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1 Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Sector 39-A, Chandigarh, 160 036, India
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
3 Department of Microbiology, Guru Nanak Dev University (GNDU), Amritsar, 143 005, India
Correspondence
S. Mayilraj
mayil{at}imtech.res.in
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ABSTRACT |
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The fatty acid compositions of strain K22-21T and the type strains of the three known Agrococcus species are available as supplementary material in IJSEM Online.
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; Rainey et al., 1994; Stackebrandt et al., 1997; Suzuki et al., 1997). At the time of writing, the genus Agrococcus comprises three species, Agrococcus jenensis (Groth et al., 1996), Agrococcus citreus (Wieser et al., 1999) and Agrococcus baldri (Zlamala et al., 2002). Strain K22-21T was isolated from a soil sample collected from the Lahaul-Spiti Valley in the Indian Himalayas by the dilution-plating technique on tryptic soy agar medium (TSA; HiMedia) and maintained as glycerol stocks at –70°C. The reference strains Agrococcus jenensis MTCC 6413T, Agrococcus citreus MTCC 6410T and Agrococcus baldri MTCC 6457T were obtained from the Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India.
Colony and cell morphology were studied according to standard methods (Murray et al., 1994). Gram reaction was determined using a Gram staining kit (HiMedia) according to the manufacturer's instructions. Growth at different temperatures, pH and NaCl concentrations was examined by growing the strain on basal TSA medium. Catalase production, oxidase activity (with N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride) and urea hydrolysis were determined as described by Cowan & Steel (1965). The following characters were analysed as described by Smibert & Krieg (1994): decomposition of casein, gelatin, Tween 20 and starch; indole and hydrogen sulfide production; methyl red and Voges–Proskauer tests; acid-fast and endospore staining; and motility. Nitrate reduction was assayed as described by Lanyi (1987). Utilization of various carbon sources was tested using Biolog GP2 MicroPlates; plates were used according to the manufacturer's instructions with the modification that TSA medium was used instead of Biolog Universal Growth agar medium. The inoculated plates were incubated for 24 h and results were read with a MicroPlate Reader using Microlog 4.2 software to perform automated reading. Acid production from various sugars was tested as described by Smith et al. (1952). Standard procedures were used to determine the cell-wall sugars (Staneck & Roberts, 1974). Preparation of cell walls and determination of peptidoglycan structure were done as described by Schleifer & Kandler (1972), with the modification that TLC on cellulose sheets (Merck) was used instead of paper chromatography. Polar lipids and menaquinones were extracted and analysed according to Minnikin et al. (1984) and Kroppenstedt (1982). The absence of mycolic acids was demonstrated by TLC (Minnikin & Goodfellow, 1976). The glycolic acid content of the bacterial cell wall was determined as described by Uchida & Aida (1984). For cellular fatty acid analysis, the strain was grown on TSA medium at 30 °C for 36 h and fatty acid methyl ester analysis was performed using the Sherlock Microbial Identification System (MIDI) as described previously (Pandey et al., 2002). Freeze-dried cells for other chemotaxonomic analyses were prepared following growth of the strains in tryptic soy broth (HiMedia) for 4 days at 30 °C. Genomic DNA extraction and amplification, 16S rRNA gene sequencing and phylogenetic analysis were performed as described previously (Mayilraj et al., 2005). DNA–DNA hybridization was performed by the membrane filter method (Tourova & Antonov, 1987). The G+C content of the genomic DNA was determined as described by Saha et al. (2005).
The organism had phenotypic properties, including chemical properties, that were consistent with its classification in the genus Agrococcus. The detailed differential physiological and biochemical properties are listed in Table 1 and further characteristics are given in the species description. The fatty acid compositions of strain K22-21T and the type strains of the three Agrococcus species are detailed in Supplementary Table S1 in IJSEM Online. To elucidate the phylogenetic position of strain K22-21T within the genus Agrococcus, an almost complete sequence of the 16S rRNA gene was determined (1454 bases). The 16S rRNA gene sequence of strain K22-21T generated in this work was aligned with those of the other Agrococcus species retrieved from GenBank. A phylogenetic tree was constructed using the 16S rRNA gene sequences of the three Agrococcus type strains and strain K22-21T; Agromyces ramosus DSM 43045T was used as an outgroup. Strain K22-21T formed a separate lineage within the genus Agrococcus (Fig. 1). 16S rRNA gene sequence similarities between strain K22-21T and Agrococcus jenensis DSM 9580T, Agrococcus baldri DSM 14215T and Agrococcus citreus DSM 12453T were 98.5, 96.8 and 96.6 %, respectively. However, the DNA–DNA relatedness value between strain K22-21T and Agrococcus jenensis was 55.1 %, which is well below the 70 % cut-off point recommended for the delineation of bacterial species (Wayne et al., 1987). DNA–DNA relatedness values between strain K22-21T and the two other Agrococcus type strains were not determined, as it has been shown that organisms with more than 3 % 16S rRNA gene sequence dissimilarity belong to different genomic species (Stackebrandt & Goebel, 1994). On the basis of the polyphasic data presented above, strain K22-21T should be placed in the genus Agrococcus as a representative of a novel species; the name Agrococcus lahaulensis sp. nov. is proposed.
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Gram-positive, non-acid-fast, aerobic bacterium. Forms lemon-coloured, circular, glistening, opaque colonies with an entire margin on TSA medium. Colony sizes are 0.7–3.0 mm. Cells are irregularly spherical, occurring in pairs and clusters. Cells vary in size (0.6–1.0 µm wide by 1.0–1.5 µm long). Does not form endospores. Non-motile. No mycelial growth phase is observed. Catalase-positive and oxidase-negative. Tolerates up to 7.0 % NaCl and grows at temperatures between 25 and 37 °C, with optimum growth at 30 °C. Growth occurs at between pH 6 and 10; optimum growth is at pH 8.0. Positive for decomposition of casein, starch and gelatin. Negative for indole, urease and hydrogen sulfide production, methyl red and Voges–Proskauer reactions, utilization of citrate, decomposition of aesculin and tyrosine and nitrate reduction. Acid is produced from L-rhamnose, D-fructose and trehalose. Acid is produced weakly from L-arabinose, but not from D-cellobiose, myo-inositol, D-mannitol, D-maltose, D-galactose, glycerol, salicin, sucrose or D-xylose. Utilization of various compounds as sole carbon sources is detailed in Table 1
. The diagnostic cell wall diamino acid is diaminobutyric acid. The peptidoglycan contains Ala, Gly, Asp, Thr and diaminobutyric acid. The cell-wall sugars are glucose, rhamnose, ribose, mannose and galactose. The predominant lipids are diphosphatidylglycerol, phosphatidylglycerol, two unknown glycolipids and some non-characterized lipids. No mycolic acids are present. Contains major amounts of anteiso-branched C15 : 0 (48.44 %) and C17 : 0 (27.61 %) fatty acids and iso-branched C15 : 0 (9.94 %) and C16 : 0 (5.80 %) fatty acids. The major menaquinones are MK-10, MK-11 and MK-12. The acyl type is acetyl.
The type strain is K22-21T (=MTCC 7154T=DSM 17612T), isolated from soil, 0.45 m below an ice glacier, 4200 m above sea level, in Lahaul Valley, Himachal Pradesh, India. The G+C content of strain K22-21T is 74.1 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. (1996). Agrococcus jenensis gen. nov., sp. nov., a new genus of actinomycetes with diaminobutyric acid in the cell wall. Int J Syst Bacteriol 46, 234–239.
Kroppenstedt, R. M. (1982). Separation of bacterial menaquinones by HPLC using reverse phase (RP 18) and a silver loaded ion exchanger as stationery phases. J Liq Chromatogr 5, 2359–2367.[CrossRef]
Lanyi, B. (1987). Classical and rapid identification methods for medically important bacteria. Methods Microbiol 19, 1–67.
Mayilraj, S., Prasad, G. S., Suresh, K., Saini, H. S., Shivaji, S. & Chakrabarti, T. (2005). Planococcus stackebrandtii sp. nov., isolated from a cold desert of the Himalayas, India. Int J Syst Evol Microbiol 55, 91–94.
Minnikin, D. E. & Goodfellow, M. (1976). Lipid composition in the classification and identification of Nocardia and related taxa. In The Biology of the Nocardiaceae, pp. 160–219. Edited by M. Goodfellow, G. H. Brownell & J. A. Serrano. London: Academic Press.
Minnikin, D. E., O'Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, K. & Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.[CrossRef]
Murray, R. G. E., Raymond, N., Doetsch, R. N. & Robinow, C. F. (1994). Determinative and cytological light microscopy. In Methods for General and Molecular Bacteriology, pp. 21–41. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology
Pandey, K. K., Mayilraj, S. & Chakrabarti, T. (2002). Pseudomonas indica sp. nov., a novel butane-utilizing species. Int J Syst Evol Microbiol 52, 1559–1567.[Abstract]
Park, Y. H., Suzuki, K., Yim, D. G. & 7 other authors (1993). Suprageneric classification of peptidoglycan group B actinomycetes by nucleotide sequencing of 5S ribosomal RNA. Antonie van Leeuwenhoek 64, 307–313.[CrossRef][Medline]
Rainey, F., Weiss, N., Prauser, H. & Stackebrandt, E. (1994). Further evidence for phylogenetic coherence of actinomycetes with group B-peptidoglycan and evidence for the phylogenetic intermixing of the genera Microbacterium and Aureobacterium as determined by 16S rDNA analysis. FEMS Microbiol Lett 118, 135–139.[CrossRef]
Saha, P., Krishnamurthi, S., Mayilraj, S., Prasad, G. S., Bora, T. C. & Chakrabarti, T. (2005). Aquimonas voraii gen. nov., sp. nov., a novel gammaproteobacterium isolated from a warm spring of Assam, India. Int J Syst Evol Microbiol 55, 1491–1495.
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
Smith, N. R., Gordon, R. E. & Clark, F. E. (1952). Aerobic spore-forming bacteria. Agricultural Monograph no. 16. Washington, DC: US Department of Agriculture.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. (1997). Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol 47, 479–491.
Staneck, J. L. & Roberts, G. D. (1974). Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography. Appl Microbiol 28, 226–231.[Medline]
Suzuki, K., Sasaki, J., Uramoto, M., Nakase, T. & Komagata, K. (1997). Cryobacterium psychrophilum gen. nov., sp. nov., nom. rev., comb. nov., an obligately psychrophilic actinomycete to accommodate ‘Curtobacterium psychrophilum’ Inoue and Komagata 1976. Int J Syst Bacteriol 47, 474–478.
Tourova, T. P. & Antonov, A. S. (1987). Identification of microorganisms by rapid DNA-DNA hybridization. Methods Microbiol 19, 333–355.
Uchida, K. & Aida, K. (1984). An improved method for the glycolate test for simple identification of the acyl type of bacterial cell walls. J Gen Appl Microbiol 30, 131–134.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Wieser, M., Schumann, P., Martin, K., Altenburger, P., Burghardt, J., Lubitz, W. & Busse, H.-J. (1999). Agrococcus citreus sp. nov., isolated from a medieval wall painting of the chapel of Castle Herberstein (Austria). Int J Syst Bacteriol 49, 1165–1170.
Zlamala, C., Schumann, P., Kämpfer, P., Rosselló-Mora, R., Lubitz, W. & Busse, H.-J. (2002). Agrococcus baldri sp. nov., isolated from the air in the ‘Virgilkapelle’ in Vienna. Int J Syst Evol Microbiol 52, 1211–1216.[Abstract]
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