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ek3ová3
1 Department of Microbiology, Faculty of Science, Masaryk University Brno, Tvrdého 14, 602 00 Brno, Czech Republic
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, 38124 Braunschweig, Germany
3 CCM – Czech Collection of Microorganisms, Masaryk University Brno, Tvrdého 14, 602 00 Brno, Czech Republic
Correspondence
Ludmila Tvrzová
lida{at}sci.muni.cz
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ABSTRACT |
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Lys–Ala2, major menaquinone MK-9(H2) and fatty acid composition confirmed assignment of the strain to the genus Arthrobacter. The results of phylogenetic analysis, DNA–DNA relatedness experiments and physiological and chemotaxonomic characteristics indicate that CCM 2783 differs from its nearest phylogenetic relative Arthrobacter psychrolactophilus and from other recognized Arthrobacter species. Therefore, a novel species, Arthrobacter stackebrandtii sp. nov., is proposed with the type strain CCM 2783T (=DSM 16005T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CCM 2783T is AJ640198.
An extended neighbour-joining tree based on 16S rRNA gene sequence analysis is available as a supplementary figure in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). During the course of a taxonomic study of arthrobacters, we found that the strain differed from the type strain of A. aurescens. In this study, we report the taxonomic characterization of strain CCM 2783 and propose to classify it as representing a novel species of the genus Arthrobacter, Arthrobacter stackebrandtii sp. nov.
Strain CCM 2783 and the closely related type strain Arthrobacter psychrolactophilus DSM 15612T were routinely cultivated on tryptone soy medium (Oxoid) at 28 °C. Standardized methods as described by Smibert & Krieg (1994) and the API CORYNE, API ZYM and Biolog systems were used for phenotypic characterization of the strains. The commercial kits were applied according to the manufacturer's instructions.
Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene sequence and purification of PCR products were carried out as described by Rainey et al. (1996). Purified PCR products were sequenced with the CEQ DTCS Quick Start kit (Beckman Coulter) as directed in the manufacturer's protocol. The CEQ 8000 Genetic Analysis System was used for electrophoresis of the sequence reaction products.
The ae2 editor (Maidak et al., 1999) was used to align the 16S rRNA gene sequence of strain CCM 2783 against those of representatives of the main bacterial lineages available from the public databases. Evolutionary distances were calculated by the method of Jukes & Cantor (1969). Phylogenetic dendrograms were constructed using the neighbour-joining algorithms (De Soete, 1983). Bootstrap analysis was used to evaluate the tree topology by performing 1000 resamplings (Felsenstein, 1993). DNA for DNA–DNA hybridization experiments was isolated by using a French pressure cell and purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA–DNA reassociation was performed under optimal conditions (2x SSC at 67 °C) and recorded using a Gilford 2600 spectrophotometer (Huß et al., 1983; Jahnke, 1992). Automated ribotyping was carried out using the RiboPrinter microbial characterization system (Qualicon; DuPont) and EcoRI and PvuII to generate restriction fragments.
The peptidoglycan structure was determined by using total (4 M HCl, 100 °C, 16 h) and partial (4 M HCl, 100 °C, 45 min) hydrolysates of purified cell walls according to Schleifer (1985). Amino acids and peptides were separated by two-dimensional ascending TLC on cellulose plates with the solvent systems of Schleifer & Kandler (1972). The molar ratio of the amino acids was determined by GC as described by MacKenzie (1987). Menaquinones were extracted according to Collins et al. (1977) and analysed by HPLC (Groth et al., 1996). Fatty acids were extracted and analysed following the instructions of the Microbial Identification System operating manual (MIDI, 1999).
Phylogenetic analysis based on the almost complete 16S rRNA gene sequence (1518 nt) confirmed that strain CCM 2783 belongs to the genus Arthrobacter and that it was distinct from A. aurescens (Fig. 1; an extended dendrogram is available as Supplementary Fig. A in IJSEM Online). The highest binary 16S rRNA gene sequence similarity was found to the type strain of Arthrobacter psychrolactophilus DSM 15612T (98·1 %). The similarity to the recently described Arthrobacter russicus GTC 863T (Li et al., 2004) was 95·3 %. DNA–DNA relatedness between strain CCM 2783 and A. psychrolactophilus DSM 15612T was only 18·7 % (24·3 % after repetition), which is clearly below the 70 % considered to be the threshold value for the delineation of genomic species (Wayne et al., 1987). The RiboPrint patterns of strain CCM 2783 (generated by EcoRI and PvuII) were different from those of the type strain of A. psychrolactophilus (data not shown).
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(according to Schleifer & Kandler, 1972) and the major menaquinone MK-9(H2) place strain CCM 2783 within the ‘Arthrobacter globiformis group’ of the genus Arthrobacter (Stackebrandt & Schumann, 2000). The peptidoglycan composition and menaquinone pattern allow differentiation of CCM 2783 from A. psychrolactophilus DSM 15612T and related species.
The peptidoglycan of strain CCM 2783 contained an Ala2 interpeptide bridge and the alpha-carboxyl group of D-glutamic acid at position 2 of the peptide subunit was replaced by D-alanine amide. Analysis of the peptidoglycan structure of A. psychrolactophilus DSM 15612T revealed type A3 with a Thr–Ala3 interpeptide bridge, type A11.28 according to the DSMZ catalogue of strains (DSMZ, 2001). The presence of threonine in the interpeptide bridge linked to the lysine residue was supported by the occurrence of the characteristic stable peptide Lys–Thr in the total peptidoglycan hydrolysate. Consequently, only reduced amounts of lysine and threonine could be detected after total hydrolysis of the peptidoglycan. This is in agreement with the relatively small amount of threonine in the peptidoglycan of the A. psychrolactophilus type strain reported by Loveland-Curtze et al. (1999). The presence of threonine in the interpeptide bridge was also reported in the phylogenetic neighbours of A. psychrolactophilus, Arthrobacter polychromogenes, Arthrobacter oxydans, Arthrobacter chlorophenolicus and Arthrobacter sulfonivorans (Keddie et al., 1986; Kodama et al., 1992; Westerberg et al., 2000; Borodina et al., 2002) and also of A. aurescens (Keddie et al., 1986). The Ala2 interpeptide bridge distinguishes CCM 2783 from all these related species and also from A. aurescens, to which this strain was affiliated by Antheunisse & Vonkeman (1975).
Along with the major menaquinone MK-9(H2), strain CCM 2783 contained MK-10(H2) as a minor component and a small amount of MK-11(H2). The strain differed in its menaquinone pattern from A. psychrolactophilus DSM 15612T (Table 1) and from A. aurescens, which was reported to possess predominant menaquinone MK-9(H2) and minor amounts of MK-8(H2), MK-7(H2) and MK-9 (Collins & Jones, 1981).
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; Kotouková et al., 2004).
Strain CCM 2783 is additionally differentiated from its closest relative A. psychrolactophilus DSM 15612T on the basis of a number of physiological and biochemical characteristics (Table 1
). Additional phenotypic characteristics of strain CCM 2783 are given in the species description below.
Description of Arthrobacter stackebrandtii sp. nov.
Arthrobacter stackebrandtii (sta.cke.brand'ti.i. N.L. gen. masc. n. stackebrandtii of/from Stackebrandt, named in honour of Erko Stackebrandt for his pioneering contributions to our insight into the phylogenetic structure of the suborder Micrococcineae and of the genus Arthrobacter in particular).
Cells are irregular club-shaped rods showing a rod–coccus cycle, 0·6–1x1–3 µm, occurring in pairs as typical V-forms. Gram-positive, non-motile and non-acid-fast, non-spore-forming. Growth occurs at 4–30 °C. Growth occurs over a pH range 5·7–9·1 and in the presence of 5 % NaCl. Obligately aerobic. Positive for catalase, urease and pyrazinamidase. Negative for oxidase, 
-glucuronidase, -galactosidase, -glucosidase, N-acetyl--glucosaminidase and pyrrolidonyl arylamidase. Nitrate is reduced to nitrite. Nitrite is not reduced; Tween 80 and aesculin are not hydrolysed. Starch and gelatin are hydrolysed. Acid production is negative from glucose, ribose, xylose, mannitol, maltose, lactose, sucrose and glycogen. The following compounds are utilized (Biolog system): -cyclodextrin, dextrin, Tween 40, L-arabinose, D-arabitol, arbutin, D-cellobiose, D-fructose, L-fucose, D-galactose, -D-glucose, -D-lactose, lactulose, maltose, maltotriose, D-mannose, D-melibiose, methyl -D-galactoside, methyl -D-galactoside, palatinose, D-psicose, D-raffinose, D-ribose, salicin, stachyose, sucrose, D-trehalose, turanose, D-xylose, -ketovaleric acid, methyl pyruvate, pyruvic acid, L-asparagine, glycerol, adenosine, inosine, thymidine and uridine. Negative reactions (Biolog) were observed with -cyclodextrin, glycogen, inulin, mannan, Tween 80, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, amygdalin, D-galacturonic acid, gentiobiose, D-gluconic acid, meso-inositol, D-melezitose, 3-methyl glucose, methyl -D-glucoside, methyl -D-glucoside, methyl -D-mannoside, L-rhamnose, sedoheptulosan, D-sorbitol, xylitol, -hydroxybutyric acid, 
-hydroxybutyric acid, p-hydroxyphenylacetic acid, -ketoglutaric acid, lactamide, D-lactic acid methyl ester, D-malic acid, monomethyl succinate, propionic acid, succinamic acid, succinic acid, N-acetylglutamic acid, alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-glutamic acid, glycyl L-glutamic acid, L-pyroglutamic acid, putrescine, 2,3-butanediol, adenosine 5'-monophosphate, uridine 5'-monophosphate, fructose 6-phosphate, glucose 1-phosphate, glucose 6-phosphate and DL-glycerophosphate.
The type strain, CCM 2783T (=DSM 16005T), was isolated from poultry litter.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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