View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by IR@NEIST - North East Institute of Science and Technology (CSIR) Antonie van Leeuwenhoek DOI 10.1007/s10482-010-9547-9 ORIGINAL PAPER Description of a novel actinobacterium Kocuria assamensis sp. nov., isolated from a water sample collected from the river Brahmaputra, Assam, India Chandandeep Kaur • Ishwinder Kaur • Revti Raichand • Tarun Chandra Bora • Shanmugam Mayilraj Received: 3 November 2010 / Accepted: 22 December 2010 Ó Springer Science+Business Media B.V. 2011 Abstract A Gram-positive, pale yellow pigmented (99.1%); however, the DNA–DNA relatedness value actinobacterium, strain S9-65T was isolated from a between strain S9-65T and K. palustris was 20.6%. water sample collected from the river Brahmaputra, On the basis of differential phenotypic characteristics Assam, India and subjected to a polyphasic taxo- and genotypic distinctiveness, strain S9-65T should nomic study. The physiological and biochemical be classified as representative of a novel species properties, major fatty acids (anteiso-C15:0 and Kocuria, for which the name Kocuria assamensis is anteiso-C17:0), estimated DNA G?C content proposed. The type strain is S9-65T (=MTCC (69.2 mol %) and 16S rRNA gene sequence analysis 10622T = DSM 23999T). showed that strain S9-65T belonged to the genus Kocuria. Strain S9-65T exhibited highest 16S rRNA Keywords DNA–DNA hybridization Á FAME Á gene sequence similarity with Kocuria palustris 16S rRNA gene sequence Institute of Microbial Technology Chandigarh and North East Introduction Institute of Science & Technology—a constituent laboratory of Council of Scientific and Industrial Research (CSIR), The genus Kocuria was proposed by Stackebrandt Government of India Chandandeep Kaur, Ishwinder Kaur, Both authors have et al. (1995) to accommodate phylogenetically contributed equally to the study. distinct Actinobacteria formerly classified in the The GenBank accession number for the 16S rDNA sequence of genus Micrococcus. The type species of the genus T Kocuria assamensis strain S9-65 is HQ018931. is Kocuria rosea. Members of the genus have been isolated from different sources such as air, fermented Electronic supplementary material The online version of this article (doi:10.1007/s10482-010-9547-9) contains sea food, mammalian skin, soil, the rhizoplane, supplementary material, which is available to authorized users. freshwater or seawater, marine sediment and desert soil (Kloos et al. 1974; Stackebrandt et al. 1995; & C. Kaur Á I. Kaur Á R. Raichand Á S. Mayilraj ( ) Kovacs et al. 1999; Reddy et al. 2003; Kim et al. Microbial Type Culture Collection (MTCC) & Gene Bank, Institute of Microbial Technology, 2004; Tvrzova´ et al. 2005; Li et al. 2006; Mayilraj Chandigarh 160 036, India et al. 2006a, b; Zhou et al. 2008; Tang et al. 2009; e-mail: [email protected] Seo et al. 2009). At present the genus Kocuria consists of 17 species with validly published names T. C. Bora Department of Biotechnology, North East Institute (http://www.bacterio.cict.fr/k/kocuria.html). Here, we of Science & Technology, Jorhat 785 005, India describe the taxonomic status of an actinobacterium, 123 Antonie van Leeuwenhoek strain S9-65T, isolated from a surface water sample Saha et al. (2005). The purified quinones were collected from the river Brahmaputra, Assam, India separated by reversed phase HPLC (SCL-10AVP, (93° 080–98° 360E and 26° 300–20° 450N) by using a Shimadzu) using the solvent system of acetonitrile polyphasic approach. and isopropanol in a ratio of 65:35 with a flow rate of 1 ml/min and monitored at a wavelength of 269 nm. For cellular fatty acid analysis, the strains were Materials and methods grown on tryptic soy agar medium at 30°C for 36 h and the fatty acid methyl ester analysis was per- Strains, cultivation and phenotypic formed by using Sherlock Microbial Identification characterization System (MIDI, USA) as described previously (Sasser 1990; Pandey et al. 2002). Extraction of polar lipids The strain S9-65T was isolated by the dilution plate was done based on the modified protocol of Bligh and technique on tryptic soy agar medium (TSA; HiMe- Dyer (1959). Two-dimensional TLC was run for dia, India) and incubated for 4 days at 30°C. To study identification of polar lipids according to procedures its phenotypic characteristics, the isolate was rou- described by Komagata and Suzuki (1987). Lipid tinely cultivated on TSA medium at 30°C and spots were detected using the following spray maintained as glycerol stocks at -70°C. The refer- reagents: molybdatophosphoric acid (5% w/v) in ence type strain Kocuria palustris strain TAGA 27T absolute ethanol, molybdenum blue spray reagent MTCC 10490T (=DSM 11925T) was obtained from (1.3% Sigma), ninhydrin (0.2% w/v) in acetone and Microbial Type Culture Collection & Gene Bank anisaldehyde reagent (Sigma) for detection of total (MTCC), Institute of Microbial Technology, Chan- lipids, phospholipids, aminolipids and glycolipids digarh, India. Colony morphology, cell morphology, respectively. The peptidoglycan structure was deter- motility and Gram’s reaction of the strain were mined by using a hydrolysate of purified cell walls determined by using standard methods (Barrow and according to Schleifer (1985). The amino acids and Feltham 1993; Murray et al. 1994; Smibert and Krieg peptides were separated by two-dimensional ascend- 1994). Phenotypic characterization was performed ing TLC as described by Schleifer and Kandler using TSA as basal medium and strains were (1972), with the modification that TLC on cellulose incubated at their optimum growth temperatures. sheets (Merck 5577) was used instead of paper Physiological tests such as growth at different chromatography. The G?C content of genomic DNA temperatures (between 10 and 45°C), pH (using was determined spectrophotometrically (Lambda 35, biological buffers; Na2HPO4/NaH2PO4,Na2CO3/ Perkin Elmer, Waltham, MA, USA) using thermal NaHCO3 for pH below 8 and Na2HPO4/NaOH for denaturation method (Mandel and Marmur 1968). pH above 8), NaCl concentrations and acid production from various carbohydrates and other biochemical Determination of 16S rRNA gene sequence, tests were performed as described (Smibert and Krieg phylogenetic analysis and genomic relatedness 1994). The API ZYM and API 20NE micro test strips were used as per the instructions of the manufacturer For 16S rRNA gene sequencing the genomic DNA (bioMe´rieux). Sensitivity of the strain to antibiotics extraction and amplification was performed as was tested by using antibiotic susceptibility discs described previously (Mayilraj et al. 2006a, b). (HiMedia, India) after incubation of 48 h. Identification of phylogenetic neighbours and the calculation of pairwise 16S rRNA gene sequence Chemotaxonomic characterization similarities were achieved using the EzTaxon server (http://www.eztaxon.org/; Chun et al. 2007). The 16S Freeze-dried cells for chemotaxonomic analysis rRNA gene sequence of S9-65T and representative (except for the fatty acids study) were prepared by closely related species were retrieved from the harvesting the bacterial cells in the late exponential Eztaxon server and aligned using the MEGA version phase following their growth in Tryptic Soy Broth 4.0 (Tamura et al. 2007). Phylogenetic trees were (TSB; HiMedia, India) at 30°C for 2 days. Isoprenoid constructed using the neighbour-joining as well as quinones were extracted and purified as described by maximum parsimony algorithms. Bootstrap analysis 123 Antonie van Leeuwenhoek was performed to assess the confidence limits of the Table 1 Characters that differentiate strain S9-65T along with branching. The G?C content of genomic DNA was the closest species K. palustris (MTCC 10490T): 1, strain S9- T determined spectrophotometrically (Lambda 35, 65 and 2, K. palustris Perkin Elmer, Waltham, MA, USA) using thermal Characteristics 1 2 denaturation method (Mandel and Marmur 1968). Growth at pH 11 ?- DNA–DNA hybridization was performed each time Growth at 12°C -? with freshly isolated genomic DNA and was repeated Casein hydrolysis -? three times by the membrane filter method (Tourova Enzyme assayed for (API ZYM) and Antonov 1987). Leucine arylamidase -? Valine arylamidase -? Results and discussion Cystine -? Trypsin -? Phenotypic characteristics a- galactosidase -? a-glucosidase -? Growth of the strain on TSA produced a yellow b-glucosidase -? pigment after incubation on TSA for 2 days. The N-acetyl-b-glucosaminidase -? detailed differential phenotypic properties are shown a-mannosidase -? in Table 1 and also mentioned in species description. Assimilation of (API 20 NE) Phenotypic data presented in the table indicated that Arabinose -? strain S9-65T differed from the closely related species N-acetyl-glucosamine -? at least by 29 characters which includes acid produc- Potassium gluconate -? tion from carbohydrates like adonitol, raffinose, Malic acid -? rhamnose, cellobiose, arbinose and xylose, casein Hydrogen sulphide production -? hydrolysis, nitrate reduction, hydrogen sulphide pro- Nitrate reduction -? duction were negative for strain S9-65T and positive Acid production from for the closely related strain K. palustris TAGA 27T. Adonitol -? There were major differences in oxidation of different Raffinose -? carbon sources using biolog. Strain S9-65T was Rhamnose -? -1 sensitive to antibiotics (lg disc ) such as nitrofuran- Cellobiose -? tion (300), norfloxacin (10), polymyxin B (300), Arabinose -? cephalothin (30) and oxacillin (5) in comparison to Xylose