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1 Division of Biology, King George VI Building, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
2 Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
3 School of Applied Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, UK
4 Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK
Correspondence
Michael Goodfellow
m.goodfellow{at}ncl.ac.uk
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Present address: Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. 
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to accommodate an unusual mycolic-acid-containing actinomycete. The taxon currently contains three recognized species, Williamsia muralis Kämpfer et al. 1999, Williamsia maris Stach et al. 2004 and Williamsia deligens Yassin and Hupfer 2006, species that encompass strains isolated from indoor building materials, deep-sea sediment and human blood, respectively. These species form a distinct 16S rRNA clade within the evolutionary radiation occupied by mycolic-acid-containing actinomycetes, i.e. by organisms classified in the suborder Corynebacterineae Stackebrandt et al. 1997 (Butler et al., 2005; Soddell et al., 2006).
The present investigation was designed to determine the taxonomic status of an actinobacterial strain, isolate MT8T, that had been isolated from a deep-sea sediment and was considered to represent a member of the genus Williamsia (Pathom-aree et al., 2006). The isolate was the subject of a polyphasic taxonomic study, which showed that it merited recognition within a novel species of the genus Williamsia.
Strain MT8T was isolated from sediment collected from the Mariana Trench in the north-west Pacific Ocean [Challenger Deep (10 898 m): 11° 19.911' N 142° 12.372' E]) using the remotely operated unmanned submersible Kaiko (Kato et al., 1997). The sample (2 ml), which was collected during dive number 74 on 21 May 1998, was transported to the UK in an insulated container at 4 °C and then stored at –20 °C. The isolate was recovered from a suspension of the sediment sample used to inoculate a raffinose–histidine agar plate (Vickers et al., 1984) supplemented with cycloheximide and nystatin. The organism was maintained on glucose–yeast extract agar (Gordon & Mihm, 1962) at room temperature and as glycerol suspensions (20 %, v/v) at –20 °C.
The phylogenetic position of isolate MT8T was determined by 16S rRNA gene sequence analysis. The organism was grown at 28 °C for 7 days in a glucose–yeast extract shake culture (Gordon & Mihm, 1962), and the resultant biomass was harvested by centrifugation and washed twice in distilled water. Isolation of chromosomal DNA, PCR amplification and direct sequencing of the purified products of the strain were carried out according to the methods of Kim et al. (2000). The almost complete 16S rRNA gene sequence (1443 nt) was aligned manually with corresponding sequences of representatives of the genera classified in the suborder Corynebacterineae, retrieved from the DDBJ/EMBL/GenBank databases, using the pairwise alignment option and 16S rRNA secondary structure information held within the PHYDIT program (available at http://plaza.snu.ac.kr/
jchun/phydit/).
Phylogenetic trees were inferred using the least-squares (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) tree-making algorithms from the PHYLIP suite of programs (Felsenstein, 1993). Evolutionary distance matrices were generated for the least-squares and neighbour-joining methods after Jukes & Cantor (1969) and the resultant unrooted tree topologies were evaluated in a bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings from the neighbour-joining dataset using the SEQBOOT and CONSENSE options from the PHYLIP package. It is evident from Fig. 1 that isolate MT8T not only falls within the zone of evolutionary radiation occupied by the genus Williamsia but forms a distinct phyletic line together with the type strain of W. muralis, an association underpinned by all of the tree-making algorithms and by a 100 % bootstrap value in the neighbour-joining analysis. The two organisms share 99.5 % 16S rRNA gene similarity, a value that corresponds to 7 nt differences over the 1450 locations available for alignment.
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) as described above. Standard procedures were used to extract and to analyse the isomeric forms of diaminopimelic acid (Staneck & Roberts, 1974), fatty acids (Sutcliffe, 2000), mycolic acids (Hamid et al., 1993), isoprenoid quinones (Collins, 1994), muramic acid type (Uchida et al., 1999), polar lipids (Minnikin et al., 1984) and whole-organism sugars (Hasegawa et al., 1983). The organism was characterized by the presence of meso-diaminopimelic acid, arabinose and galactose in whole-organism hydrolysates (wall chemotype IV sensu Lechevalier & Lechevalier, 1970), N-glycolyl muramic acid residues, dihydrogenated menaquinones with nine isoprene units as the sole isoprenologue, major proportions of straight-chain, unsaturated and tuberculostearic acids (fatty acid type 1b; Kroppenstedt, 1985), phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as major polar lipids (phospholipid type II sensu Lechevalier et al., 1977) and by mycolic acids that co-migrated with those of W. muralis DSM 44343T. This chemical profile is consistent with the classification of the isolate in the genus Williamsia (Goodfellow & Maldonado, 2006; Yassin & Hupfer, 2006).
The fatty acid profile was dominated by palmitic acid (C16 : 0, 30 % of total), a hexadecenoic acid (C16 : 1, 22 %), oleic acid (C18 : 1, 15 %) and tuberculostearic acid (10-methyl octadecanoate, 32 %). This profile is generally consistent with that reported for the type strain of W. muralis (Kämpfer et al., 1999), except that the latter organism was reported to contain significant quantities of palmitoleic acid (C16 : 1cis9) whereas we detected a fatty acid provisionally identified as a hexadecenoic acid but clearly resolved from a palmitoleic acid standard. In this respect it is notable that W. muralis DSM 44343T was reported to contain a minor amount of C16 : 1cis11 (1.4 %; Kämpfer et al., 1999). The fatty acid profiles of MT8T and W. muralis DSM 44343T are thus both notably distinct from that reported for the type strain of W. deligens as the latter contained only minor amounts of C16 : 1 and significant quantities (40 %) of longer chain (
C20) saturated and unsaturated fatty acids (Yassin & Hupfer, 2006).
DNA–DNA hybridization experiments were carried out between isolate MT8T and the type strains of W. maris and W. muralis using DNA prepared following the methods of Kim et al. (1998). The analyses were performed using the microplate method, as described by Ezaki et al. (1989). Mean DNA–DNA relatedness values were calculated from triplicate hybridization experiments. The G+C content of the DNA of isolate MT8T was determined by reversed-phase HPLC (Tamaoka & Komagata, 1984). The mean levels of DNA–DNA relatedness found between isolate MT8T and W. maris DSM 44693T and W. muralis DSM 44343T were 10±2.8 and 11±0 %, respectively, values well below the cut-off point recommended for the assignment of bacterial strains to the same genomic species (Wayne et al., 1987). The DNA G+C content of strain MT8T was 65.2 mol%.
Isolate MT8T was examined for a range of phenotypic markers using established procedures (Stach et al., 2004). It is evident from Table 1 that it can be differentiated readily from the type strains of the three recognized Williamsia species. It is particularly interesting that only isolate MT8T grows at 4 °C and in the presence of high concentrations of sodium chloride.
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Description of Williamsia marianensis sp. nov.
Williamsia marianensis (ma.ri.an.en'sis. N.L. fem. adj. marianensis pertaining to the Mariana Trench, the source of the type strain).
Non-acid–alcohol-fast actinomycete that forms short rods and coccoid-like elements, and produces round, entire, convex, orange colonies with smooth matt surfaces on glucose–yeast extract agar after 5 days incubation at 28 °C. Grows well on trypticase soy agar, and between 4 and 30 °C, with an optimum temperature for growth around 28 °C. Neither aesculin nor arbutin are hydrolysed. Casein, cellulose, hypoxanthine, starch and uric acid are degraded, but not gelatin, guanine, Tween 80, L-tyrosine or xanthine. D(+)-Fructose, D(+)-mannitol, D(+)-mannose, sucrose, D(+)-sorbitol and xylitol are used as sole carbon sources for energy and growth, but not L(–)-arabitol, D(+)-cellobiose, dextran, dextrin, D(+)-glycerol, glycogen, D(+)-melezitose, D(+)-raffinose, D(+)-salicin or L(+)-sorbose. Additional phenotypic properties are given in Table 1
. Exhibits chemical markers characteristic of the genus Williamsia. The predominant fatty acid components are palmitic and tuberculostearic acids. The G+C content of the DNA is 65.4 mol%.
The type and only strain, MT8T (=DSM 44944T=NCIMB 14085T), was isolated from sediment collected from the Challenger Deep of the Mariana Trench in the north-west Pacific Ocean.
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ACKNOWLEDGEMENTS |
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