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1 Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA
2 Harbor Branch Oceanographic Institution, Division of Biomedical Marine Research, 5600 US 1 North, Fort Pierce, FL 34946, USA
3 Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
Correspondence
Julie B. Olson
jolson{at}bama.ua.edu
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ABSTRACT |
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The fatty acid profiles of strain K362T and other species of the genus Tsukamurella are presented in a supplementary table with the online version of this paper.
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MAIN TEXT |
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, the type species being Tsukamurella paurometabola. This taxon currently contains seven species (in addition to the type species) with validly published names. Species recovered from clinical specimens include Tsukamurella inchonensis (Yassin et al., 1995), Tsukamurella pulmonis (Yassin et al., 1996), Tsukamurella strandjordii (Kattar et al., 2001) and Tsukamurella tyrosinosolvens (Yassin et al., 1997). The remaining species were associated with activated sludge foaming and include Tsukamurella pseudospumae (Nam et al., 2004) and Tsukamurella spumae (Nam et al., 2003). These species form a distinct clade within the evolutionary radiation of the mycolate actinomycetes and share very high 16S rRNA gene sequence similarity values (Kattar et al., 2001; Nam et al., 2003).
Strain K362T was isolated from a deep-water marine hexactinellid sponge with associated zoanthids collected off the coast of Curaçao (the Netherlands Antilles), at a depth of 220 m, using the Harbor Branch Oceanographic Institution's Johnson-Sea-Link II submersible. A small section of the sponge (approx. 15 g wet weight, including both the pinacoderm and mesohyl regions) was gently rinsed in sterile natural seawater, cut into smaller pieces and then homogenized at low speed (5000 r.p.m.) with an ethanol-sterilized high-speed homogenizer (VirTis). The sponge suspension was heat-treated (70 °C for 15 min) and plated onto maltose-seawater agar (Olson et al., 2000). Strain K362T was isolated after 28 days incubation in the dark at ambient temperature (20–25 °C). Colonies were transferred to fresh plates of the isolation media and ultimately maintained on slants of marine agar 2216 (Becton Dickinson). Morphological observations were made with a light microscope (BH-2; Olympus), using cultures grown in marine broth [5 g peptone, 1 g yeast extract, 1 ml trace metal solution (Olson et al., 2000), 1 l artificial seawater (Sieburth, 1979)].
Tolerance of various temperatures (10, 25, 30, 37, 46 and 55 °C) was tested using marine agar. Tolerance of salt (NaCl at 0, 0.5, 1.0, 2.0, 3.0 and 4.0 %, w/v; KCl at 0, 1.0, 2.0, 3.0 and 4.0 %, w/v) was determined using modified marine broth (5 g peptone, 1 g yeast extract, 1 ml trace metal solution, 1 l distilled H2O) at 22 °C with agitation.
The ability of the organism to grow on a range of sole carbon sources was examined using the basal medium of Boiron et al. (1993) and the methods employed by Nam et al. (2003, 2004). Fatty acid and mycolic acid analyses were performed on a fee-for-service basis by Microbial ID, Inc. (USA), using strain K362T grown on nutrient agar at room temperature for 48–72 h.
Extraction of genomic DNA and PCR amplification using universal bacterial primers 8F and 1492R were performed as described by Olson et al. (2002). Amplified fragments were ligated into plasmid pCR 2.1 (TA cloning kit; Invitrogen) and used to transform Escherichia coli according to the manufacturer's instructions. Plasmids with inserts of the correct size were sequenced at the Macrogen (Korea) sequencing facility.
Total genomic DNA from strains K362T, T. pulmonis IMMIB D-1321T and T. strandjordii BAA-173T was purified using the procedure described by Ausubel et al. (1987). Genomic DNA (500 µg) from each strain was sonicated to generate DNA fragments of 400–600 bp. The concentration and purity of the resulting DNA were determined from the A260 and the A260/A280 ratio.
Melting temperatures (Tm) were determined using previously described procedures (De Ley et al., 1970; Gillis et al., 1970). DNA (30 µg) from each of the three strains was denatured in 16x SSC buffer (pH 7.0) by increasing the temperature of the sample from 32 to 98 °C (at a rate of 0.7–1.0 °C min–1); optical density measurements were then recorded at 260 nm. The melting-temperature analysis was confirmed in 16x SSC buffer (pH 7.0) by using SYBR Green I (Roche Applied Science) double-stranded DNA-binding dye and a Cepheid Smart Cycler instrument (Panicker et al., 2004). The Tm values for each DNA sample were recorded from the fluorescent readings obtained with the SYBR Green I dye following 50 % dissociation of the DNA. The DNA G+C content was determined by using the equation of Xu et al. (2000) with the value for G+C content (mol%) for T. pulmonis as reported by Yassin et al. (1996).
DNA–DNA hybridization was performed by using DNA reassociation kinetics as described previously (De Ley et al., 1970; Johnson, 1985). Purified, sonicated genomic DNA (65 µg) from each strain was used according to the protocol of Pikuta et al. (2005).
On marine agar, strain K362T formed dry, matte, cream-coloured colonies with irregular spreading margins and raised, wrinkled, rough centres. Wet mounts prepared from cells grown in marine broth showed large clusters of non-motile short rods (1x2–5 µm). Strain K362T showed growth at 0–4 % KCl and NaCl (w/v), the optimum salt concentration being 0–1 % (w/v).
A comparison of substrate utilization patterns showed strain K362T to be distinct from the recognized species of the genus Tsukamurella. Some characteristic and differential properties of Tsukamurella species are given in Table 1. The fatty acid composition of strain K362T was found to be typical of those of members of the genus Tsukamurella (see the comparison with other species presented in Supplementary Table S1, available in IJSEM Online) and was composed of C18 : 1
9c (40.7 %), C16 : 0 (27.7 %), summed feature 3 (C16 : 17c and/or C15 iso 2-OH; 10.3 %), C20 : 19c (8.5 %), C14 : 0 (3.8 %), C18 : 0 (3.0 %), tuberculostearic acid (1.5 %), C20 : 0 (1.1 %), C17 : 0 (0.86 %), summed feature 6/7 (C19 : 19c and/or C19 : 111c; 0.70 %), C17 : 18c (0.66 %), C15 : 0 (0.56 %), C16 : 19c (0.54 %) and C12 : 0 (0.25 %). Analysis of the mycolic acids indicated that those present in strain K362T (number of carbon atoms: 58–75) were also typical of those present in other tsukamurellae and that the size range of the mycolic acids was representative of that of recognized Tsukamurella strains (64–78 carbon atoms; Collins et al., 1988).
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The Tm of the genomic DNA of strain K362T was 75±0.85 °C (mean±SD, n=3), whereas it was 73±0.3 °C for T. pulmonis IMMIB D-1321T and 62±1.6 °C for T. strandjordii BAA-173T. No significant differences in the Tm values determined with the spectrophotometer and the Cepheid Smart Cycler were noticed. The G+C content of the DNA of strain K362T is 74.6 mol%.
DNA–DNA hybridization between strain K362T and closely related strains (selected on the basis of 16S rRNA gene sequence similarity) was performed. The levels of relatedness between strain K362T and T. pulmonis IMMIB D-1321T [48±1.3 % (mean±SD, n=3)], strain K362T and T. strandjordii BAA-173T (44±1.2 %), and T. pulmonis IMMIB D-1321T and T. strandjordii BAA-173T (41±0.8 %) indicate that strain K362T can be considered as a novel taxon.
On the basis of phenotypic and genotypic characteristics (cellular and colonial morphology, salt tolerance, the substrate utilization pattern, the fatty acid and mycolic acid profiles, 16S rRNA gene sequence and DNA–DNA hybridization data), strain K362T represents a novel species of the genus Tsukamurella, for which the name Tsukamurella spongiae sp. nov. is proposed.
Description of Tsukamurella spongiae sp. nov.
Tsukamurella spongiae (spon'gi.ae. L. gen. n. spongiae of a sponge, referring to the source of isolation, a deep-water sponge).
Aerobic, Gram-positive, non-motile, non-spore-forming actinomycete. Cells are straight to slightly curved rods. Growth is observed on brain-heart infusion agar, nutrient agar and marine agar after 24–48 h incubation at ambient temperature (approx. 25 °C). Optimal temperature for growth is 25–37 °C. No growth occurs at 10 °C or above 45 °C. On all media, colonies are dry, matte and cream-coloured with irregular spreading margins and raised, wrinkled, rough centres. Colonies range in size from 2 to 5 mm in diameter and show irregular elevation. Utilizes (+)-D-galactose, (+)-D-glucose, (+)-D-mannose, 
-L-rhamnose, (+)-D-sucrose and (+)-D-trehalose as sole carbon sources, in addition to those listed in Table 1
. Does not utilize amyl alcohol (1 %, v/v) or methanol (1 %, v/v). Tolerates NaCl concentrations up to 4 %, but growth is enhanced at lower salt concentrations. The major cellular fatty acids of the type strain are C18 : 19c (40.7 %), C16 : 0 (27.7 %) and C16 : 17c and/or C15 iso 2-OH (10.3 %). Tuberculostearic acid (1.5 %) is also present. Contains mycolic acids with 58–75 carbon atoms.
The type strain, strain K362T (=DSM 44990T=NRRL B-24467T), was isolated from a deep-water sponge collected off the coast of Curaçao in the Netherlands Antilles, at a depth of 220 m.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Boiron, P., Provost, F. & Duport, B. (1993). Laboratory Methods for the Diagnosis of Nocardiosis, pp. 107–126. Paris: Institut Pasteur.
Collins, M. D., Smida, J., Dorsch, M. & Stackebrandt, E. (1988). Tsukamurella gen. nov., harboring Corynebacterium paurometabolum and Rhodococcus aurantiacus. Int J Syst Bacteriol 38, 385–391.
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurements of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
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Kattar, M. M., Cookson, B. T., Carlson, L. D., Stiglich, S. K., Schwartz, M. A., Nguyen, T. T., Daza, R., Wallis, C. K., Yarfitz, S. L. & Coyle, M. B. (2001). Tsukamurella strandjordae sp. nov., a proposed new species causing sepsis. J Clin Microbiol 39, 1467–1476.
Nam, S.-W., Chun, J., Kim, S., Kim, W., Zakrzewska-Czerwinska, J. & Goodfellow, M. (2003). Tsukamurella spumae sp. nov., a novel actinomycete associated with foaming in activated sludge plants. Syst Appl Microbiol 26, 367–375.[CrossRef][Medline]
Nam, S.-W., Kim, W., Chun, J. & Goodfellow, M. (2004). Tsukamurella pseudospumae sp. nov., a novel actinomycete isolated from activated sludge foam. Int J Syst Evol Microbiol 54, 1209–1212.
Olson, J. B., Lord, C. C. & McCarthy, P. J. (2000). Improved recoverability of microbial colonies from marine sponge samples. Microb Ecol 40, 139–147.[Medline]
Olson, J. B., Harmody, D. K. & McCarthy, P. J. (2002). Alpha-proteobacteria cultivated from marine sponges display branching rod morphology. FEMS Microbiol Lett 211, 169–173.[Medline]
Panicker, G., Vickery, M. C. & Bej, A. K. (2004). Multiplex PCR detection of clinical and environmental strains of Vibrio vulnificus in shellfish. Can J Microbiol 50, 911–922.[CrossRef][Medline]
Pikuta, E. V., Marsic, D., Bej, A., Tang, J., Krader, P. & Hoover, R. B. (2005). Carnobacterium pleistocenium sp. nov., a novel psychrotolerant, facultative anaerobe isolated from permafrost of the Fox Tunnel in Alaska. Int J Syst Evol Microbiol 55, 473–478.
Sieburth, J. M. (1979). In Sea Microbes, p. 125. Oxford University Press: New York.
Xu, H.-X., Kawamura, Y., Li, N., Zhao, L., Li, T.-M., Li, Z.-Y., Shu, S. & Ezaki, T. (2000). A rapid method for determining the G+C content of bacterial chromosomes by monitoring fluorescence intensity during DNA denaturation in a capillary tube. Int J Syst Evol Microbiol 50, 1463–1469.[Abstract]
Yassin, A. F., Rainey, F. A., Brzezinka, H., Burghardt, J., Lee, H. J. & Schaal, K. P. (1995). Tsukamurella inchonensis sp. nov. Int J Syst Bacteriol 45, 522–527.
Yassin, A. F., Rainey, F. A., Brzezinka, H., Burghardt, J., Rifai, M., Seifert, P., Feldmann, K. & Schaal, K. P. (1996). Tsukamurella pulmonis sp. nov. Int J Syst Bacteriol 46, 429–436.
Yassin, A. F., Rainey, F. A., Burghardt, J., Brzezinka, H., Schmitt, S., Seifert, P., Zimmermann, O., Mauch, H., Gierth, D. & other authors (1997). Tsukamurella tyrosinosolvens sp. nov. Int J Syst Bacteriol 47, 607–614.
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