International Journal of Systematic Bacteriology (1999),49, 167-1 73 Printed in Great Britain

Kocuria palustris sp. nov, and 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 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 , 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 kristinae and . 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 , type strain DSM 11926l.

Keywords : Kocuria palustris, Kocuria rhizophila, phylogeny, classification

INTRODUCTION species were shown to form an individual cluster within the Arthrobacter- 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 TAGA27T.Strain TAGA27Tgrew in up to 7 % (w/v) NaC1, while strain TA6STgrew well Chemotaxonomic characterization. Purified cell-wall prep- % arations were obtained as described by Schleifer & Kandler at 10 NaCl, sparsely at 15 YONaCl, but no growth (1972). Amino acids and peptides in cell-wall hydrolysates was detected above 15 YONaC1. Both strains grew well were analysed by two-dimensional TLC on cellulose plates between pH 5.7-7-5, but growth was very poor at using the solvent systems described by Schleifer & Kandler pH 8.0 and no growth occurred at pH 4.5 or 8.5. (1972). The molar ratios of cell-wall amino acids were Growth was never observed under anaerobic con- determined by GC and GC-MS of N-heptafluorobutyryl ditions.

168 International Journal of Systematic Bacteriology 49 Kocuriu pulustris and Kocuriu rhizophilu spp. nov

Table 1. Phylogenetic distance as determined by 165 rDNA analysis (upper right triangle) and DNA-DNA similarities as determined by hybridization (lower left triangle) of the isolates and three type strains of the genus Kocuria I Taxon Strain TAGA2F Strain TA6gT K. vavians K. kristinae K. vosea I Strain TAGA27T - 97.0 96.9 95.8 97.0 Strain TA68T 30.8 - 98.6 95.8 96.9 K. varians 42.2 52-6 - 95.8 96.6 K, kristinae 37.0 46.6 44.0 - 95.7 K. rosea 46.3 43.0 ND 34.4 - m, Not determined.

Morphological characteristics - Strain TA68T (DSM 11926T) On nutrient agar the cells of TA68T and TAGA27T - Strain TAGA27T (DSM 11 925T) were spherical (1.0-1.5 pm in diameter), occurring in Kocuria kristinae DSM 20032T pairs, tetrads and packets. Rarely, larger cells 2-0 pm -80 in diameter were also found. Cells were non-motile. Kocuria erythromyxa DSM 11630T Endospores were not detected. 95 Kocuria rosea DSM 20447T 7 100 Rothia dentocariosa DSM 46363T Staining reactions - Stomatococcus mucilaginosus DSM 20746T Strains TA68T and TAGA27T stained Gram-positive 7 Nesterenkonia halobia DSM 20541T and no lysis was observed following treatment with - Micrococcus luteus DSM 2O03OT 3 % (w/v) KOH. Cells were not acid-fast. D 100 Derma coccus nish inom iyaensis S M 20U8T Phylogenetic analysis 1 Yo Almost complete 16s rDNA sequences comprising 1447 nucleotides for strain TAGA27T and 1471 Fig. 7. 165 rDNA sequence based phylogenetic dendrogram nucleotides for strain TA68T [951 and 95-3%, re- constructed from evolutionary distances (De Soete, 1983) showing the phylogenetic position of isolates TA68T and spectively, of the Escherichia coli sequence according TAGA27T within the radiation of Kocuria species. Numbers at to Brosius et al. (1978)l were determined. Similarity branching points refer to bootstrap values (500 trees values were calculated for these strains and a rep- resampled). Scale bar, inferred nucleotide substitutions per 100 resentative selection of actinomycete species (not nucleotides. shown). The similarity value between the two strains was 97-6YO, while the similarity values determined for Maintenance the two strains and their closest relatives, which were the type strains of members of the genus Kocuriu Serial transfers were done at 4-week intervals on ranged between 95-8 and 98.6 YO(Table 1, upper-right nutrient agar followed by storage at 4 "C. Long-term triangle). The two isolates contained all the signature preservation was achieved by freeze-drying, and main- nucleotides that define the family Micrococcuceae to tenance in the vapour phase above liquid nitrogen. which the genus Kocuriu belongs phylogenetically

Table 2. Fatty acid composition of the isolates and the type strains of the genus Kocuria

TA6gT 1.1 2.4 13.8 48.4 59 2.6 1.2 21.1 TAGA27' 2.2 3.1 1.0 59.2 1.0 3.3 2.1 97 3.0 4.6 1.4 K. kristinaet 2.6 1 .O 1.6 701 12.1 1.7 9.3 K. varianst 1.4 2.9 1.3 603 1.3 11.1 9.6 12.1 K. roseat 143 1.5 7.6 70.9 1.6 1.6 5.9 4.3 3.0 K. eryihromyxa 1.2 1.8 14.1 63.9 1.4 1.2 6.7 2.4 1.9 1.4

*Values less than 1 % not shown. i, iso; ai, anteiso. t Data from Stackebrandt et al. (1995).

International Journal of Systematic Bacteriology 49 169 G. Kovacs and others

Table 3. Differentiation of the isolates from Kocuria species based on biochemical and physiological characteristics ., . , . . , ...... , ...... , ...... , ...... , ...... , ...... , ...... , ...... , . , ...... , ...... + , Positive; (+), weak positive; -, negative. Physiological reaction TA6ST TAGA27T K. varians" K. kristinae* K. rosea* I Gelatinase + + Oxidase - Starch hydrolysis Growth on Simmons' citrate + (+) Urease - + + Phosphatase + Tween 80 hydrolysis + Aesculin hydrolysis - + Arginine dihydrolase H,S production (+) - NO; from NO; + + Growth in presence of: 10 'YO NaCl + + 15 'YO NaCl (+> - Acid production from: D-Mannose + - + + Galactose - + - Lactose + +t - Maltose - + Saccharose + + D-Xylose L-Arabinose - Glycerol + Mannitol - Sorbitol + Arbutin + Salicin + Trehalose + Melezitose + Amidon + D-Turanose + Ribose + - P-Gentiobiose + - Utilization of: Dextrin + + Glycogen + + Tween 40 + + Tween 80 + + N-Acetyl-D-galactosamine - + N-Acetyl-D-glucosamine - - Adoni t ol + + L-Arabinose + + L-Fucose + + meso-Inositol - + Maltose + + D-Mannitol - + D-Mannose + + D-Meli biose + - Sorbitol - + + Turanose + + + Xylitol + + + Methyl-pyruvate + + - Inosine - + - Glycerol - + L-Glutamic acid + L- Alanine + * Data from Stackebrandt et al. (1995). t Reaction different from published data (Stackebrandt et aE., 1995).

170 lnterna tional Journal of Systematic Bacteriology 49 Kocuria palustris and Kocuria rhizophila spp. nov

(Stackebrandt et al., 1997). The phylogenetic dendro- Physiological characteristics gram shown in Fig. 1 was constructed from evol- K. rosea, K. varians utionary distances by the distance matrix method (De For comparison, the type strains of Rubrobacter and K. kristinae were included in the determination of Soete, 1983). The 16s rDNA sequence of K. radiotolerans was used to determine the root. A total of physiological properties of TA68T and TAGA27T. erythromyxa was not included because of its high 1350 nucleotides present in all strains between K. rosea positions 41 and 1458 (E. coli positions) were used for phylogenetic relatedness to (Fig. 1). Except for two reactions, the physiological tests of the type the analysis. Strains TA68T and TAGA27T grouped Kocuria. strains of the three Kocuria species gave results amongst species of the genus While strain identical with those published in the literature (sum- TA68T was a phylogenetic neighbour of K. varians et al., (98.6 % sequence similarity), strain TAGA27T was marized by Stackebrandt 1995). The two differences were a negative oxidase reaction in K. more distinct, showing between 95.8 and 97% se- kristinae Kocuria. strain DSM 20032T, and acid production quence similarity to representatives of from lactose in K. varians strain DSM 20033T. Strains TA68T and TAGA27T shared the following DN A-D NA hy br i d i za t io n physiological properties with strains of the other three Kocuria species studied : growth under strictly aerobic To verify the phylogenetically distinct position of conditions ; benzidine and catalase tests and growth on strains TA68T and TAGA27T as determined by 16s inorganic nitrogen were positive ; indole-negative, oxi- rDNA sequence analysis, the degree of DNA-DNA dase test and phenylalanine deaminase reaction were similarity was determined for these two strains and the negative; acid production from D-glucose and D- type strains of three Kocuria species (Table 1, lower left ; K. erythromyxa fructose utilization of cellobiose, D-trehalose and triange). was not included because, on glucuronamide was negative. The results of physio- the basis of the high 16s rDNA similarity, it can be K. rosea. logical tests in which the isolates differed from each considered a close relative of The DNA other and/or from the type strains of three species of -DNA similarity values obtained confirmed the Kocuria are shown in Table 3. slightly higher degree of relatedness between strain TA68T and K. varians DSM (52.6 % similarity) but all Susceptibility tests to antibiotics gave the following DNA similarities were clearly below 70% which is results: the two isolates and the type strains of the considered to be the threshhold value for the de- three species of Kocuria were susceptible to penicillin, lineation of genospecies (Wayne et al., 1987). sulbactam/ampicillin, amoxicillin, piperacillin, mero- penem, cefalexine, cefamandol, cefoperasone, chloramphenicol, erythromycin, clindamycin, netil- Chemotaxonomic characteristics mycin, vancomycin, trimethroprim ; all strains were resistant to nitrofurantoin. Strain TA68Twas resistant Strains TA68T and TAGA27T contained the peptido- to ciprofloxacin while strain TAGA27T was mod- glycan amino acids Lys, Glu and Ala at the molar ratio erately sensitive. 1.2: 1-0:4.5. From this ratio and from the occurrence of characteristic peptides in the partial hydrolysates of cell walls (data not shown), it was concluded that both Description of two new Kocuria species strains displayed variation A3a (Schleifer & Kandler, Phylogenetic and chemotaxonomic evidence indicate 1972) with three Ala residues as the interpeptide that the two isolates from the rhizoplane of Typha bridge. The isoprenoid quinones of strain TA6gT were angustifolia represent two new lineages within the MK-7(H2), MK-8(H2), MK-6(H2) and MK-9(H2) genus Kocuria. Low to moderate DNA-DNA re- (peak area ratio, 5 1 : 39 :4 : 1). Strain TAGA27T con- association values obtained for the two isolates and tains the menaquinones MK-7(H2), MK-6(H2) and between them and the type strains of the three of the MK-8(H2) (peak area ratio, 87: 7 :2). The polar lipid four currently recognized Kocuria species confirm their patterns of both strains consisted of phosphatidyl- species status. Differentiation of the two new species glycerol, diphosphatidylglycerol and one unknown from each other and from the other Kocuria species phospholipid. Additionally, strain TA68T contained (little information is available for the moderately one and strain TAGA27T contained three unknown radiation-resistant species K. erythromyxa ; Brooks & glycolipids. The cellular fatty acid profiles of both Murray, 1981) is achieved by the primary structure of strains were of the branched-chain saturated iso- 16s rDNA, composition of fatty acids (Table 2) and anteiso type and showed anteiso-C,, : , as the pre- physiological properties (Table 3). dominating component (Table 2). Strain TA68T is characterized by the additional occurrence of anteiso- C,,:, and iso-C,,,, in high amounts, By comparison, Description of Kocuria palustris sp. nov. the type strains of K. rosea and K. erythromyxa both Kocuria palustris (pa.lus’tris. L.fem. adj. palustris have fatty acid profiles with remarkably high amounts marshy, swampy). of iso-C,,:, and C,,:, fatty acids. The latter fatty acid could not be detected in the profiles of the other The cells are spherical, occurring in pairs, tetrads and Kocuria species (Table 2). packets, 14-1.5 pm in diameter, Gram-positive and

International Journal of Systematic Bacteriology 49 171 G. Kovacs and others non-motile. Endospores not produced. Not acid-fast. amidon. Utilization of adonitol, L-arabinose, D-fruc- The colonies are pale-yellow, 24---3-0mm in diameter, tose, L-fucose, D-glucose, turanose, xylitol, methyl- opaque, smooth with irregular edges with pale-yellow pyruvate, glucuronamide, dextrin, glycogen, Tween pigmentation. Aerobic. Chemo-organotrophic. No 40, Tween 80 and N-acetyl-D-glucosamine. No utili- growth above 30°C. Good growth at pH 5.7-7.7. zation of cellobiose, D-trehalose, N-acetyh-galacto- Catalase, urease benzidine and growth on inorganic samine, meso-inositol, maltose, D-mannitol, D- nitrogen are positive. Phosphatase, Tween 80 hydroly- melibiose, sorbitol, inosine, glycerol, L-glutamic acid sis and aesculin hydrolysis are negative. Oxidase, and L-alanine. Good growth at 10% NaCl. Pepti- gelatinase, starch hydrolysis, growth on Simmons’ doglycan contains L-lysine as the diagnostic diamino citrate, indole, aesculin hydrolysis, arginine di- acid. The interpeptide bridge consists of three alanine hydrolase and phenylalanine deaminase reaction are residues (variation A3a). The major menaquinones are negative. H2Sreaction weak. Nitrate reduced to nitrite. MK-7(H2) and MK-8(H2). The major fatty acids are Acid production from D-glucose,D-fructose, galactose, ai-C,, : o, ai-C,,: and i-Ci5 :o. Predominant polar lipids lactose and saccharose. No acid production from are phosphatidylglycerol and diphosphatidylglycerol. glycerol, mannitol, sorbitol, ribose, D-xylose, L- The DNA base composition is 69.4 mol% G+C. arabinose, D-mannose, maltose, P-gentiobiose, D- Isolated from the rhizosphere of Typha angustifolia turanose, arbutine, salicine, trehalose, melezitose or from a floating mat on the Soroksar tributary of the amidon. Utilization of adonitol, glucuronamide, ino- river Danube, Hungary. The type strain is strain sine, Tween 40 or Tween 80. No utilization of TA68T. It has been deposited at the DSMZ as strain cellobiose, turanose, xyli t 01, methyl-pyruvate, dextrin, DSM 11926T. glycogen, N-acetyl-D-glucosamine, D-trehalose, L- arabinose, D-fructose, L-fucose, N-acetyl-D-galacto- REFERENCES samine, meso-inositol, maltose, D-mannitol, D- mannose, D-melibiose, sorbitol, glycerol, L-glutamic Brosius, 1. M., Palmer, L., Kennedy, P. J. & Noller, H. F. (1978). acid or L-alanine. Growth at 7 % NaC1, but no growth Complete nucleotide sequence of the 16s ribosomal RNA gene at 10% NaCl. Peptidoglycan contains L-lysine as the from Escherichia coli. Proc Nut1 Acad Sci USA 75, 48014805. diagnostic diamino acid. The interpeptide bridge Brooks, B. W. & Murray, R. G. E. (1981). Nomenclature for consists of three alanine residues (variation A3a). The “Micrococcus radiodurans” and other radiation-resistant major menaquinone is MK-7(H2). The major fatty cocci : Deinococcaceae fam. nov. and Deinococcus gen. nov., acid is ai-C15:o. Predominant polar lipids are including five species. Znt J Syst Bacteriol31, 353-360. phosphatidylglycerol and diphosphatidylglycerol. The Cashion, P., Holder-Franklin, M. A., McCully, 1. & Franklin, M. DNA base composition is 69. 6 mol YOG + C. Isolated (1977). A rapid method for the base ratio determination of from the rhizosphere of Typha angustifolia from a bacterial DNA. Anal Biochem 81, 461466. floating mat in the Soroksar tributary of the river Christensen, W. B. (1946). Urea decomposition as means of Danube, Hungary. The type strain is strain TAGA27T. differentiating Proteus and paracolon cultures from each other It has been deposited at the DSMZ-German Collection and from Salmonella and Shigella types. JBacteriol52,461466. of Microorganisms and Cell Cultures as strain DSM Collins, M. D. & Jones, D. (1980). Lipids in the classification and 11925T. identification of coryneform containing peptidoglycans based on 2,4-diaminobutyric acid. J Appl Bacterio148,459470. Description of Kocuria rhizophila sp. nov. Cowan, S. T. & Steel, K. J. (1974). Manual for the Identification of Medical Bacteria, 2nd edn. Cambridge : Cambridge University Kocuria rhizophila (rhi.zo’phi.la. Gr. n. rhiza a root; Press. Gr. adj.philos loving; M.L.adj. rhizophila root-loving). Deibel, R. H. & Ewans, J. B. (1960). Modified benzidine test for The cells are spherical, occurring in pairs, tetrads and the detection of cytochrome-containing respiratory systems in packets, 1.0-15 pm in diameter, Gram-positive and microorganisms. J Bacteriol79, 356360. non-motile. Endospores not produced. Not acid-fast. De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative The colonies are 1.5-2.5 mm in diameter, opaque, measurement of DNA hybridisation from renaturation rates. smooth with irregular edges with yellow pigmentation. Eur J Biochem 12, 133-142. Aerobic. Chemo-organotrophic. No growth above DeSoete, G. (1983). A least square algorithm for fitting additive 40°C. Good growth between pH 5.7-7.7. Catalase, trees to proximity data. Psychometrika 48, 621-626. benzidine and growth on inorganic nitrogen positive. Escara, J. F. & Hutton, 1. R. (1980). Thermal stability and Gelatinase, phosphatase, Tween 80 hydrolysis, growth renaturation of DNA in dimethylsulphoxide solutions : ac- on Simmons’ citrate are positive. Oxidase, starch celeration of renaturation rate. Biopolymers 19, 13 15-1327. hydrolysis, indol, urease, aesculin hydrolysis, arginine Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. dihydrolase and phenylalanine deaminase reaction are (1996). Agrococcusjenensis gen. nov., sp. nov., a new genus of negative. 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172 International Journal of Systematic Bacteriology 49 Kocuria palustris and Kocuria rhizophila spp. nov

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