94.8 %), Nocardia beijingensis (94.7 %), Nocardia elegans (94.9 %), Nocardia takedensis (94.5 %) and Nocardia paucivorans (94.5 %). Twelve of 16 signature nucleotides of the 16S rRNA gene in Nocardiaceae (Stackebrandt et al., 1997) were identified in the 16S rRNA gene sequence of strain Hou_blueT. The gyrB gene-based phylogenetic tree is available as Supplementary Fig. S2 in IJSEM Online.
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. The amino acids in an acid hydrolysate of the cell walls were identified by using two-dimensional descending chromatography on cellulose TLC plates (Tokyo Kasei), following the method of Harper & Davis (1979), and by HPLC as their phenylthiocarbamoyl derivatives, with LC-10AD HPLC apparatus (Shimadzu) equipped with a Wakopak WS-PTC column (Wako Pure Chemical Industries, 1989). Whole-cell sugars were analysed using the method of Becker et al. (1964). The hydrolysate of whole cells of strain Hou_blueT contained the sugars arabinose, galactose, glucose and fucose, whereas the hydrolysate of the cell wall contained meso-diaminopimelic acid, alanine, glutamic acid and glycine in a molar ratio of approximately 0.5 : 3 : 1 : 2. This combination of cell-wall diamino acid and sugar indicated chemotype IV sensu Lechevalier & Lechevalier (1970) and variation A1 of Schleifer & Kandler (1972). The murein acyl type was determined by using a modification of the colorimetric method of Uchida & Aida (1977). In contrast to the original procedure, the whole-cell hydrolysate was neutralized by being passed through an ion-exchange column (Analytichem Bond Elut SCX; Varian). As expected for a member of the family Nocardiaceae, the sugars of the peptidoglycan were glycolated. Polar lipids were extracted, examined by two-dimensional TLC and identified using published procedures (Minnikin et al., 1977). The polar lipids were composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and some unspecified glycolipids. This pattern matched quite well with those reported by Minnikin et al. (1977) for members of the family Nocardiaceae. To determine the DNA base composition, DNA was extracted and purified by using a Genomic-tip and buffer set (Qiagen). Total DNA was digested with P1 nuclease using a GC kit (Yamasa Shoyu). The DNA G+C content was measured using HPLC (Tamaoka & Komagata, 1984).
Quinones were extracted with 5 ml chloroform/methanol (2 : 1, v/v) from 10 mg freeze-dried cells that were grown in 300 ml 1/10 trypticase soy broth (TSB). After centrifugation, the supernatant was evaporated to dryness and suspended in 10 µl acetone. An aliquot of the acetone solution was analysed with an HPLC/PDA/APCI-MS/MS system using a C30 Develosil column (1.0 mm i.d.x150 mm; Nomura Chemical). The crude extract was eluted at a rate of 0.1 ml min–1 with methanol/2-propanol (8 : 2, v/v). Mass spectra were monitored in the mass range m/z 200–1200 with the LCQ Advantage system. The capillary temperature was set at 150 °C, the APCI vaporizer temperature was held at 400 °C, the capillary voltage was optimized to 23 V and the nitrogen gas flow was set to 28 (arbitrary units). HPLC peaks showing the characteristic UV spectra of naphthoquinones (Kroppenstedt, 1985) were identified by comparison with the MS data of authentic menaquinones (MK-7, 8, 9 and 10). Two major menaquinones were detected by using the HPLC/PDA/APCI-MS/MS system. One had the same patterns of UV and MS/MS as those of MK-8(H4, -cycl), which is diagnostic for Nocardia, the other having a UV pattern of MK type with MS/MS data that have not been found previously. Several milligrams of these two quinones were obtained from a 10 l liquid culture, and instrumental analyses using NMR and HRMS indicated the structures to be those of novel cyclic menaquinones, smaragdiquinone A-8(H4, -cycl) and smaragdiquinone B-8(H4, dicycl) (Adachi and others, unpublished data). Smaragdiquinones A and B are abbreviated to SQA and SQB. SQA-8(H4, -cycl) is different from MK-8(H4, -cycl) in the structure of the -ring (structures are available as Supplementary Fig. S3 in IJSEM Online).
For analysis of the fatty acid and mycolic acid composition, strain Hou_blueT was grown on TSA for 4 days at 28 °C. For other chemotaxonomic analyses, cells were grown in TSB for 4 days at 28 °C on a rotary shaker, harvested by centrifugation and washed twice with distilled water and lyophilized. Fatty acid methyl esters were obtained by saponification, methylation and extraction, using the method of Miller (1982) with minor modifications (Kuykendall et al., 1988), from 40 mg cells that had been scraped from Petri dishes. The fatty acid methyl ester mixture was separated by using the Sherlock Microbial Identification System (MIS; Microbial ID) consisting of a 5980 gas chromatograph fitted with a 5 % phenylmethyl silicone capillary column (0.2 mmx25 m), a flame-ionization detector, a 7673A automatic sampler and a Kayak XA computer (Hewlett Packard). The peaks were integrated automatically and the fatty acids present and their content (%) were calculated using the MIS software. The gas chromatographic parameters were as follows: carrier gas, ultra-high-purity hydrogen; column head pressure, 60 kPa; injection volume, 2 µl; column split ratio, 100 : 1; septum purge, 5 ml min–1; column temperature, 170–270 °C at 5 °C min–1; injection port temperature, 250 °C; and detector temperature, 300 °C. The fatty acid pattern of strain Hou_blueT did not match any of the patterns found previously for members of the genus Nocardia. Interestingly, only very small amounts of 10-methyl-branched fatty acids were present in strain Hou_blueT (10-methyl-17 : 0, 0.17 %) and, unlike all other nocardiae, tuberculostearic acid (10-methyl-18 : 0) was not present. In addition, a significant amount of iso-16 : 0 (8.12 %) was synthesized by strain Hou_blueT; in contrast, this fatty acid, if present at all, has only been found previously in trace amounts among nocardiae and all other members of the suborder Corynebacterineae. Half of the fatty acid methyl ester extract (0.3 ml) was mixed with 0.1 ml N-methyl-N-(trimethylsilyl)-heptafluorobutyramide (MSHFBA) and trimethylchlorosilane (TMCS) [10 : 1 (v/v); Macherey and Nagel], yielding trimethylsilylated derivatives (TMS-MAME) of the -OH group of mycolic acid methyl ester (MAME) at room temperature (Klatte et al., 1994). A TMS-MAME analysis was carried out using a 5890A gas chromatograph (Hewlett Packard) equipped with a flame-ionization detector and a 7673A automatic sampler (Hewlett Packard). Chromatographic data were sampled and processed by using a Vectra Series 4 computer (Hewlett Packard) with MIS version 3.3 software package (Microbial ID). The TMS-MAME analysis was performed using a 12 m HT5 column (SGE) of 0.32 mm i.d. and 0.1 µm film thickness using H2 as the carrier gas and a split ratio of 50 : 1. The oven temperature was increased from 210 to 400 °C, at 10 °C min–1, the final temperature being held for 10 min. The MIS apparatus was calibrated with a series of saturated TMS-MAMEs derived from Corynebacterium bovis DSM 20582T, Rhodococcus erythropolis DSM 43066T, Rhodococcus rhodnii DSM 43336T and Gordonia sputi DSM 44019. The chain length of the mycolic acids of strain Hou_blueT was shorter (C43–C49) than that found in other nocardiae (C50–C62), the pattern being very complex, i.e. highly unsaturated mycolic acids. It was therefore difficult to identify the numbers of the double bond. A detailed comparison of the chemotaxonomic data of strain Hou_blueT with those of other mycolic acid-containing genera is given in Table 1. Results of phenotypic characterization by using Biolog and API ZYM assays are given in the species description.
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Description of Smaragdicoccus gen. nov.
Smaragdicoccus [Sma.rag.di.coc'cus. L. n. smaragdus malachite; N.L. masc. n. coccus (from Gr. masc. n. kokkos), grain; N.L. masc. n. Smaragdicoccus malachite(-coloured) coccus].
Gram-positive, non-spore-forming cocci. The type of cell-wall diamino acid and sugar is chemotype IV and variation A1. Sugars of the peptidoglycan are glycolated. Polar lipids are composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and some unspecified glycolipids. Carbon-chain length of mycolic acid is C43–C49. Major fatty acids are straight-chain saturated and unsaturated fatty acids, whereas 10-methyl-branched fatty acids are present in trace amounts or absent. Two menaquinone species, SQA-8(H4, -cycl) and SQB-8(H4, dicycl), are present. 16S rRNA and gyrB gene sequence analyses indicate that the genus is a member of the family Nocardiaceae. The type species is Smaragdicoccus niigatensis.
Description of Smaragdicoccus niigatensis sp. nov.
Smaragdicoccus niigatensis (ni.i.ga.ten'sis. N.L. masc. adj. niigatensis, pertaining to the Niigata Prefecture of Japan, the source of the soil from which the organism was isolated).
Cells are coccoid without branching (0.86x0.86 µm). Utilizes the following carbon sources after incubation for 14 days at 30 °C: D-fructose, D-glucose, sodium n-butyrate and hexadecane. After prolonged incubation (1 month) growth occurs with sucrose. Better growth is observed on W-medium containing 0.1 than 1 % of these carbon sources. The following carbon sources are not used for growth: L-arabinose, myo-inositol, D-mannitol, L-rhamnose, raffinose and D-xylose. Positive for activities of esterase (C4), esterase lipase (C8), lipase (C4), leucine arylamidase and naphthol-AS-BI-phosphohydrolase (API ZYM).
The DNA G+C content of the type strain is 63.7 mol%. The type strain is Hou_blueT (=MBIC 06267T=DSM 44881T), which was isolated from soil samples from an oil spring in Niigata, Japan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Butler, W. R., Floyd, M. M., Brown, J. M., Toney, S. R., Daneshvar, M. I., Cooksey, R. C., Carr, J., Steigerwalt, A. G. & Charles, N. (2005). Novel mycolic acid-containing bacteria in the family Segniliparaceae fam. nov., including the genus Segniliparus gen. nov., with descriptions of Segniliparus rotundus sp. nov. and Segniliparus rugosus sp. nov. Int J Syst Evol Microbiol 55, 1615–1624.
Cole, J. R., Chai, B., Farris, R. J., Wang, Q., Kulam, S. A., McGarrell, D. M., Garrity, G. M. & Tiedje, J. M. (2005). The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res 33, D294–D296.
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.
Guindon, S. & Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52, 696–704.[CrossRef][Medline]
Harper, J. J. & Davis, G. H. G. (1979). Two-dimensional thin-layer chromatography for amino acid analysis of bacterial cell walls. Int J Syst Bacteriol 29, 56–58.
Kämpfer, P., Andersson, M. A., Rainey, F. A., Kroppenstedt, R. M. & Salkinoja-Salonen, M. (1999). Williamsia muralis gen. nov., sp. nov., isolated from the indoor environment of a children's day care centre. Int J Syst Bacteriol 49, 681–687.
Katsuta, A., Adachi, K., Matsuda, S., Shizuri, Y. & Kasai, H. (2005). Ferrimonas marina sp. nov. Int J Syst Evol Microbiol 55, 1851–1855.
Klatte, S., Kroppenstedt, R. M. & Rainey, F. A. (1994). Rhodococcus opacus sp. nov., an unusual nutritionally versatile Rhodococcus-species. Syst Appl Microbiol 17, 355–360.
Kroppenstedt, R. M. (1985). Fatty acid and menaquinone analysis of actinomycetes and related organisms. In Chemical Methods in Bacterial Systematics, pp. 173–199. Edited by M. Goodfellow & D. E. Minnikin. No. 20 SAB Technical Series. London: Academic Press.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5, 150–163.
Kuykendall, L. D., Roy, M. A., O'Neill, J. J. & Devine, T. E. (1988). Fatty acids, antibiotic resistance, and deoxyribonucleic acid homology groups of Bradorhizobium japonicum. Int J Syst Bacteriol 38, 358–361.
Lechevalier, H. A. & Lechevalier, M. P. (1970). A critical evaluation of the genera of aerobic actinomycetes. In The Actinomycetales, pp. 393–405. Edited by H. Prauser. Jena: VEB Gustav Fisher Verlag.
Miller, L. T. (1982). Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids. J Clin Microbiol 16, 584–586.
Minnikin, D. E., Patel, P. V., Alshamaony, L. & Goodfellow, M. (1977). Polar lipid composition in the classification of Nocardia and related bacteria. Int J Syst Bacteriol 27, 104–117.
Peng, X., Misawa, N. & Harayama, S. (2003). Isolation and characterization of thermophilic bacilli degrading cinnamic, 4-coumaric, and ferulic acids. Appl Environ Microbiol 69, 1417–1427.
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.
Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. (1997). Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol 47, 479–491.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Uchida, K. & Aida, K. (1977). Acyl type of bacterial cell wall: its simple identification by a colorimetric method. J Gen Appl Microbiol 23, 249–260.[CrossRef]
Wako Pure Chemical Industries (1989). Technical note on the system of PTC-amino acid analysis. Osaka: Wako Pure Chemical Industries (in Japanese).
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