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1 School of Food Biosciences, University of Reading, Reading, UK
2 Swedish Match North Europe, Maria Skolgata 83 1tr, S-11885 Stockholm, Sweden
3 Culture Collection, Department of Clinical Bacteriology, University of Göteborg, Sweden
4 Department of Botany and Microbiology, University of Oklahoma, Norman, OK, USA
Correspondence
Paul A. Lawson
paul.lawson{at}ou.edu
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, type L-lysine–L-glutamic acid), synthesized long-chain cellular fatty acids of the straight-chain saturated and monounsaturated types (with C16 : 1
9, C16 : 0 and C18 : 19 predominating) and possessed a DNA G+C content of 46 mol%. Based on morphological, biochemical and chemical characteristics, the coccus-shaped organism did not conform to any presently recognized taxon. Comparative 16S rRNA gene sequencing studies confirmed the distinctiveness of the unknown coccus, with the bacterium displaying sequence divergence values of greater than 7 % with other recognized Gram-positive taxa. Treeing analysis reinforced its distinctiveness, with the unidentified organism forming a relatively long subline branching at the periphery of an rRNA gene sequence cluster which encompasses the genera Alloiococcus, Allofustis, Alkalibacterium, Atopostipes, Dolosigranulum and Marinilactibacillus. Based on phenotypic and molecular phylogenetic evidence, it is proposed that the unknown organism from tobacco be classified as a new genus and species, Atopococcus tabaci gen. nov., sp. nov. The type strain of Atopococcus tabaci is CCUG 48253T (=CIP 108502T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CCUG 48253T is AJ634917.
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). Some of the newly described genera have arisen due to taxonomic revision of existing genera (e.g. Abiotrophia, Enterococcus and Lactococcus from Streptococcus; Oenococcus from Leuconostoc) but the majority of new genera have come to light due to improved taxonomic methodologies used for characterizing and identifying these organisms (e.g. Aguirre & Collins, 1992; Aguirre et al., 1993; Collins et al., 1992, 1997, 1999a, b). Today, over 15 genera of Gram-positive, catalase-negative cocci are recognized. However, despite the rapid increase in the number of recognized genera of Gram-positive, catalase-negative cocci, it is evident that much diversity remains to be discovered. In the course of an ongoing study exploring the diversity of taxonomically problematic catalase-negative cocci, we have characterized a hitherto unknown organism from tobacco which does not correspond to any recognized genus. Based on the results of a polyphasic taxonomic study, we describe yet another new genus and species, Atopococcus tabaci, within the Gram-positive, catalase-negative cocci.
An unknown coccus-shaped organism (designated CCUG 48253T) originating from moist snuff tobacco was received by CCUG, Sweden, for identification. The unidentified coccus was cultured on Columbia blood agar base supplemented with 5 % horse blood at 37 °C under aerobic conditions. The organism was biochemically characterized using the API Rapid ID 32Strep, API Coryne and API ZYM test systems according to the manufacturer's instructions (bioMérieux). The G+C content of DNA was determined by HPLC according to Mesbah et al. (1989). Cell-wall murein was prepared by the mechanical disruption of cells and the analysis of acid hydrolysates as described by Schleifer & Kandler (1972), Schleifer (1985) and MacKenzie (1987). For long-chain cellular fatty acid analysis, the organism was grown statically in BHI broth and fatty acids were analysed using the MIDI system). For isoprenoid quinone analysis, the organism was grown statically in BHI broth and in BHI broth with shaking. Isoprenoid quinones were extracted as described by Collins et al. (1977) and analysed by TLC and HPLC as described by Collins (1994) and Groth et al. (1997). For phylogenetic analysis, 16S rRNA gene sequences were amplified by PCR and directly sequenced using a Taq Dye-Deoxy terminator cycle sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 373A; Applied Biosystems). The closest known relatives of the new isolate were determined by performing database searches using the FASTA program (Pearson & Lipman, 1985). These sequences and those of other related strains were retrieved from GenBank and aligned with the newly determined sequence using the SEQtools program (Rasmussen, 2002). The resulting multiple sequence alignment was corrected manually using the GeneDoc program (Nicholas et al., 1997) and a phylogenetic tree was constructed according to the neighbour-joining method (Saitou & Nei, 1987) with the programs SEQtools and TREEVIEW (Page, 1996). The stability of the groupings was estimated by bootstrap analysis (1000 replications) using the same programs.
The unidentified coccus-shaped organism originating as a contaminant of tobacco stained Gram-positive and was non-motile. It grew aerobically, but not under strictly anaerobic conditions. The organism was catalase-negative. Using the API Rapid ID 32Strep system, the organism produced acid from lactose, maltose, ribose, sucrose and trehalose, but not from any of the other carbohydrates in this test gallery. The organism hydrolysed hippurate and was Voges–Proskauer negative. It displayed activity for 
-glucosidase, -galactosidase, pyroglutamic acid arylamidase and N-acetyl--glucosaminidase. All other enzyme tests included in this system were negative. Using the API ZYM test system, the organism showed activity for alkaline phosphatase, acid phosphatase, esterase C4, -glucosidase, leucine arylamidase and valine arylamidase. All other enzyme tests in the API ZYM system were negative. Employing the API Coryne test kit, the organism produced acid from glucose, maltose, lactose, ribose (weak reaction) and sucrose. It hydrolysed aesculin and displayed activity for -galactosidase, -glucosidase, N-acetyl--glucosaminidase and pyrrolydonyl arylamidase. The long-chain cellular fatty acids of the organism were found to be primarily of the unbranched saturated and monounsaturated types, with iso- and anteiso-methyl-branched forms present in very small amounts. The quantitative fatty acid data corresponded to: C10 : 0 (0·7 %), C12 : 0 (2·6 %), C14 : 0 (8·3 %), C14 : 1 (4·5 %), C16 : 19c (41·9 %), C16 : 0 (15·8 %), iso-C17 : 1 (5·3 %), C18 : 19c (9 %) and iso-C19 : 1 (1·9 %). No respiratory quinones were detected using the methods employed. Analysis of the cell wall murein of isolate CCUG 48253T showed the presence of an A4 murein based on L-lysine type, L-lys–L-Glu. The amino acids lysine, alanine and glutamic acid were detected in approximate molar ratios of 1·0 Lys : 1·9 Ala : 2·4 Glu. The partial hydrolysate contained the peptides L-ala–D-glu and L-lys–D-Ala. Dinitrophenylation revealed that glutamic acid represented the N terminus of the interpeptide bridge. Since the peptide D-ala–D-Glu was not detected, it is likely that the N terminal glutamic acid is of the L-configuration. The DNA G+C composition of the unidentified strain was found to be 46 mol%.
In order to investigate the phylogenetic affinities of the isolate, an almost complete 16S rRNA gene sequence (1498 nucleotide bases, from positions 37 to 1510, Escherichia coli numbering system) was determined. Sequence database searches revealed that the strain was a member of the low-G+C Clostridium subphylum of the Gram-positive bacteria. From the comparative 16S rRNA gene sequence analysis, it was evident that the unidentified, catalase-negative, coccus-shaped organism isolated from tobacco represented an unknown taxon. Phylogenetically, the unidentified isolate formed a distinct subline branching at the periphery of a distinct rRNA gene sequence cluster which embraced Alkalibacterium olivoapovliticus, Alloiococcus otitis, Allofustis seminis, Atopostipes suicloacale, Dolosigranulum pigrum and Marinilactobacillus psychrotolerans. The branching of the unknown organism at the base of this group was supported by a bootstrap resampling value of 95 % (Fig. 1). Amongst the aforementioned taxa, the unidentified organism displayed the highest sequence similarity to Alkalibacterium olivoapovliticus (92·6 %), Alloiococcus otitis (89·9 %), Allofustis seminis (87·5 %), Atopostipes suicloacale (90·4 %), Dolosigranulum pigrum (86·8 %) and Marinilactobacillus psychrotolerans (92·6 %). The relatively high divergence values (>7 %), together with the deep branching position of the unidentified organism, showed that the unidentified bacterium was only distantly related to these taxa and merited classification at a similar taxonomic rank (i.e., Genus).
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). Similarly, the unidentified isolate differed from Alloiococcus otitis, Allofustis seminis and Dolosigranulum pigrum in having a growth temperature optimum of 30 °C. Alloiococcus otitis, Allofustis seminis and Dolosigranulum pigrum all grow optimally at 37 °C (Aguirre & Collins, 1992; Aguirre et al., 1993; Collins et al., 2003). In addition, unlike the unidentified isolate, Alloiococcus otitis is nutritionally very fastidious. Dolosigranulum pigrum differed from the unknown organism in having a L-lys–D-Asp type cell wall murein (Aguirre & Collins, 1992; Aguirre et al., 1993; Miller et al., 1996). Allofustis seminis further differed from the unknown organism by displaying a rod-shaped cellular morphology and by possessing an L-Lys-directly cross-linked cell wall murein (Collins et al., 2003). Therefore, based on sequence divergence values of >7 % with its nearest named phylogenetic relatives and the distinct and deep subline formed by the novel bacterium and in concert with its quite distinct phenotypic characteristics, we are of the opinion that the unknown bacterial contaminant from tobacco merits assignment to a new genus and novel species, for which the name Atopococcus tabaci is proposed.
Description of Atopococcus gen. nov.
Atopococcus (A.to.po.coc'cus. Gr. adj. atopos having no place, strange; Gr. n. coccus a grain or berry; N. L. masc. n. Atopococcus a strange coccus).
Cells stain Gram-positive, are coccoid in shape and occur in pairs or short chains. Non-endospore-forming and non-motile. Aerobic. Catalase-negative. Acid is produced from glucose and some other sugars. Pyroglutamic acid arylamidase is produced. Arginine dihydrolase is not produced. The cell wall murein contains L-lysine (type L-lys–L-Glu). Respiratory menaquinones are not detected. The major long-chain fatty acids are of the straight-chain and monounsaturated types. The DNA G+C content is 46 mol%. The type species is Atopococcus tabaci.
Description of Atopococcus tabaci sp. nov.
Atopococcus tabaci (ta.ba'ci. N.L. gen. neut. n. tabaci of/from tobacco).
Displays the following properties in addition to those given in the genus description. -Haemolytic on horse blood agar. Grows aerobically but not under anaerobic conditions. Optimal growth temperature on tryptic soy agar is 30 °C. Halotolerant, growing in 8–9 % NaCl. Using API test kits, acid is produced from glucose, lactose, maltose, ribose, sucrose and trehalose but not from L-arabinose, D-arabitol, cyclodextrin, glycogen, mannitol, melibiose, melezitose, methyl -D-glucopyranoside, pullulan, raffinose, sorbitol, tagatose or D-xylose. Aesculin and hippurate are hydrolysed but not gelatin. Activity is detected for acid phosphatase, esterase C4, -glucosidase, -galactosidase, leucine arylamidase, valine arylamidase, pyroglutamic acid arylamidase and N-acetyl--glucosaminidase. Activity may or may not be detected for alkaline phosphatase, -glucosidase and urease. Alanine phenylalanine proline arylamidase, arginine dihydrolase, ester lipase C8, lipase C14, chymotrypsin, trypsin, phosphoamidase, cystine arylamidase, -glucuronidase, glycyl trytophan arylamidase, -fucosidase, -mannosidase and -mannosidase are not detected. Nitrate is not reduced and the Voges–Proskauer test is negative. The predominant long-chain fatty acids are C14 : 0, C16 : 19c, C16 : 0 and C18 : 19c.
The type strain, CCUG 48253T (=CIP 108502T), was isolated from moist powdered tobacco.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Aguirre, M., Morrison, D., Cookson, B. D., Gay, F. W. & Collins, M. D. (1993). Phenotypic and phylogenetic characterization of some Gemella-like organisms from human infections: description of Dolosigranulum pigrum gen. nov., sp. nov. J Appl Bacteriol 75, 608–612.[Medline]
Collins, M. D. (1994). Isoprenoid quinones. In: Chemical Methods in Prokaryotic Systematics, pp. 265–309. Edited by M. Goodfellow & A. G. O'Donnell. Chichester: Wiley.
Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, 221–230.
Collins, M. D., Aguirre, M., Facklam, R. R., Shallcross, J. & Williams, A. M. (1992). Globicatella sanguis gen. nov., sp. nov., a new gram-positive catalase-negative bacterium from human sources. J Appl Bacteriol 73, 433–437.[Medline]
Collins, M. D., Falsen, E., Lemozy, J., Åkervall, E., Sjödén, B. & Lawson, P. A. (1997). Phenotypic and phylogenetic characterization of some Globicatella-like organisms from human sources: description of Facklamia hominis gen. nov., sp. nov. Int J Syst Bacteriol 47, 880–882.
Collins, M. D., Lawson, P. A., Monasterio, R., Felsen, E., Sjödén, B. & Facklam, R. R. (1999a). Ignavigranum ruoffiae gen. nov., sp. nov., isolated from human clinical specimens. J Clin Microbiol 37, 1161–1164.
Collins, M. D., Rodriguez Jovita, M., Hutson, R. A., Falsen, E., Sjödén, B. & Facklam, R. R. (1999b). Dolosicoccus paucivorans gen. nov., sp. nov., isolated from human blood. Int J Syst Bacteriol 49, 1439–1442.
Collins, M. D., Higgins, R., Messier, S., Fortin, M., Hutson, R. A., Lawson, P. A. & Falsen, E. (2003). Allofustis seminis gen. nov., sp. nov., a novel Gram-positive, catalase-negative rod-shaped bacterium from pig semen. Int J Syst Evol Microbiol 53, 811–814.
Facklam, R. R. & Elliott, J. A. (1995). Identification, classification, and clinical relevance of catalase-negative, gram-positive cocci, excluding the streptococci and enterococci. Clin Microbiol Rev 8, 479–495.[Abstract]
Groth, I., Schumann, P., Rainey, F. A., Martin, K., Schuetze, B. & Augsten, K. (1997). Demetria terragena gen. nov., sp. nov., a new genus of actinomycetes isolated from compost soil. Int J Syst Bacteriol 47, 1129–1133.
Ishikawa, M., Nakajima, K., Yanagi, M., Yamamoto, Y. & Yamasato, K. (2003). Marinilactobacillus psychrotolerans gen. nov., sp. nov., a halophilic and alkaliphilic marine lactic acid bacterium isolated from marine organisms in temperate and subtropical areas of Japan. Int J Syst Evol Mcrobiol 53, 711–720.
Lipman, D. J. & Pearson, W. R. (1985). Rapid and sensitive protein similarity searches. Science 227, 1435–1441.
MacKenzie, S. L. (1987). Gas chromatographic analysis of amino acids as the N-heptafluorobutyryl isobutyl esters. J Assoc Off Anal Chem 70, 151–160.[Medline]
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Miller, P. H., Facklam, R. R. & Miller, J. M. (1996). Atmospheric growth requirements of Alloiococcus species and related gram-positive cocci. J Clin Microbiol 34, 1027–1028.[Abstract]
Nicholas, K. B., Nicholas, H. B., Jr & Deerfield, D. W., II (1997). GeneDoc, analysis and visualization of genetic variation. EMBNEW News 4, 14.
Page, R. D. (1996). TREEVIEW, an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358.
Rasmussen, S. W. (2002). SEQtools, a software package for analysis of nucleotide and protein sequences. http://www.seqtools.dk
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Schleifer, K. H. (1985). Analysis of the chemical composition and primary structure of murein. Methods Microbiol 18, 123–156.
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.
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