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Int J Syst Evol Microbiol 56 (2006), 979-982; DOI  10.1099/ijs.0.63831-0
© 2006 International Union of Microbiological Societies

Rhodococcus kroppenstedtii sp. nov., a novel actinobacterium isolated from a cold desert of the Himalayas, India

S. Mayilraj1, S. Krishnamurthi1, P. Saha1 and H. S. Saini2

1 Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh 160 036, India
2 Department of Microbiology, Guru Nanak Dev University (GNDU), Amritsar 143 005, India

Correspondence
S. Mayilraj
mayil{at}imtech.res.in


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The taxonomic position of an actinomycete, strain K07-23T, isolated from a cold desert of the Himalayas, India, was established by a polyphasic approach. The strain exhibited phenotypic characters that were typical of the genus Rhodococcus. 16S rRNA gene sequence (1467 bases) comparisons confirmed that strain K07-23T belongs to the genus Rhodococcus. 16S rRNA sequence similarity studies showed that the isolate is very closely related to Nocardia corynebacterioides DSM 20151T (98.6 %), which has been recently reclassified as Rhodococcus corynebacterioides. It showed 94.4–96.6 % sequence similarity with other species of the genus Rhodococcus. However, genomic relatedness between strain K07-23T and R. corynebacterioides as revealed by DNA–DNA hybridization was low (62 %). Based on polyphasic analysis, strain K07-23T could be clearly distinguished from other species. It is proposed that strain K07-23T (=MTCC 6634T=DSM 44908T=JCM 13011T) represents a novel species of Rhodococcus, Rhodococcus kroppenstedtii sp. nov.

Published online ahead of print on 16 December 2005 as DOI 10.1099/ijs.0.63831-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Rhodococcus kroppenstedtii strain K07-23T is AY726605.


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In this study, the taxonomic position of an actinobacterium, which was isolated during our study of microbial diversity of Lahaul-Spiti Valley, a cold desert of the Himalayas, India, was examined by a polyphasic approach. Analysis of strain K07-23T showed that it belongs to the genus Rhodococcus, but is quite distinct from other species within the genus. Strain K07-23T was isolated from a soil sample collected from the Lahaul-Spiti Valley by a dilution plating technique on tryptic soy agar (TSA; HiMedia) medium and maintained as glycerol stocks at –70 °C. The reference strain Rhodococcus corynebacterioides MTCC 699T (=JCM 3376T=DSM 20151T) was taken from the Microbial Type Culture Collection (MTCC), Chandigarh, India.

Strain K07-23T and R. corynebacterioides MTCC 699T were tested for a range of phenotypic properties (Table 1Go). Gram reaction was determined using a HiMedia Gram Staining kit according to the manufacturer's instructions. Physiological tests such as growth at different temperatures, pH and NaCl concentrations were examined by growing the strain on basal tryptic soy broth (TSB) medium. Catalase activity and urea hydrolysis were determined as described by Cowan & Steel (1965)Go. Hydrolysis of casein, gelatin, Tween 80 and starch, methyl red/Voges–Proskauer tests and oxidase activity (using N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride) were determined as described by Smibert & Krieg (1994)Go. Nitrate reduction was determined as described by Lanyi (1987)Go. Growth on sole carbon sources was examined on carbon utilization agar (Shirling & Gottlieb, 1966Go). The ability to use sole nitrogen sources was studied according to Williams et al. (1983)Go with the modification that agarose (1.5 %) was used instead of agar. Antibiotic susceptibility was examined as described by Groth et al. (2004)Go using antibiotic discs (HiMedia). For cellular fatty acid analysis, strains were grown on TSA medium at 30 °C for 36 h and fatty acid methyl ester analysis was performed using the Sherlock Microbial Identification System (MIDI) as described previously (Pandey et al., 2002Go). Freeze-dried cells for other chemotaxonomic analyses were prepared following growth of the strains in TSB for 4 days at 30 °C. Standard procedures were used to determine the diagnostic cell wall sugars and amino acids (Staneck & Roberts, 1974Go). Phospholipids and menaquinones were extracted and analysed as described by Minnikin et al. (1984)Go and Kroppenstedt (1982)Go. The presence of mycolic acids was demonstrated by TLC (Minnikin & Goodfellow, 1976Go) and the mycolic acid pattern was determined according to Klatte et al. (1994)Go. The glycolic acid content of the bacterial cell wall was determined by the colour reaction method of Uchida & Aida (1984)Go. Genomic DNA extraction, amplification, sequencing of the 16S rRNA gene and phylogenetic analysis were performed as described previously (Mayilraj et al., 2005Go). DNA–DNA hybridization was performed using the membrane filter method (Tourova & Antonov, 1987Go) as described by Reddy et al. (2000)Go. The G+C content of genomic DNA was determined by HPLC (Groth et al., 1996Go).


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  		Table 1. Comparison of phenotypic characters and G+C content of strain K07-23T and Rhodococcus corynebacterioides MTCC 699T

Both strains are orange–red-pigmented, do not produce endospores and are catalase-positive. Both produceacid from D-fructose, mannitol, D-glucose and sucrose; acid is not produced from L-rhamnose, D-galactose, trehalose, D-lactose, inulin, D-maltose, adonitol, L-arabinose, D-melibiose or dulcitol. Both are positive for hydrolysis of adenine and testosterone and negative for urea, casein, elastin, aesculin, gelatin, guanine, hypoxanthine, tyrosine and xanthine. +, Positive; –, negative; W, weakly positive.

 
Strain K07-23T had chemical and phenotypic properties consistent with its classification in the genus Rhodococcus. The detailed differential phenotypic properties and G+C content are listed in Table 1Go and other characters are given in the species description. The diagnostic cell wall amino acid is meso-A2pm; the diagnostic cell wall sugars are arabinose and galactose (cell wall chemotype IV; Lechevalier & Lechevalier, 1970Go). The non-diagnostic sugars glucose and ribose were also found in the whole-cell hydrolysate. The acyl type was acetyl. The isolate had phospholipid pattern type PII, according to Lechevalier et al. (1977)Go. Mycolic acids with a chain length of 42–50 carbon atoms were present. Under similar growth conditions, quantitative differences in fatty acid composition were found between strain K07-23T and its closest relative. Like all rhodococci, strain K07-23T had a fatty acid pattern of type 1b (Kroppenstedt, 1985Go). Compared to R. corynebacterioides, strain K07-23T had higher proportions of C16 : 0 (42.1 vs 36.5 % for K07-23T and R. corynebacterioides, respectively) and C18 : 1{omega}9c (28.5 vs 22.7 %), and lower proportions of C16 : 1{omega}7c (10.1 vs 13.8 %), 10-Me C18 : 0 (6.1 vs 9.9 %), C17 : 1{omega}8c (6.8 vs 9.0 %) and C14 : 0 (5.8 vs 8.2 %).

To determine the phylogenetic position of strain K07-23T, the 16S rRNA gene sequence (1467 nt) was compared with those of type strains of species of the genus Rhodococcus retrieved from GenBank. Strain K07-23T is closely related to R. corynebacterioides (98.6 %) and distantly related to all other species studied (94.4–96.6 %). It is evident from the phylogenetic tree (Fig. 1Go) based on the neighbour-joining method that strain K07-23T and R. corynebacterioides formed a separate phylogenetic branch along with Rhodococcus triatomae and Rhodococcus equi. Genomic relatedness, as shown by DNA–DNA hybridization of strain K07-23T with the closely related strain R. corynebacterioides, was only 62 %. Other species of the genus can be excluded from DNA–DNA hybridization studies because of the dissimilarity (3–4 %) in 16S rRNA gene sequences (Stackebrandt & Goebel, 1994Go). This is consistent with the conclusion drawn by Yassin (2005)Go that representatives of Rhodococcus species with 16S rRNA gene sequence similarities greater than 98 % may share whole genomic DNA relatedness values well below the 70 % cut-off point recommended for the delineation of bacterial species (Wayne et al., 1987Go). The DNA G+C content of strain K07-23T is 69.7 mol%. Based on phenotypic and genotypic data, strain K07-23T is very clearly distinguishable from its closest phylogenetic relative and other species of the genus Rhodococcus. It is therefore concluded that strain K07-23T represents a novel species of the genus Rhodococcus; the name Rhodococcus kroppenstedtii sp. nov. is proposed.


Figure 1
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  		Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences (1467 bases) showing the phylogenetic relationship between strain Rhodococcus kroppenstedtii K07-23T and other species of the genus Rhodococcus. Corynebacterium diphtheriae was used as an outgroup. Bootstrap values (expressed as percentage of 1000 replications) greater than 50 % are given at the nodes. Bar, 1 % sequence variation.

 
Description of Rhodococcus kroppenstedtii sp. nov.
Rhodococcus kroppenstedtii (krop.pen.sted'ti.i. N.L. gen. n. kroppenstedtii of Kroppenstedt, to honour Reiner Michael Kroppenstedt, a German microbiologist, for his enormous contributions to the taxonomy of actinomycetes).

Cells are Gram-positive, acid-fast, non-motile, non-spore-forming, rod- and coccoid-like, occurring in groups. Colonies on TSA medium are orange–red-pigmented, small, smooth, glistening and convex. Catalase is produced, oxidase is not produced. Capable of growing in up to 9 % NaCl; grows weakly at 10 % NaCl, but not at 12 % NaCl. Temperature range for growth is 10–37 °C, with optimum growth at 30 °C; does not grow at 42 °C. pH range for growth is 5.0–11.0, with optimum growth at pH 8.0. Positive for hydrolysis of Tween 20, 40, 60, 80 and testosterone, but negative for hydrolysis of starch, casein, gelatin and urea. Acid is produced from raffinose, meso-inositol, D-lactose and L-rhamnose, but not from D-maltose, L-arabinose, D-galactose, D-glucose, D-xylose, sucrose, D-fructose or salicin. Negative for methyl red and Voges–Proskauer reactions and positive for nitrate reduction. Positive for utilization of L-arginine, L-serine, L-methionine, L-histidine, L-proline, L-phenylalanine and potassium nitrate as sole nitrogen sources. Positive for utilization of sodium lactate, sodium gluconate and p-hydroxybenzoate as sole carbon sources. Positive for tolerance to sodium azide (0.01 %) and negative for crystal violet (0.0001 %). Diagnostic cell wall amino acid is meso-A2pm. Diagnostic cell wall sugars are arabinose and galactose, wall type IV; the non-diagnostic sugars ribose and glucose are also found. The acyl type is acetyl. Type II phospholipid. The strain synthesizes mycolic acids with a chain length of 42–50 carbon atoms showing 1–3 double bonds in the chain. Tuberculostearic acid (10-methyloctadecanoic acid, 6.1 %) is present. Major fatty acids are C14 : 0 (5.8 %), C16 : 0 (42.07 %), C16 : 1{omega}7c (10.1 %), C17 : 1{omega}8c (6.8 %) and C18 : 1{omega}9c (28.5 %). Major menaquinone is MK-8(H2).

The type strain is K07-23T (=MTCC 6634T=DSM 44908T=JCM 13011T), isolated from soil, 1.5 feet below an ice glacier, 4200 m above sea-level, in Kibber village of the Spiti Valley, Himachal Pradesh, India. The DNA G+C content of strain K07-23T is 69.7 mol%.


   ACKNOWLEDGEMENTS
 
We would like to thank Dr Peter Schumann, DSMZ, for performing G+C analysis, Ms Gabi Poetter for the chemotaxonomic analysis and Mr Ganesan and Mr Malkit Singh for their excellent technical assistance. P. S. and S. K. are the recipients of CSIR research fellowships. Financial assistance from CSIR and DBT, Government of India is duly acknowledged. This is IMTECH communication number 15/2005.


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