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Kocuria Palustris Sp. Nov, and Kocuria Rhizophila Sp. Nov., Isolated from the Rhizoplane of the Narrow-Leaved Cattail (Typha Angustifolia)

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Kocuria Palustris Sp. Nov, and Kocuria Rhizophila Sp. Nov., Isolated from the Rhizoplane of the Narrow-Leaved Cattail (Typha Angustifolia) International Journal of Systematic Bacteriology (1999),49, 167-1 73 Printed in Great Britain Kocuria palustris sp. nov, and Kocuria rhizophila sp. nov., isolated from the rhizoplane of the narrow-leaved cattail (Typha angustifolia) Gabor KOV~CS,’Jutta Burghardt,’ Silke Pradella,’ Peter Schumann,’ Erko Stackebrandt’ and KAroly Mhrialigeti’ Author for correspondence: Erko Stackebrandt. Tel: +49 531 2616 352. Fax: +49 531 2616 418. e-mail : [email protected] Department of Two Gram-positive, aerobic spherical actinobacteria were isolated from the Microbiology, Edtvds rhizoplane of narrow-leaved cattail (lypha angustifolia) collected from a Lordnd University, Budapest, Hungary floating mat in the Soroksdr tributary of the Danube river, Hungary. Sequence comparisons of the 16s rDNA indicated these isolates to be phylogenetic 2 DSMZ-German Collection of Microorganisms and neighbours of members of the genus Kocuria, family Micrococcaceae, in which Cell Cultures GmbH, they represent two novel lineages. The phylogenetic distinctness of the two Mascheroder Weg 1b, organisms TA68l and TAGA27l was supported by DNA-DNA similarity values of 38124 Braunschweig, Germany less than 55% between each other and with the type strains of Kocuria rosea, Kocuria kristinae and Kocuria varians. Chemotaxonomic properties supported the placement of the two isolates in the genus Kocuria. The diagnostic diamino acid of the cell-wall peptidoglycan is lysine, the interpeptide bridge is composed of three alanine residues. Predominant menaquinone was MK-7(H2). The fatty acid pattern represents the straight-chain saturated iso-anteiso type. Main fatty acid was anteiso-C,,,,. The phospholipids are diphosphatidylglycerol, phosphatidylglycerol and an unknown component. The DNA base composition of strains TA68l and TAGA27l is 69.4 and 69-6 mol% G+C, respectively. Genotypic, morphological and physiological characteristics are used to describe two new species of Kocuria, for which we propose the names Kocuria palustris, type strain DSM 11925l and Kocuria rhizophila, type strain DSM 11926l. Keywords : Kocuria palustris, Kocuria rhizophila, phylogeny, classification INTRODUCTION species were shown to form an individual cluster within the Arthrobacter-Micrococcus line of descent The genus Kocuria (Stackebrandt et al., 1995) currently (Stackebrandt et al., 1980, 1995; Koch et al., 1994); a contains four species, i.e. the type species Kocuria cluster later described as the family Micrococcaceae rosea, and Kocuria varians, Kocuria kristinae and (Stackebrandt et al., 1997). Based on 16s rDNA Kocuria erythromyxa. All were originally placed in the analysis and the presence of a unique pattern of genus Micrococcus. While the first three species had chemotaxonomic properties the three Micrococcus been recognized as individual species, K. erythromyxa, species were transferred to a new genus Kocuria. Later, strain UWO 1045, was first called ‘Sarcina erythro- Deinococcus erythromyxa was added to this genus as myxa’, then recognized as a strain of Micrococcus K. erythromyxa when information on the 16s rDNA roseus and subsequently described as Deinococcus sequence became available (Rainey et al., 1997). This erythromyxa, species incertae sedis (Brooks & Murray, species is highly related to K. rosea, to which it was 1981). Following 16s rDNA analysis the Micrococcus originally affiliated. ,. , . , . , . , . .. .. .. In this communication we report the description of The EMBL accession numbers for the sequences of DSM 11925T and DSM two new species of Kocuria isolated from the rhizo- 11 926T are Y16264 and Y 16263, respectively. plane of Typha angustifolia in the Danube river. 00816 0 1999 IUMS 167 G. Kovacs and others METHODS amino acid isobutyl esters (MacKenzie, 1987). Cellular fatty acids extracted according to Korn-Wendisch et al. (1989) Bacterial strains and cultural conditions. Strains TA68T and were analysed by GC (Groth et al., 1996). Menaquinone TAGA27T were isolated from the rhizoplane of the narrow- profiles were examined by HPLC (Stackebrandt et al., 1995). leaved cattail (Typha angustifolia) inhabiting a floating mat Polar lipids were extracted as described by Minnikin et al. on the Soroksir tributary of the Danube river, by plating (1979) and identified by two-dimensional TLC followed by root mass serially diluted on medium B (Luedeman, 1971) spraying with specific reagents (Collins & Jones, 1980). agar plates. The strains were grown on nutrient agar at 28 "C. Strains used for phenotypic comparisons were the DNA isolation and determination of G + C content of DNA. type strains of the species Kocuria rosea DSM 20447T, The DNA was isolated as described by Cashion et al. (1977). Kocuria kristinae DSM 20032T and Kocuria varians DSM The G+C content of the DNA was determined by high- 20033T.K. erythromyxa DSM 11630T was included in fatty performance liquid chromatography as described by acid analyses. Mesbah et al. (1989). Media. The following media were used in liquid or semisolid DNA-DNA hybridization. DNA hybridization was carried state; nutrient agar consisting (1-l) of peptone 5 g, meat out according to De Ley et al. (1970) with modifications as extract 3 g and agar (Oxoid) 15 g, adjusted to pH 7.0 and described by HUBet al. (1983) and Escara & Hutton (1980) medium B, consisting of starch 20 g, yeast extract 10 g and using a Gilford System 2600 spectrophotometer equipped agar (Oxoid) 15 g, adjusted to pH 7.0. with a Gilford 2527-R thermoprogrammer and plotter. Morphological studies. Cell and aggregate morphology was Renaturation rates were computed by the program studied by phase-contrast microscopy and phase-contrast TRANSFER.BAS (Jahnke, 1992). microphotography of material from surface growth on agar, 165 rDNA sequence determination and phylogenetic analy- and by direct observation on the agar surface. sis. Genomic DNA extraction, PCR mediated amplification Staining procedures. Gram-staining and acid-fast staining of the 16s rDNA and sequencing of the PCR products were were performed according to Murray et al. (1994). as described by Rainey et al. (1996). The sequence reaction products were electrophoresed using a model 373A auto- Physiological characterization. The temperature for all matic DNA sequencer (Applied Biosystems). The 16s rDNA physiological tests was 28 "C except for studies concerning sequences were manually aligned with those of members of the temperature range. The relation to oxygen was studied the class Actinobacteria using the ae2 editor (Maidak et al., using stab cultures and agar slant cultures placed in an 1996). Evolutionary distances were calculated by the method anaerobic chamber. Oxidase activity was checked by the of Jukes & Cantor (1969). Phylogenetic dendrograms were benzidine (Deibel & Ewans, 1960) and tetramethyl-p- reconstructed using the treeing algorithm of De Soete (1983). phenylendiamine (Tarrand & Groschel, 1982) tests. Carbo- hydrate degradation and acid production from carbo- Nucleotide sequence accession numbers. Accession numbers hydrates and alcohols was determined via the OF-test for sequences used in the construction of the phylogenetic according to Hugh & Leifson (1953) and by use of API tree are: Kocuria rosea DSM 20447*, X87556; Kocuria 50CH test strips (bioM6rieux). Utilization of various com- varians DSM 20033T, X87754; Kocuria kristinae DSM pounds as carbon sources was tested with BIOLOG GP- 20032T,X80749; Kocuria erythromyxa ATCC 187T,Y 11329; plate, System Release 3.50, following the manufacturer's Nesterenkonia halobia DSM 20541T,X80747 ;Stomatococcus instructions. Catalase production was demonstrated on mucilaginosus DSM 20746T, X87756; Rothia dentocariosa slides by the formation of bubbles after mixing a suspension ATCC 1993lT, M59055 ; Dermacoccus nishinomiyaensis of the organism with a drop of 3 YO(v/v) hydrogen peroxide DSM 20448T,X87757 ;Kvtococcus sedentarius DSM 20547T, solution. Urease activity, nitrate reduction, aesculin hy- X87755 and Rubrobacter radiotolerans JCM 21 53T,U65647. drolysis, and hydrolysis of Tween 80 were tested according The 16s rDNA sequence of Micrococcus luteus was retrieved to published methods (Christensen, 1946; Cowan & Steel, from the Ribosomal Database Project (Maidak et al., 1996). 1974). Decomposition of an emulsion of UV-sterilized cinefilm strips incubated in phosphate buffer was used to verify gelatin hydrolysis. Casein hydrolysis was determined RESULTS AND DISCUSSION on streak-inoculated agar mixed from 6ml liquefied agar Cultural characteristics and 2 ml steamed skimmed milk, DNA hydrolysis on Bacto DNase test agar (Difco), and hydrolysis of starch on Optimal growth for strains TA6ST and TAGA27Twas inorganic salts starch agar (Difco) after flooding the agar obtained on nutrient agar at 28 "C under aerobic surface with Lugol's solution containing 0-1YO (w/v) iodine conditions and visible growth was observed 12 h after and 0.2% (w/v) potassium iodide. H,S production was inoculation. Colonies were opaque, smooth with ir- determined as described by Cowan & Steel (1974). NaCl regular edges, and yellow and pale-yellow pigmen- tolerance was checked by adding NaCl to nutrient broth at final concentrations of 2,5,7-5, 10 and 15 % (w/v) of NaC1. tation for strains TA6ST and TAGA27T, respectively. Antibiotic resistance tests were performed by measuring Temperature range for growth on nutrient agar was diameters of inhibition zones on Mueller-Hinton agar plates between 10 and 40 "C for strain TA6STand between 10 containing antibiotic disks (SANOFI Diagnostic, Pasteur). and 30 "C for strain
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