Tsukamurella spongiae sp. nov., a novel actinomycete isolated from a deep-water marine sponge Julie B. Olson, 1 Dedra K. Harmody, 2 Asim K. Bej 3 and Peter J. McCarthy 2 Correspondence Julie B. Olson jolson@bama.ua.edu 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 A Gram-positive, rod-shaped, non-spore-forming bacterium (strain K362 T ) was isolated from a deep-water marine sponge collected off the coast of Curac ¸ ao in the Netherlands Antilles. On the basis of 16S rRNA gene sequence similarities, strain K362 T was shown to belong to the genus Tsukamurella, being most closely related to Tsukamurella pulmonis (99.2 %), Tsukamurella tyrosinosolvens (98.9 %), Tsukamurella strandjordii (98.8 %), Tsukamurella pseudospumae (98.8 %) and Tsukamurella spumae (98.8 %). A combination of the substrate utilization patterns, the fatty acid and mycolic acid profiles and the DNA–DNA hybridization results supported the affiliation of strain K362 T to the genus Tsukamurella and enabled the genotypic and phenotypic differentiation of strain K362 T from the seven recognized Tsukamurella species. Strain K362 T therefore represents a novel species of the genus Tsukamurella, for which the name Tsukamurella spongiae sp. nov. is proposed. The type strain is K362 T (=DSM 44990 T =NRRL B-24467 T ). The genus Tsukamurella was proposed by Collins et al. (1988), 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 K362 T was isolated from a deep-water marine hexactinellid sponge with associated zoanthids collected off the coast of Curac ¸ao (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 uC for 15 min) and plated onto maltose-seawater agar (Olson et al., 2000). Strain K362 T was isolated after 28 days incubation in the dark at ambient temperature (20–25 u 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 uC) 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 H 2 O) at 22 u 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 The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain K362 T is AY714239. The fatty acid profiles of strain K362 T and other species of the genus Tsukamurella are presented in a supplementary table with the online version of this paper. 1478 64837 G 2007 IUMS Printed in Great Britain International Journal of Systematic and Evolutionary Microbiology (2007), 57, 1478–1481 DOI 10.1099/ijs.0.64837-0