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Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Sector-39A, Chandigarh –160 036, India
Correspondence
Rakesh K. Jain
rkj{at}imtech.res.in
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ABSTRACT |
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Scanning electron micrographs of strain RKJ300T and a table showing fatty acid compositions are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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, 1999). At the time of writing, the genus Rhodococcus consists of more than 30 recognized species (http://www.bacterio.cict.fr/qr/rhodococcus.html). Rhodococci are ubiquitous in the environment, but occur primarily in soils and sludges. As these micro-organisms exhibit broad metabolic diversity, particularly with respect to hydrophobic compounds, they have attracted increased interest with regard to the biochemical and genetic characterization of their metabolic capabilities, and they have also been considered to be excellent candidates for bioremediation treatments (Finnerty, 1992; Warhurst & Fewson, 1994). In this work, a polyphasic approach was used to determine the taxonomic position of bacterial strain RKJ300T, which is capable of utilizing toxic and recalcitrant compounds such as p-nitrophenol and 2,4-dinitrophenol as sole sources of carbon and energy. Strain RKJ300T was isolated from a pesticide-contaminated site in Punjab State, India, on tryptone soya agar (TSA; HiMedia) at 30 °C by using the dilution plating technique. Subcultivation was done on TSA at 30 °C for 48 h and the bacterial isolate was maintained as a glycerol stock at –70 °C. The reference strains Rhodococcus wratislaviensis MTCC 6421T, Rhodococcus opacus MTCC 6420T, Rhodococcus percolatus MTCC 6422T and Rhodococcus koreensis MTCC 6425T were obtained from the Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India.
The colony morphology was examined following growth of the strain on TSA at 30 °C for 3 days. The cell morphology was investigated using light microscopy (Zeiss) at x1000 magnification and scanning electron microscopy (Stereoscan 260; Leica). Cells showing different morphological stages of the growth cycle in liquid (tryptone soya broth) (TSB; HiMedia) and solid (TSA) media were processed for scanning electron microscopy as described previously (Richard & Wilson, 1993). Motility was checked using the method described by Skerman (1967). The Gram reaction was determined by using the HiMedia Gram staining kit according to the manufacturer's instructions. Physiological tests such as growth at different temperatures, pH values and NaCl concentrations were performed as described by Goodfellow (1986). Catalase activity and urea hydrolysis were determined as mentioned by Cowan & Steel (1965). The ability to hydrolyse arbutin, casein, gelatin, elastin, Tween 80 and starch was examined by following the protocol of Smibert & Krieg (1994). Nitrate reduction was determined as described by Lanyi (1987). The following tests were performed as described by Goodfellow (1986): Voges–Proskauer, methyl red, oxidation/fermentation, growth on citrate agar and MacConkey agar and the production of H2S and indole. Oxidase activity was determined from the oxidation of tetramethyl-p-phenylenediamine dihydrochloride (Sigma) (Smibert & Krieg, 1994). Acid production from various carbohydrates was determined after 3 days incubation at 30 °C, using bromocresol purple as an acid/base indicator (Gordon et al., 1974). Tests for the utilization of various substrates as sole carbon (1 %, w/v) (Shirling & Gottlieb, 1966) and sole nitrogen (0.1 %, w/v) (Williams et al., 1983) sources were carried out and the utilization results were observed over a period of 1 week. The utilization of substrates as sole carbon and energy sources was also determined by using the Biolog GP2 MicroPlate system. The plate was used according to the manufacturer's instructions, with the modification that TSA medium was used instead of Biolog Universal Growth agar medium. The inoculated plates were incubated for 24 h and the results were read with a MicroPlate Reader, using Microlog 4.2 computer software to perform automated reading. The strains were examined for their ability to degrade p-nitrophenol and 2,4-dinitrophenol by using the method described by Prakash et al. (1996) following incubation at 30 °C for 3 days. The sensitivity of the strain to various antibiotics was tested using antibiotic-susceptibility discs supplied by HiMedia.
Freeze-dried cells for chemotaxonomic analyses (excepting fatty acid studies) were prepared following growth of the strain in TSB for 4 days at 30 °C. The diagnostic cell-wall amino acids and sugars were determined using TLC as described by Staneck & Roberts (1974). Polar lipids were extracted, examined by two-dimensional TLC and identified using the method described by Minnikin et al. (1984). Menaquinones were extracted (Minnikin et al., 1984) and separated by reversed-phase HPLC (Kroppenstedt, 1982). Mycolic acids were extracted and analysed according to the protocol of Minnikin et al. (1975). For cellular fatty acid analysis, the strains were grown on TSA at 30 °C for 36 h, and fatty acid methyl esters were analysed by using the Sherlock Microbial Identification System (MIDI) as described previously (Pandey et al., 2002). Genomic DNA was isolated using a Qiagen genomic DNA isolation kit. DNA–DNA relatedness was studied by using the membrane filter method (Tourova & Antonov, 1987) as described by Shivaji et al. (1992); this was performed three times starting from the isolation of genomic DNA. The G+C content of the genomic DNA was determined spectrophotometrically (Lambda 35; Perkin Elmer) using the thermal denaturation method (Mandel & Marmur, 1968).
PCR amplification of the 16S rRNA gene was performed with universal primers 27f (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492r (5'-TACGGYTACCTTGTTACGACTT-3') (Escherichia coli 16S rRNA numbering system; Brosius et al., 1978). The reaction mixture contained 70 ng chromosomal DNA, 1 U Deep Vent DNA polymerase, 1x ThermoPol reaction buffer, 200 µM each dNTP (New England Biolabs) and 20 pmol each primer (BioBasic). The PCR cycling parameters consisted of an initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min and extension at 75 °C for 2 min and a final extension for 10 min at 75 °C. An amplicon of approximately 1.5 kb was separated by gel electrophoresis, eluted using the Qiaquick gel extraction kit (Qiagen) and cloned into electrocompetent E. coli DH5
cells using the SmaI-digested cloning vector pBluescript II KS(+). Transformants were selected on the basis of the blue/white screening procedure (Sambrook & Russell, 2001). Recombinant plasmids were isolated using the QIAprep spin miniprep kit (Qiagen) and the insert was completely sequenced using five forward primers and four reverse primers, namely KS (5'-TCGAGGTCGACGGTATC-3'), 27f, 357f, 704f, 1114f, 685r, 1110r, 1492r and SK (5'-CGCTCTAGAACTAGTGGATC-3') (KS and SK, vector-specific primers) (Johnson, 1994), using the dideoxy chain-termination method as described previously (Pandey et al., 2002). The 16S rRNA gene sequences of closely related taxa with validly published names were retrieved from the GenBank database using BLASTN (Altschul et al., 1997) and aligned using the CLUSTAL X program (Thompson et al., 1997); the alignment was edited manually. For the neighbour-joining analysis (Saitou & Nei, 1987), the distances between sequences were calculated on the basis of the method of Jukes & Cantor (1969). A bootstrap analysis of 1000 replications was performed to assess the confidence limits of the branching (Felsenstein, 1985).
Scanning electron micrographs revealed that the cells of strain RKJ300T formed filaments or showed preliminary branching at an early phase of growth (12 h) and fragmented into short rods during the exponential phase (32 h); most cells appeared as cocci in stationary phase (64 h) (see Supplementary Fig. S1 available in IJSEM Online). Growth of the strains on minimal agar medium supplemented with p-nitrophenol (0.5 mM) or 2,4-dinitrophenol (0.5 mM) as the sole source of carbon and energy is indicated in Table 1. Cells of strain RKJ300T were resistant to cephaloridin (30 µg per disc) and ampicillin (10 µg per disc). They were sensitive to the following antibiotics (µg per disc): rifampicin (5), tetracycline (30), chloramphenicol (10), chlorotetracycline (30), gentamicin (10), streptomycin (10), neomycin (30), tobramycin (10), norfloxacin (10), vancomycin (30) and oleandomycin (15). Most of the chemotaxonomic properties of the strain (presented in the species description) were typical of members of the genus Rhodococcus. No significant qualitative differences in the fatty acid profiles were found when strain RKJ300T was compared with phylogenetically close taxa (see Supplementary Table S1 available in IJSEM Online). The DNA G+C content of strain RKJ300T was estimated to be 72 mol% (mean of three replications; Table 1), a value within the range (63–72 mol%) for the genus Rhodococcus (Takeuchi & Hatano, 1998).
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), showing the highest level of sequence similarity with R. wratislaviensis NCIMB 13082T (99.3 %), followed by R. opacus DSM 43205T (98.8 %), R. percolatus MBS1T (98.6 %) and R. koreensis DNP505T (98.1 %).
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that representatives of Rhodococcus species with 16S rRNA gene sequence similarity greater than 98 % share whole genomic relatedness values well below the 70 % cut-off point recommended for the delineation of bacterial species (Wayne et al., 1987). In view of this, we did not investigate the DNA–DNA relatedness of strain RKJ300T with respect to species of Rhodococcus that showed 16S rRNA gene sequence similarities below 98 %. The characteristics that differentiate strain RKJ300T from closely related species are summarized in Table 1
. On the basis of the data presented in this study, we conclude that strain RKJ300T should be assigned to a novel species, for which the name Rhodococcus imtechensis sp. nov. is proposed.
Description of Rhodococcus imtechensis sp. nov.
Rhodococcus imtechensis [im.tech.en'sis. N.L. masc. adj. imtechensis pertaining to the Institute of Microbial Technology (IMTECH), where the type strain was characterized].
Gram-positive, aerobic, non-motile, catalase-positive, oxidase-negative actinobacterium that shows a hypha–rod–coccus life cycle. On TSA plates, colonies are circular, glistening, opaque, convex, creamish pink in colour and have smooth margins. Growth occurs at 15–37 °C (optimum 30 °C), at pH 5.0–10.0 (optimum pH 7.0) and at 2.5–5.0 % (w/v) NaCl. No growth occurs on citrate agar or MacConkey agar. Tween 80 and urea are hydrolysed, but casein, gelatin, starch, elastin and arbutin are not. Methyl red, Voges–Proskauer and oxidation/fermentation tests give negative results. p-Nitrophenol and 2,4-dinitrophenol are degraded. Nitrate and nitrite are not reduced. H2S and indole are not produced. Utilization of carbon and nitrogen sources and acid production from carbohydrates under aerobic conditions are shown in Table 1. The diagnostic cell-wall amino acid is meso-diaminopimelic acid, and arabinose and galactose are the major cell-wall sugars, indicating cell-wall chemotype IV. The major isoprenoid quinone is MK-8(H2); MK-9(H2) is present in minor amounts. The cellular polar lipid content is of type II, comprising phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, phosphatidylglycerol and traces of diphosphatidylglycerol. The predominant fatty acids are C16 : 0 (34.14 %), C16 : 1
7c (21.18 %), C17 : 18c (15.54 %), C18 : 19c (8.72 %), 10-methyl C18 : 0 (1.68 %) and 10-methyl C17 : 0 (1.55 %). Mycolic acids are present. The DNA G+C content is 72 mol%.
The type strain, RKJ300T (=MTCC 7085T=JCM 13270T), was isolated from a pesticide-contaminated soil in Punjab State, India.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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-dextrin, D-fructose 6-phosphate, 
50 %, based on a neighbour-joining analysis of 1000 resampled datasets. GenBank accession numbers are given in parentheses. Bar, 1 substitution per 100 nucleotide positions.