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1 Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn, 53127 Bonn, Germany
2 Kekulé-Institut für Organische Chemie und Biochemie der Universität Bonn, 53121 Bonn, Germany
Correspondence
A. F. Yassin
yassin{at}mibi03.meb.uni-bonn.de
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to accommodate Rhodococcus maris (Nesterenko et al., 1982). Dietzia strains are widely distributed in nature. Dietzia maris was originally isolated from halibut by Harrison (1929) and later from soil, skin and intestinal tracts of carp (Nesterenko et al., 1982), deepest sea mud (Takami et al., 1997) and sediments (Colquhoun et al., 1998), and from a drain of a fish-product-processing plant (Yumoto et al., 2002). D. maris has also been implicated in human diseases. Bemer-Melchior et al. (1999) were the first to report a case of bacteraemia due to D. maris infection associated with a catheter in an immunocompromised patient presenting with septic shock and pneumothorax. D. maris has also been isolated from a bone biopsy specimen from a hospitalized patient associated with a total hip prosthesis replacement (Pidoux et al., 2001). In this paper, a bacterial strain, designated IMMIB RIV-399T, isolated from a perianal swab of a human with a bone marrow transplant is described. Based on phylogenetic and phenotypic evidence, it is proposed that this strain should be classified as a representative of a novel species of the genus Dietzia for which the name Dietzia cinnamea sp. nov. is proposed.
Strains D. maris DSM 43672T, Dietzia natronolimnaea DSM 44860T and Dietzia psychralcaliphila DSM 44820T were obtained from the DSMZ. All strains were cultured on Columbia agar supplemented with 5 % sheep blood agar and brain-heart infusion agar to determine their morphological characteristics. Pigment production was determined during growth at 37 °C for 7 days and observations were made at 24 h intervals. Air-dried smears at 24, 48 and 72 h intervals were Gram-stained to determine the Gram reaction and cell morphology. The Ziehl–Neelsen method as described by Kubica & Kent (1985) was used to determine acid-fastness. Growth temperature was determined by incubating cells at 27, 37 and 42 °C. The oxidase reaction was performed on filter paper moistened with a 1 % (w/v) aqueous solution of N,N,N',N'-tetramethyl-p-phenylenediamine. Physiological properties of the strain were assessed using tests to determine hydrolysis of complex substrates as described previously (Gordon, 1966, 1967; Gordon & Mihm, 1957), as well as tests to determine carbon source utilization according to Yassin et al. (1995). The isomeric form of diaminopimelic acid was determined by the methods of Becker et al. (1964) and whole-cell sugars were determined according to Lechevalier (1968). The acyl type of muramic acid was determined by the colorimetric method (Uchida & Aida, 1977). Lipids were extracted using acid methanolysis and mycolic acids were detected by TLC as described by Minnikin et al. (1980); pyrolysis GC of mycolates was performed according to Yassin et al. (1993a). Non-hydroxylated fatty acids were purified, identified and quantified by GC as described by Yassin (1988). Phospholipids were extracted, purified and identified as described previously (Yassin et al., 1993b). Menaquinones were extracted and purified according to Collins et al. (1977). Analyses of the menaquinones were recorded in positive ion mode on a Q-TOF 2 MS (Micromass) equipped with a nanospray source. Analytes were dissolved in acetonitrile and injected into the MS by glass capillaries (long type; Protona) using a capillary voltage of 950 V and a source block temperature of 80 °C. Instrument calibration was done with a mixture of sodium iodide and caesium iodide dissolved in 50 % aqueous 2-propanol. Collision energy was 35–45 eV at 0·7 bar. For the compounds under study, the major ions observed with electrospray were protonated pseudo-molecular ions, [M+Na]+. The identity of menaquinones was verified by observing the diagnostic ion at m/z 187, which represents the 2-methylnaphthoquinone core.
DNA was isolated as described previously (Yassin et al., 2000). The isolated DNA was then purified by chromatography on hydroxyapatite using the method of Cashion et al. (1977). G+C contents were determined by HPLC (Mesbah et al., 1989) using 8 phage as reference. DNA–DNA hybridization was determined spectrophotometrically from renaturation rates (De Ley et al., 1970; Huß et al., 1983). Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and the purification of PCR products were carried out using procedures described previously (Rainey et al., 1996). Purified PCR products were sequenced using a Taq DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems) according to the manufacturer. An Applied Biosystems 310 DNA Genetic Analyser was used for electrophoresis of the sequence reaction products. The 16S rRNA gene sequences of D. maris DSM 43672T, D. natronolimnaea DSM 44860T and D. psychralcaliphila DSM 44820T determined in this study, as well as those of some representatives of the mycolic-acid-containing taxa retrieved from GenBank, were added to the ARB database (Ludwig et al., 2004) and aligned using the respective tools of the ARB package. The resulting alignment was corrected manually and evolutionary trees were inferred using maximum-parsimony (Kluge & Farris, 1969), neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1981). An evolutionary distance matrix was calculated using the corrections of Jukes & Cantor (1969). Topologies of the resultant trees were evaluated by bootstrap analyses (Felsenstein, 1985) of the neighbour-joining database on 500 resamplings using the ARB package.
The almost complete 16S rRNA gene sequences of strain IMMIB RIV-399T [1486 nt; 96·3 % of the Escherichia coli sequence (Brosius et al., 1978)], D. maris DSM 43672T (1483 nt), D. natronolimnaea DSM 44860T (1484 nt) and D. psychralcaliphila DSM 44820T (1485 nt) were determined in this study; the latter three were identical to sequences of the same strains available from public databases under accession numbers X79270, X92157 and AB159036, respectively, and so the database sequences of these species were used in the comparative analyses. 16S rRNA gene sequence comparison showed clearly that isolate IMMIB RIV-399T is a member of the suborder Corynebacterineae (Stackebrandt et al., 1997) and it contained all signature nucleotides expected for this suborder. Furthermore, in our alignment, strain IMMIB RIV-399T contained 11 of the 14 signature nucleotides defined for this family, whereas D. natronolimnaea DSM 44860T and D. psychralcaliphila DSM 44820T contained 12 of the 14 signature nucleotides defined for the family Dietziaceae. The isolation of a fourth Dietzia species allows signature nucleotides to be highlighted: the pattern of the 16S rRNA gene signatures for the genus Dietzia in the family Dietziaceae Rainey et al. 1997 now consists of 11 signature nucleotide rather than 14 signatures (Stackebrandt et al., 1997). These are nucleotides at positions 70–98 (U–A), 293–304 (G–U), 307 (U), 418–425 (U–A), 661–744 (A–U), 771–808 (A–U), 824–876 (C–G), 825–875 (G–C), 843 (C), 1049–1198 (U–A) and 1122–1151 (A–U). Strain IMMIB RIV-399T, D. natronolimnaea DSM 44860T and D. psychralcaliphila DSM 44820T share the same nucleotides at position 508 (C). D. natronolimnaea DSM 44860T and D. psychralcaliphila DSM 44820T have the same nucleotides at positions 614–626 (C–G). Strain IMMIB RIV-399T differs from other Dietzia species in that it possesses U rather than G at position 631.
The phylogenetic tree (Fig. 1) shows the position of strain IMMIB RIV-399T within the radiation of representative phylogenetic groups of the suborder Corynebacterineae. The phylogenetic position of this organism, determined by the different tree algorithms (maximum-parsimony, neighbour-joining and maximum-likelihood), reveals that strain IMMIB RIV-399T is closely associated with the genus Dietzia and represents a distinct subline within its members (Fig. 1). The determined sequence displays similarities of 97·5, 97·7 and 98·4 % to D. natronolimnaea DSM 44860T, D. psychralcaliphila DSM 44820T and D. maris DSM 43672T, respectively. This result suggests that strain IMMIB RIV-399T belongs to a genetically distinct Dietzia species that is closely related to D. maris (1·6 % sequence divergence). In view of the high levels of 16S rRNA gene sequence similarity between isolate IMMIB RIV-399T and D. natronolimnaea DSM 44860T, D. psychralcaliphila DSM 44820T and D. maris DSM 43672T, chromosomal DNA–DNA hybridization studies were performed to establish whether strain IMMIB RIV-399T represents a distinct species. Strain IMMIB RIV-399T displayed low levels of DNA–DNA reassociation with D. natronolimnaea DSM 44860T (34·2 %), D. psychralcaliphila DSM 44820T (35·7 %) and D. maris DSM 43672T (40·3 %), results that are below the cut-off point recommended for the circumscription of bacterial genomic species by Wayne et al. (1987) and that confirm the separation of isolate IMMIB RIV-399T from D. natronolimnaea, D. psychralcaliphila and D. maris.
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). The muramic acid residues of the peptidoglycan are N-acetylated. One-dimensional TLC of whole-cell acid methanolysates of the organism revealed the presence of two lipid spots on the chromatogram. The lower one corresponded to mycolic acids, as identified by RF value (0·48), and the higher spot corresponded to non-hydroxylated fatty acids. Pyrolysis GC of the purified mycolic acid methyl esters from strain IMMIB RIV-339T released fatty acid methyl esters of C14 : 0, C15 : 0, C16 : 0, C17 : 0, C18 : 1 and C18 : 0 as pyrolysis cleavage products. GC analyses of the non-hydroxylated fatty acid methyl esters revealed the presence of tetradecanoate (0·8 % total fatty acids), pentadecanoate (8·3 %), cis-hexadecenoate (2·8 %), hexadecanoate (28·9 %), 10-methyl hexadecanoate (3·2 %), heptadecenoate (5·3 %), heptadecanoate (11·7 %), 10-methyl heptadecanoate (11·1 %), octadecenoate (4·8 %), octadecanoate (2·3 %) and tuberculostearic acid (10-methyl octadecanoate, 28·8 %) as the major cellular fatty acid methyl esters. Polar lipid analysis showed that the organism contains phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as the characteristic phospholipids (i.e. phospholipid type PII sensu Lechevalier et al., 1977). MS analysis of the main component from strain IMMIB RIV-399T shows a strong peak at m/z 741·55 attributable to [M+Na]+ in the high mass region. This corresponds to a dihydrogenated menaquinone with eight isoprene units [MK-8(H2)]. The second band shows a strong peak at m/z 673·50, attributable to [M+Na]+ in the high mass region. This corresponds to a dihydrogenated menaquinone with seven isoprene units [MK-7(H2)]. The results of triplicate determinations gave a DNA G+C content for strain IMMIB RIV-399T of 72·3±0·4 mol%. It is apparent from the genotypic and phenotypic data that strain IMMIB RIV-399T represents a novel species of the genus Dietzia, for which the name Dietzia cinnamea is proposed.
Description of Dietzia cinnamea sp. nov.
Dietzia cinnamea (cin.na.me'a. L. fem. adj. cinnamea of/from cinnamon referring to the colour of the cellular biomass).
Forms smooth, yellow-pigmented colonies on agar media. Cells are rod-shaped, exhibit snapping division and produce V-forms. Gram-positive and not acid-fast. Aerobic, catalase-positive, oxidase-negative. Grows at temperatures of 22–45 °C. Has the salient chemotaxonomic characteristics of the genus Dietzia. The glycan moiety of the cell walls contains N-acetyl residues (N-acetylmuramic acid). Cleavage of mycolic acids by pyrolysis releases fatty acids of C14 : 0, C15 : 0, C16 : 0, C17 : 0, C18 : 1 and C18 : 0 as the major products. The fatty acid profile mainly consists of straight-chain saturated, unsaturated and 10-methyl branched fatty acids. MK-8(H2) is the major menaquinone component and MK-7(H2) is the minor one. The phospholipid type is PII, with phosphatidylethanolamine as diagnostic phospholipid. Hydrolyses testosterone and urea, but not adenine, casein, elastin, aesculin, gelatin, guanine, hypoxanthine, tyrosine or xanthine. Assimilates acetate, D-glucose, maltose and 1,2-propanediol as carbon sources, but not adonitol, adipate, iso-amylalcohol, L-arabinose, 2,3-butanediol, cellobiose, citrate, meso-erythritol, D-galactose, gluconate, m-hydroxybenzoate, p-hydroxybenzoate, myo-inositol, lactate, lactose, mannitol, melezitose, raffinose, rhamnose, D-sorbitol, sucrose, trehalose or D-xylose. Unable to utilize acetamide, L-alanine, arginine, gelatin, ornithine, proline or serine as simultaneous carbon and nitrogen sources.
The type strain of Dietzia cinnamea, IMMIB RIV-399T (=DSM 44904T=CCUG 50875T), was isolated from a perianal swab of a patient with a bone marrow transplant.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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