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1 Departamento de Microbiología y Genética, Edificio Departamental, Lab. 209, Campus Miguel de Unamuno, Universidad de Salamanca, 37007 Salamanca, Spain
2 DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany
Correspondence
Martha E. Trujillo
mett{at}usal.es
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Published online ahead of print on 25 November 2005 as DOI 10.1099/ijs.0.63745-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Kribbella lupini sp. nov. strain LU14T is AJ811962.
A supplementary table detailing cellular fatty acid profiles for strain LU14T and Kribbella sandramycini is available in IJSEM Online.
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) includes six species that form a homogeneous group within the family Nocardioidaceae. While most Kribbella species have been isolated from soil, the recent description of Kribbella solani (Song et al., 2004) recovered from potato tubers suggests that soil is not the only ecological niche where these bacteria may be found. In the present paper, we report on the isolation of a novel actinomycete strain, LU14T, from the root nodules of Lupinus angustifolius, a native plant from the Mediterranean.
Strain LU14T was isolated from root nodules of L. angustifolius growing near a former uranium mine in Salamanca, Spain. The root samples were washed twice with sterile distilled water and surface sterilized using HgCl2 (1 %, w/v) for 2 min. The samples were rinsed five times with sterile distilled water and crushed using a sterile glass rod. The macerate was inoculated on yeast extract-mannitol agar (Vincent, 1970) and the plates were incubated for 3 weeks at 28 °C. Mycelium formation, colony colour and other growth characteristics were examined for 3 weeks on various culture media including SA1 (Trujillo et al., 2005), ISP 2 (Shirling & Gottlieb, 1966) and nutrient agar. Gram- and acid-fast-staining procedures were performed on 3 day cultures (Doetsch, 1981).
For chemotaxonomic analyses, strain LU14T was grown in tryptic soy broth in flasks on a rotary shaker at 90 r.p.m. and 28 °C. Biomass was harvested, washed in distilled water and freeze-dried. Cell walls were prepared according to the method of Schleifer (1985). Peptidoglycan structure was studied in whole- and partial-cell-wall hydrolysates using TLC on cellulose (Schleifer & Kandler, 1972). Analysis of sugars in the purified cell walls was carried out as described by Staneck & Roberts (1974). Menaquinones were extracted and purified by the method of Minnikin et al. (1984) and analysed by HPLC (1100; Hewlett Packard). Methyl esters of cellular fatty acids for LU14T and Kribbella sandramycini DSM 15626T were prepared from cells grown for 24 h on trypticase soy agar cultures (28 °C) and analysed by GLC (Schröeder et al., 1997). Polar lipids were extracted and identified by two-dimensional TLC (Minnikin et al., 1984).
Catalase activity was determined by observing bubble formation in a culture after the addition of 3 % hydrogen peroxide. Oxidase activity was determined by oxidation of 1 % tetramethyl-p-phenylenediamine. Hydrolysis of arbutin, aesculin, starch, tyrosine and xylan was determined according to Trujillo et al. (2005). Tests for the utilization of various substrates as sole carbon and energy sources were performed as described by Williams et al. (1983). Antibiotic resistance was examined as described by Trujillo et al. (2005). Determination of temperature and pH growth rates and tolerance of NaCl were performed on nutrient agar. Strain LUP14T was also characterized using miniaturized API 20NE, API Coryne and API ZYM tests (bioMérieux) following the manufacturer's instructions. The reference strains Kribbella antibiotica DSM 15501T, Kribbella flavida KACC 20248T, Kribbella koreensis IMSNU 50530T and K. sandramycini DSM 15626T were also included in the physiological studies for comparison.
Genomic DNA extraction, PCR amplification of the 16S rRNA gene and sequencing parameters were as described previously (Rivas et al., 2003). An almost-complete sequence was obtained and aligned against members of the family Nocardioidaceae using CLUSTAL_X (Thompson et al., 1997). A phylogenetic tree was constructed according to the neighbour-joining method (Saitou & Nei, 1987) using the Kimura two-parameter distance matrix and compared with an additional tree obtained using the maximum-parsimony method (Fitch, 1971). In both cases, 1000 resamplings were used for bootstrap analysis. All analyses were carried out with the MEGA2 program (Kumar et al., 2001).
Genomic DNA for hybridization studies was isolated using a French pressure cell (Thermo Spectronic) and purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA–DNA hybridization [in 2x SSC plus 10 % (v/v) DMSO at 67 °C] was performed between strain LU14T and its closest neighbour, K. sandramycini DSM 15626T, as described by De Ley et al. (1970) and modified by Huß et al. (1983), using a Cary 100 Bio UV/VIS-spectrophotometer (Varian). The DNA G+C content was determined using the thermal melting method (Mandel & Marmur, 1968).
On all media tested, colonies were white to cream coloured with a pasty texture and lichenous shapes. No diffusible pigments were observed on any of the media. Substrate mycelium was extensively branched and penetrated into the agar and fragmented into rods; white aerial mycelium was produced abundantly and fragmented into non-motile rods.
A 16S rRNA gene sequence of 1458 nt was obtained for isolate LU14T. The results of the phylogenetic analysis (Fig. 1) revealed that the isolate formed a separate line of descent in the phylogenetic cluster of the genus Kribbella. The closest neighbour of strain LU14T was K. sandramycini KACC 20249T, with 98·7 % 16S rRNA gene sequence similarity, followed by K. flavida NBRC 14399T with 98·6 % similarity. The sequence similarities with other species of the genus were in the range 98·2–97·1 %. A very similar tree was obtained using the maximum-parsimony method (data not shown).
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peptidoglycan (Schleifer & Kandler, 1972). Galactose and one unidentified component, which is neither arabinose, glucose, mannose, rhamnose, ribose nor xylose, were detected in the purified cell wall of isolate LU14T. Galactose was also reported in the whole cell sugar patterns of the remaining recognized Kribella species (Song et al., 2004; Li et al., 2004), with the exception of K. antibiotica DSM 15501T (Li et al., 2004), which contains glucose, ribose and xylose. In the case of K. sandramycini KCTC 9609T and K. flavida KACC 20248T, glucose, mannose and galactose were reported (Li et al., 2004), while these three sugars and ribose were found in K. koreensis IMSNU 50530T, Kribbella jejuensis JCM 12204T and Kribbella solani JCM 12205T. These data suggest that the cell-wall sugar composition can be used as an important marker to differentiate species of the genus Kribbella.
The main menaquinones found were MK-9(H4) (93 %) and MK-9(H2) (7 %). The fatty acid profiles of strain LU14T and its closest phylogenetic neighbour, K. sandramycini DSM 15626T, are presented in Supplementary Table S1 in IJSEM Online. Strain LU14T differed from K. sandramycini DSM 15626T mainly in the presence of remarkable amounts of iso-16 : 0, 17 : 1
8c and 10-methyl branched fatty acids. The phospholipids found in strain LU14T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and phosphatidylinositol, which correspond to phospholipid type III according to Lechevalier et al. (1977).
Various differentiating physiological characteristics between strain LU14T and other Kribbella type strains are shown in Table 1. The ability of strain LU14T to tolerate 7 % NaCl deserves special mention, as the remaining species do not tolerate NaCl concentrations above 3 %. It should also be noted that, in the original and emended descriptions of the genus Kribbella (Park et al., 1999; Sohn et al., 2003), production of urease and oxidase are reported as characteristic of the genus, but as more species have been described, the results for these properties seem to vary. The novel isolate showed resistance to ampicillin (2 µg), penicillin G (10 U) and rifampicin (2 µg) after 7 days of incubation and was susceptible to amoxycillin (30 µg), gentamicin (10 µg), neomycin (5 µg), novobiocin (5 µg), oxytetracycline (30 µg), streptomycin (300 µg), tobramycin (10 µg), vancomycin (30 µg) and tetracycline (30 µg). Other results for the novel isolate are given in the species description.
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). Various authors have reported that certain genera, including actinomycetes (Wink et al., 2003), show less than 70 % DNA–DNA reassociation values at sequence similarities higher than 98·5 %, enabling this correlation between genomic and gene sequence similarities to be used to delineate novel species provided that DNA–DNA reassociation data exist for most of the other species (Romanenko et al., 2004). This indeed, appears to be the case for the presently described species of the genus Kribbella. Based on the overall results, we propose that strain LU14T represents a novel species, Kribbella lupini sp. nov.
Description of Kribbella lupini sp. nov.
Kribbella lupini (lu'pin.i. L. gen. n. lupini of lupin, isolated from Lupinus angustifolius).
Gram-positive, non-acid-fast, strictly aerobic actinomycete. Colonies are white to cream, irregular and show lichenous shapes. Well-developed substrate mycelium with hyphae that branch extensively and fragment into coccoid to rod elements. Abundant white aerial mycelium is produced that fragments into rod-shaped elements. No diffusible pigments observed. Grows between 12 and 37 °C, with an optimum temperature of 28 °C. Grows between pH 6–9. Grows in the presence of 7 % NaCl. Catalase and oxidase are produced. Arbutin, aesculin, casein and gelatin are degraded, but not starch, urea or xylan. Acid is produced from glucose. The following substrates are used as carbon sources: adipate, L-alanine, L-arabinose, L-arginine, D-cellobiose, D-galactose, gluconate, D-glucose, L-histidine, L-lysine, malate, maltose, D-mannitol, D-mannose, D-melibiose, N-acetylglucosamine, L-proline, D-raffinose, L-rhamnose, D-sorbitol, L-sorbose, sucrose, D-trehalose, L-tyrosine, xylitol and DL-valine. Caprate, citrate, D-melezitose and L-serine are not used as carbon sources. Production of arginine dihydrolase, alkaline phosphatase, esterase lipase (weak), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, acid phosphatase, -galactosidase, 
-galactosidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. Does not produce esterase (C4), naphthol-AS-BI phosphohydrolase or -glucuronidase. Cell wall contains LL-Dpm. Major fatty acids are anteiso-15 : 0 (28 %) and iso-16 : 0 (21 %). The DNA G+C content is 68 mol%.
The type strain, LU14T (=DSM 16683T=LMG 22957T), was isolated from root nodules of Lupinus angustifolius.
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ACKNOWLEDGEMENTS |
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