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Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, PO Box 124, SE-221 00 Lund, Sweden
Correspondence
Rajni Hatti-Kaul
rajni.hatti-kaul{at}biotek.lu.se
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. The G+C content of the genomic DNA was 69.0 mol%. Sequence analysis of 16S rRNA gene sequence of strain DSM 17733T placed the isolate in the genus Nesterenkonia. DNA–DNA hybridization of DSM 17733T with those organisms with the closest phylogenetic affiliation, i.e. Nesterenkonia halobia, Nesterenkonia lacusekhoensis and Nesterenkonia xinjiangensis, gave relatedness values of 48.5 %, 63.7 % (repetition, 57.2 %) and 35.7 % (repetition, 29.3 %), respectively. On the basis of both phenotypic and phylogenetic criteria and the low levels of DNA–DNA relatedness with the phylogenetically closest species N. xinjiangensis and N. halobia, it is proposed that the isolate be classified in a novel species, Nesterenkonia aethiopica sp. nov. The type strain is DSM 17733T (=CCUG 48939T).
Electron micrographs of strain DSM 17733T are available as supplementary material in IJSEM Online.
Present address: PROIMI-CONICET, Av. Belgrano y Pasaje Caseros, 4000 Tucumán, Argentina. 
Present address: Department of Biology, Science Faculty, Addis Ababa University, PO Box 1176, Addis Ababa, Ethiopia.
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and initially included only one species, Nesterenkonia halobia, originally classified as Micrococcus halobius (Onishi & Kamekura, 1972). The type strain of this species was isolated from unrefined solar salt from Noda, Japan (Onishi & Kamekura, 1972). Recently, the species Nesterenkonia lacusekhoensis, isolated from a hypersaline lake in eastern Antarctica (Collins et al., 2002), Nesterenkonia xinjiangensis and Nesterenkonia halotolerans, from hypersaline soil in China (Li et al., 2004), Nesterenkonia sandarakina, from soil in the eastern desert of Egypt, and Nesterenkonia lutea, from saline soil in China (Li et al., 2005), have been described. All Nesterenkonia species are Gram-positive, strictly aerobic and moderately halophilic or halotolerant (and some species are alkaliphilic or alkalitolerant), contain peptidoglycan of the A4 type and have DNA G+C contents in the range 64–72 mol% (Li et al., 2005). In this paper, we describe the characterization and classification of the alkaliphilic, moderately halophilic strain DSM 17733T, which was isolated from a soiled feather sample collected on the shore of Lake Abjata in Ethiopia (7° 60' N 38° 62' E).
Strain DSM 17733T was grown at 37 °C, with shaking at 200 r.p.m., in complex YP medium (Mota et al., 1997). Catalase and oxidase activities and sensitivity to antibiotics were tested, according to Smibert & Krieg (1994), using YP medium, utilized previously for the taxonomic characterization of N. halobia (Mota et al., 1997). The colony characteristics of strain DSM 17733T were studied on solid Luria–Bertani (LB) medium at pH 8 at 30 °C (Li et al., 2005). The optimum temperature for growth was determined by using incubation at 20, 30, 37, 40 and 45 °C in liquid ISP 5 medium (Tang et al., 2003). The pH tolerance was also investigated in ISP 5 medium at pH values ranging from 5 to 12, according to the method of Li et al. (2004, 2005). Halotolerance was tested in ISP 5 medium with 0–20 % (w/v) NaCl at 37 °C. Hydrolysis of xylan and dextran was tested as described by Smibert & Krieg (1994). Hydrolysis of starch and casein was tested in solid YP medium containing 1 % (w/v) soluble starch or 1.5 % (w/v) casein, respectively. Carbon-source utilization by strain DSM 17733T was determined as described previously (Li et al., 2004). Other biochemical characteristics were screened by using the API 20E system (bioMérieux), as described by Logan & Berkeley (1984).
Cell size and morphology were examined under a Nixon Optiphot-2 phase-contrast microscope at x1000 magnification during various different stages of cell growth in YP and ISP 5 media at 37 °C. Gram staining was performed using a Difco Gram-stain set. Scanning electron microscopy (JSM-5600 LV; JEOL) was performed at x10 000–15 000 magnification, using cells grown in YP medium at 37 °C for 12–18 h. Cells were then harvested, washed twice with water and dehydrated with increasing concentrations of ethanol (from 40 to 80 %, v/v) followed by increasing concentrations of isopropanol (20–100 %, v/v), mounted on 12 mm cover slips, dried in a vacuum desiccator for 12 h and, finally, coated with gold/palladium (80 : 20).
Peptidoglycan was purified from the cell wall and its composition was determined at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) as described by Schleifer & Kandler (1972) and Schleifer (1985).
Genomic DNA was extracted and purified as described by Sambrook et al. (1989). Universal primers corresponding to positions (Escherichia coli numbering) 8–27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492–1509R (5'-GGTTACCTTGTTACGACTT-3') were used to amplify the 16S rRNA gene sequence of the novel isolate by PCR (Weisburg et al., 1991). The PCR products were purified and sequenced as described previously (Quillaguamán et al., 2004).
An almost complete sequence (1449 bp) of the 16S rRNA gene of strain DSM 17733T was obtained. Sequences belonging to type strains of the genus Nesterenkonia available through the public databases (GenBank and Ribosomal Database Project II) were aligned and a similarity matrix was calculated (Maidak et al., 2000). 16S rRNA gene sequence analysis was performed with the aid of the DNAMAN 4.03 software package by using the neighbour-joining method and the Jukes–Cantor distance correction method (Saitou & Nei, 1987); the stability of the grouping was estimated by bootstrap analyses (500 replications). Only unambiguously aligned positions from all sequences were used, and gaps were not included in the match/mismatch count.
In order to calculate G+C content, DNA was isolated by chromatography on hydroxyapatite by the procedure of Cashion et al. (1977). The G+C content was calculated from the ratio of deoxyguanosine to thymidine, according to the method of Mesbah et al. (1989), by the Identification Service of the DSMZ.
DNA–DNA hybridization was carried out at the DSMZ, as described by De Ley et al. (1970) with the modification described by Huß et al. (1983) and Escara & Hutton (1980), using a Gilford System model 2600 spectrometer equipped with a Gilford model 2527-R thermoprogrammer and plotter. Renaturation rates were computed with the TRANSFER.BAS program (Jahnke, 1992).
16S rRNA gene sequence analysis placed the novel isolate in the family Micrococcaceae with a close relationship to members of Nesterenkonia (Fig. 1). The closest sequence similarities were found with N. xinjiangensis YIM 70097T (98 %; AY226510) and N. halobia DSM 20541T (97 %; X80747).
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. Strain DSM 17733T produced circular, convex, smooth, entire, opaque colonies that were white in colour but turned yellow in old cultures grown on solid LB medium. We have observed a similar change in the colour of colonies of N. xinjiangensis grown on LB medium. Microscopic examination of strain DSM 17733T at various stages of growth showed the cells to be non-motile, non-endospore-forming, non-capsule-forming, short rods around 0.7 µm in width and 1.2 µm in length (see Supplementary Fig. S1 available in IJSEM Online). In unstained preparations, cells occur singly, in pairs and in irregular clumps (Supplementary Fig. S1).
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Like N. halobia, the isolate exhibited sensitivity to ampicillin, chloramphenicol, nalidixic acid, streptomycin and kanamycin, but showed resistance to erythromycin and tetracycline (Stackebrandt et al., 1995). The minimal inhibitory concentrations of the antibiotics were as follows: ampicillin, 10 µg; tetracycline, 30 µg; chloramphenicol, 30 µg; nalidixic acid, 30 µg; kanamycin, 10 µg; novobiocin, 30 µg; streptomycin, 10 µg; erythromycin, 50 µg.
Analysis of the cell wall showed the presence of murein of the type L-lys–gly–L-Glu, as in N. halobia (Stackebrandt et al., 1995) and N. xinjiangensis (Li et al., 2004); this differed from the L-lys–L-Glu reported for N. lacusekhoensis (Collins et al., 2002) and the L-lys–gly–D-Asp found in N. halotolerans (Li et al., 2004), N. lutea and N. sandarakina (Li et al., 2005).
As shown in Table 1
, the DNA G+C content of strain DSM 17733T was found to be 69.0 mol%, which is within the range (64–72 mol%) reported in the emended description of the genus Nesterenkonia (Li et al., 2005). Furthermore, DNA–DNA hybridization analysis of strain DSM 17733T revealed 48.5 % relatedness to N. halobia DSM 20541T, 63.7 % (repetition, 57.2 %) to N. lacusekhoensis DSM 12544T and 35.7 % (repetition, 29.3 %) to N. xinjiangensis DSM 15475T. These values were below 70 %, the threshold value recommended for the delineation of genomic species (Wayne et al., 1987).
On the basis of notable differences in the phenotypic and phylogenetic features, the G+C content and the relatively low levels of DNA–DNA relatedness to species belonging to the genus Nesterenkonia, we conclude that strain DSM 17733T represents a novel alkaliphilic, moderately halophilic Nesterenkonia species, for which the name of Nesterenkonia aethiopica sp. nov. is proposed.
Description of Nesterenkonia aethiopica sp. nov.
Nesterenkonia aethiopica (L. fem. adj. aethiopica pertaining to Ethiopia).
Cells are Gram-positive, non-motile, non-capsule-forming, non-endospore-forming, short rods around 0.7 µm in width and 1.2 µm in length. Catalase- and oxidase-positive and urease-negative. Strictly aerobic and mesophilic: exhibits good growth at 25–40 °C, with an optimum between 30 and 37 °C; unable to grow at 45 °C. Alkaliphilic and moderately halophilic. Optimal growth occurs at initial pH 9 (range pH 7–11) and at 3 % (w/v) NaCl (range 3–12 %). Carbohydrates utilized as sole carbon sources are D-glucose, D-fructose and other sugars listed in Table 1. Acid is produced in medium supplied with D-glucose, D-fructose, L-arabinose and glycerol, but acidification is not detected with D-trehalose, D-galactose, D-lactose, D-mannitol or D-xylose. Starch, casein, tyrosine and gelatin are hydrolysed. Indole is not produced and the Voges–Proskauer test is negative. The DNA G+C content is 69.0 mol% (determined by HPLC). Cell wall has murein of the type L-lys–gly–L-Glu.
The type strain, DSM 17733T (=CCUG 48939T), was isolated from a soiled feather sample collected at the shore of Lake Abjata, Ethiopia.
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ACKNOWLEDGEMENTS |
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