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1 Division of Nematology, Indian Agricultural Research Institute, New Delhi 110012, India
2 DSMZ – German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstrasse 7b, 38124 Braunschweig, Germany
Correspondence
Rüdiger Pukall
rpu{at}dsmz.de
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) differ from each other mainly in menaquinone type, cell wall sugars, G+C content of the DNA and physiochemical features (Morais et al., 2004). The genus Leucobacter was proposed to accommodate Gram-positive rods with a distinct peptidoglycan with an unusual amino acid, 
-aminobutyric acid, a high percentage of menaquinone 11 (MK-11) and moderate 16S rRNA gene sequence similarities with other members of the family. Diagnostic sugars were absent from the cell wall of Leucobacter komagatae, the only species available at the time of the study (Takeuchi et al., 1996). In 2004, three new members, Leucobacter chromiireducens, Leucobacter aridicollis (Morais et al., 2004) and Leucobacter albus (Lin et al., 2004), and in 2006 two new members, Leucobacter alluvii and Leucobacter luti (Morais et al., 2006), were added to the genus on the basis of the aforementioned properties.
Members of the genus Leucobacter have been isolated from diverse environments, such as activated sludge from a treatment plant containing chromium-contaminated wastewater (L. chromiireducens, L. aridicollis and L. luti) (Morais et al., 2004, 2006) and from soil in Thailand (L. albus) (Lin et al., 2004), while L. komagatae was isolated as a contaminant from an ampoule containing ‘Pseudomonas riboflavina’ (=Devosia riboflavina) (Takeuchi et al., 1996) and L. alluvii from sediment of the Alviela river, Portugal. Here we report the isolation and characterization of a novel Leucobacter species from the bacterial flora associated with the entomopathogenic nematode Steinernema thermophilum (Ganguly & Singh, 2000), recovered from soil in India.
Strain 40T was one of the isolates found in the course of identifying bacteria from infective juveniles of the entomopathogenic nematode Steinernema thermophilum by using a procedure established by Akhurst (1980) with the modifications described by Somvanshi et al. (2006). The type strains of recognized Leucobacter species were obtained from the DSMZ and CRBIP, Paris. Cultural characteristics of strain 40T are indicated in the species description.
Genomic DNA was extracted using a DNeasy Tissue kit (Qiagen) following the manufacturer's instructions. The 16S rRNA gene was amplified as described by Rainey et al. (1996). The PCR products were purified using a QIAquick PCR purification kit (Qiagen) and sequenced directly by using a CEQ Dye Terminator Cycle sequencing kit. The products were separated on a CEQ 8000 Genetic Analysis system. The 16S rRNA gene sequences were aligned with corresponding sequences from the database of DSMZ using the ae2 editor (Maidak et al., 1997). Evolutionary distances were calculated using the method of Jukes & Cantor (1969). A phylogenetic tree based on distance analysis, reconstructed using the neighbour-joining algorithm (Saitou & Nei, 1987), and bootstrap values were determined by analysing 1000 resamplings (Felsenstein, 1993).
The 16S rRNA gene sequence of strain 40T (1508 nt) showed 97.3–98.5 % similarity to those of type strains of recognized species of the genus Leucobacter, the closest relative being L. aridicollis (Fig. 1). However, these values were lower than those determined for certain strain pairs, such as L. albus and L. aridicollis (98.9 %), L. albus and L. komagatae (98.8 %) and L. komagatae and L. aridicollis (98.7 %). As previous studies have confirmed DNA–DNA reassociation values of less than 70 % for the latter two strain pairs with high 16S rRNA gene similarities [40 % (Lin et al., 2004) and 38 % (Morais et al., 2004, 2006), respectively], we refrained from testing this parameter and consider strain 40T to represent a distinct genospecies (see also Stackebrandt & Ebers, 2006).
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. Strain 40T was also characterized by using an API 20NE strip. Biolog GP plates (Oxoid) were incubated for 24 h before being read. Wells showing a photometric value above 20 or 30 % of the highest value of an individual strain were scored as weak or positive, respectively. Conventional biochemical tests were performed according to standard methods (Smibert & Krieg, 1994). Cultural characteristics such as colony size, shape and colour were determined after 24 h incubation at 28 °C on CASO agar plates (Merck). The temperature range for growth was determined by incubating inoculated slant cultures (trypticase soy agar, pH 7.0) at 4, 25, 30, 37 and 45 °C. Growth was also assessed in trypticase soy broth (Difco) at pH 5–9. pH values of the test medium of less than 7.5 were obtained by adding HCl and pH values greater than 7.5 by addition of sterilized sodium sesquicarbonate buffer (1 M). The ability to grow in the presence of NaCl was determined using trypticase soy broth containing NaCl (5, 7 or 10 %). Growth characteristics are given in the species description.
On the basis of the results of the physiological tests, it was concluded that strains of the genus Leucobacter react negatively towards most of the substrates provided on the API 50 CH panel. There was no common substrate on the API 50 CH test panels that was acidified by all members of the genus (Table 1). According to Morais et al. (2004), many tests were scored ‘weak’ for L. chromiireducens and L. aridicollis in the original description whereas, according to our results, they were negative for all the respective substrates even when read after 5 days. The Biolog GP test plates were more suitable for distinguishing Leucobacter strains (Table 2). These data reveal a clear preference of the strains for utilization of amino acids over carbohydrates. Strain 40T differed from the other strains in utilization of N-acetylglucosamine and the weak acidification of 5-ketogluconate and salicin within the API 50 CH substrate panels. In addition, a change of the indicator dye was observed for fructose (Table 1). The reactions of some strains towards certain substrates differed between the Biolog and API substrate panels (Tables 1 and 2).
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Cellular fatty acid analysis was carried out for all the type strains except L. komagatae IAM 1093T, L. luti CIP 108818T and L. alluvii CIP 108819T, for which data were taken from Takeuchi et al. (1996) and Morais et al. (2006) (Table 3). The major fatty acids of strain 40T were anteiso-C15 : 0 (41.9 %), anteiso-C17 : 0 (33.6 %) and iso-C16 : 0 (15.5 %). These results are in accord with the fatty acid compositions of other members of the genus, confirming the affiliation of strain 40T with the genus.
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. Amino acids and peptides in cell wall hydrolysates were analysed by two-dimensional ascending thin layer chromatography on cellulose plates using previously described systems (Schleifer & Kandler, 1972). The N-terminal amino acid of the inter-peptide bridge was determined by dinitrophenylation as described by Schleifer (1985). Strain 40T possessed glutamic acid (1.0), L-diaminobutyric acid (0.5), alanine (1.5), glycine (0.9) and threonine (0.7), a composition that confirmed the presence of a B-type cross-linked peptidoglycan. As revealed by denitrophenylation, alanine was the N-terminal amino acid. The dipeptide Gly–Glu was detected after partial hydrolysis. 4-Aminobutyric acid, as detected in some other Leucobacter strains (L. komagatae, L. albus), was not observed. The N-terminal alanine, the presence of threonine and the Gly–Glu dipeptide corresponds to the information reported for L. chromiireducens, L. luti and L. alluvii (Morais et al., 2006). Lipoquinone and polar lipid analyses were done by using previously described methods (Groth et al., 1996). The dominant menaquinone was MK-11, consistent with the results obtained with the type strains of other Leucobacter species. MK-10 and MK-9 were present in minor amounts, while MK-12 occurred in trace amounts only. The polar lipids were phosphatidylglycerol and diphosphoglycerol. Several unidentified components occurred, i.e. a phospholipid, a glycolipid and an aminophospholipid. In conclusion, based upon the phylogenetic and chemotaxonomic evidence, strain 40T represents a novel member of the genus Leucobacter, family Microbacteriaceae, for which the name Leucobacter iarius sp. nov. is proposed.
Description of Leucobacter iarius sp. nov.
Leucobacter iarius [i.a'ri.us. N.L. masc. adj. iarius (arbitrary name) formed from the acronym IARI (Indian Agricultural Research Institute, New Delhi, India) to commemorate its centenary year].
Cells are Gram-positive, straight, non-motile rods (1.66–2.91x0.37–0.49 µm). Neither mycelium nor spores are detected. Colonies are round, 0.4–1 mm in diameter with smooth edges, low-convex, opaque, whitish in colour, finely granular and odourless. Optimum temperature for growth is 30 °C; growth does not occur at temperatures higher than 37 °C or at 4 °C. Growth occurs at pH 5.0–9.0. Growth is reduced in the presence of 5–7 % NaCl; no growth occurs with 10 % NaCl. Urease-negative, nitrate is not reduced, D-glucose is not fermented, and aesculin and PNPG (
-galactosidase) are positive on API 20NE strips. Gelatinase-negative. Utilizes N-acetylglucosamine and weakly utilizes 5-ketogluconate, inositol and salicin. Additional biochemical and physiological characteristics are given in Tables 1 and 2
. MK-11 is the major menaquinone; MK-10 and MK-9 are present in minor amounts while MK-12 occurs in trace amounts only. Polar lipids are phosphatidylglycerol and diphosphoglycerol. Several unidentified components occur, i.e. a phospholipid, a glycolipid and an aminophospholipid. Cross-linkage of peptidoglycan is of the B-type; cell wall amino acids are L-diaminobutyric acid, alanine, glycine, threonine and glutamic acid. 4-Aminobutyric acid is absent. Major fatty acids are anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0; iso-C15 : 0 and C16 : 0 are present in minor amounts.
The type strain is strain 40T (=DSM 17402T=CIP 108831T), which was isolated from crushed infective juveniles of Steinernema thermophilum collected from soil of the Indian Agricultural Research Institute, New Delhi, India.
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ACKNOWLEDGEMENTS |
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