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1 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
2 The Key Laboratory for Microbial Resources of Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan 650091, PR China
Correspondence
Peter Schumann
psc{at}dsmz.de
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ABSTRACT |
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L-lys–L-ser–D-Glu and major menaquinone MK-9, as well as in its DNA base composition (57 mol% G+C), to its phylogenetic neighbour Jonesia denitrificans. Differences in phenotypic characteristics and the phylogenetic distance (96·6 % 16S rRNA gene sequence similarity) from J. denitrificans justify the proposal of a second species of the genus Jonesia, Jonesia quinghaiensis sp. nov., with the type strain QH3A7T (=DSM 15701T=CGMCC 1.3459T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain QH3A7T (=DSM 15701T=CGMCC 1.3459T) is AJ626896.
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MAIN TEXT |
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), resulting in the exclusion of Listeria denitrificans Prévot 1961 from the genus. Major differences in the DNA G+C value (mol%), peptidoglycan structure, lipid pattern, isoprenoid quinone and the 16S rRNA oligonucleotides supported the taxonomic placement of J. denitrificans within the order Actinomycetales. The description of the genus Jonesia is based on a single isolate. In the present study, a coryneform strain was isolated from soda lake mud in China which showed Jonesia-specific characteristics, such as peptidoglycan structure and menaquinone pattern. Based on phenotypic differences and phylogenetic distance from J. denitrificans, a second species of the genus Jonesia, Jonesia quinghaiensis sp. nov., is proposed.
Strain and culture conditions
Strain QH3A7T was isolated from a mud sample of a soda lake (approx. pH 9) in the west of China. Isolation was done at 28 °C by dilution plating on Bacto marine broth agar (MBA), pH 7·2. The reference strain J. denitrificans DSM 20603T was inoculated on tryptic soy broth agar (TSBA, containing 1·5 % Difco agar; Difco) and on MBA, and all morphological and physiological studies were performed with cells grown on these media. For chemotaxonomic analyses, the organism was grown in tryptic soy broth in flasks on a rotary shaker at 90 r.p.m. and 28 °C. The biomass was harvested by centrifugation, washed twice with distilled water and freeze-dried.
Microscopy and cultural characteristics
The cells were stained after 3–5 days cultivation for Gram's test according to the procedure described by Doetsch (1981). Cells were observed and photographed with a phase-contrast microscope (ZEISS Axiophot) equipped with a Plan-Neofluar objective (100/1·3, oil) and a camera (Sony 3CCD). A micrograph of the Gram-positive cells is shown in Fig. 1.
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. Electrophoresis of sequencing reaction products was done by using a Beckman CEQ 2000 sequencer according to the manufacturer's protocols. The resulting 16S rRNA gene sequence was compared to sequences from the GenBank database to find the most closely related species. CLUSTAL_X (Thompson et al., 1997) was used to align the QH3A7T sequence together with those of representatives of each family in the suborder Micrococcineae and all members of the family Jonesiaceae (Stackebrandt et al., 1997). The resulting 16S rRNA gene alignment was then manually checked by using BioEdit 5.0.9 (Hall, 1999). A dataset of 16 sequences with 1425 nucleotide positions that could be aligned unambiguously was used for calculating the distance matrix corrected by using Kimura's 2-parameter method (Kimura, 1980). Phylogenetic trees were inferred using the neighbour-joining method (Saitou & Nei, 1987), and bootstrap analysis was based on 1000 resamplings.
Phylogenetic analyses of the almost-complete 16S rRNA gene sequence of strain QH3A7T (1425 nucleotides) revealed this strain to be closely related to J. denitrificans DSM 20603T (96·6 % sequence similarity). The 16S rRNA gene sequence of strain QH3A7T contained the signature nucleotides of the family Jonesiaceae (Stackebrandt & Schumann, 2000). Similarity values with sequences of neighbouring taxa were significantly lower (91·2–93·2 %). A dendrogram of relationships (Fig. 2) confirms strains QH3A7T and DSM 20603T to be phylogenetic neighbours.
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The results of the physiological tests of strain QH3A7T and J. denitrificans DSM 20603T are shown in Table 1. The two strains differ from each other in several physiological properties.
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. Peptidoglycan structure was elucidated by analyses of cell wall hydrolysates employing the following methods: qualitative analysis of amino acids and peptides by two-dimensional TLC on cellulose plates using solvent systems described previously by Schleifer & Kandler, (1972), quantitative amino acid analysis by GC and GC-MS (MacKenzie, 1987; Groth et al., 1996) and dinitrophenylation of N-terminal amino acids of the interpeptide bridge (Schleifer, 1985). Sugar analysis of the purified cell wall was performed as described by Staneck & Roberts (1974). Polar lipids extracted by the method of Minnikin et al. (1979) were identified by two-dimensional TLC and spraying with specific reagents (Collins & Jones, 1980). Menaquinones were analysed by HPLC as described previously by Groth et al. (1996). Fatty acid methyl esters were extracted and prepared according to the standard protocol of the Microbial Identification System (MIDI; Microbial ID). Extracts were analysed by using a Hewlett Packard model HP6890A gas chromatograph equipped with a flame-ionization detector as described previously (Kämpfer & Kroppenstedt, 1996).
The cellular fatty acid pattern consists of 52·8 % ai-C15 : 0, 13·9 % i-C16 : 0, 11·5 % C16 : 0, 5·9 % ai-C17 : 0, 4·8 % i-C14 : 0, 3·8 % C14 : 0, 3·7 % i-C15 : 0, 1·8 % ai-C15 : 1, 0·6 % ai-C13 : 0, 0·6 % C15 : 0, 0·3 % C12 : 0 and 0·3 % i-C17 : 0. Menaquinones were MK-9, MK-8 and MK-7 (peak area ratio, 76 : 10 : 10, respectively). Phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and two unknown phospholipids were found as polar lipids. Galactose was detected as the only cell wall sugar. The peptidoglycan of strain QH3A7T contained the amino acids Lys, Ser, Ala and Glu (1·0 : 0·9 : 2·1 : 2·3 mol, respectively). Glu was found to represent the N terminus of the interpeptide bridge. From these data and from the occurrence of characteristic di- and tri-peptides in the partial hydrolysate of the peptidoglycan (data not shown), the peptidoglycan type A4 L-lys-L-ser-D-Glu, A11.48 (DSMZ, 2001) was concluded. This peptidoglycan type is identical to that found in J. denitrificans. Similarities also exist in the cellular fatty acid, menaquinone and polar lipid patterns (Rocourt et al., 1987; Bernard et al., 1991).
Determination of DNA base composition
DNA was isolated using a French pressure cell and purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). The G+C content was determined by reversed-phase HPLC of nucleosides according to Mesbah et al. (1989). The G+C value of 57·3 mol% is within the range of the DNA base content of the genus Jonesia (Rocourt et al., 1987).
On the basis of phylogenetic, chemotaxonomic and phenotypic results, we propose strain QH3A7T to represent a novel species of the genus Jonesia, Jonesia quinghaiensis sp. nov.
Description of Jonesia quinghaiensis sp. nov.
Jonesia quinghaiensis (quing.hai.en'sis, N.L. fem. adj. pertaining to Qinghai, Western province of China, where the type strain was isolated).
Gram-positive, non-acid-fast. On TSBA medium rhizoidal colonies are 4 mm in diameter after 7 days incubation at 28 °C. On MBA medium light-yellowish colonies (about 0·5 mm in diameter) with rhizoid appearance (diameter 4–5 mm). Cells are rod-shaped (0·5x1·5–2 µm) and non-motile (Fig. 1
). Optimal growth temperature is 20
30 °C; optimal pH is 79; optimal salt concentration is 2·0–7·5 % (w/v) NaCl. The peptidoglycan type is A4 L-lys-L-ser-D-Glu. MK-9 is the predominant menaquinone. The polar lipids consist of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and two unknown phospholipids. The major cellular fatty acids are ai-C15 : 0, i-C16 : 0 and C16 : 0. Physiological properties are indicated in Table 1. Isolated from mud of a soda lake in China.
Type strain is QH3A7T (=DSM 15701T=CGMCC 1.3459T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cashion, P., Holder-Franklin, M. A., McCully, J. & Franklin, M. (1977). A rapid method for the base ratio determination of bacterial DNA. Anal Biochem 81, 461–466.[CrossRef][Medline]
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-gentobiose, D-turanose and 5-ketogluconate. Neither strain utilized (
, negative reaction also in the Biolog GP2 substrate panel): erythritol, D-arabinose, ribose, L-xylose, adonitol, methyl