| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Republic of Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
to accommodate a single genus and single species, Microsphaera multipartita, which had been described by Yoshimi et al. (1996). M. multipartita is an aerobic, Gram-positive and coccoid-shaped actinomycete that belongs to the suborder Frankineae (Yoshimi et al., 1996; Stackebrandt et al., 1997). The chemical markers characterizing the genus Microsphaera include the presence of meso-diaminopimelic acid in the peptidoglycan, MK-8(H4) as the predominant menaquinone, iso-C15 : 0, iso-C16 : 0 and C18 : 1 as the major fatty acids and a DNA G+C content of 67.5 mol% (Yoshimi et al., 1996). The bacterial genus name Microsphaera was recently proven to be illegitimate because of priority of the fungal genus name Microsphaera (Tao et al., 2004). Accordingly, the genus name Microsphaera was replaced by Nakamurella and the family name Microsphaeraceae was replaced by Nakamurellaceae (Tao et al., 2004). In this study, we describe a Gram-positive, spherical, bacterial strain, DS-52T, which was considered to be phylogenetically related to the genus Nakamurella based on 16S rRNA gene sequence comparisons. The aim of the present study was to determine the exact taxonomic position of strain DS-52T by using a polyphasic approach.
Strain DS-52T was isolated from soil samples collected in Dokdo (37° 14' 12'' N 131° 52' 07'' E), Korea, using the standard dilution plating technique at 25 °C on 10x diluted nutrient agar (NA; Difco). Nakamurella multipartita DSM 44233T, which was used as a reference strain, was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. The morphological, physiological and biochemical characteristics of strain DS-52T were investigated using routine cultivation on NA at 25 °C. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. The presence of flagella was determined via transmission electron microscopy using cells from exponentially growing cultures. For transmission electron microscopic observation, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and, following air-drying, the grids were examined with a Philips CM-20 transmission electron microscope. The Gram reaction was determined using the bioMérieux Gram stain kit according to the manufacturer's instructions. Growth at various temperatures (4–40 °C) was measured on NA. To investigate tolerance to NaCl, trypticase soy broth was prepared according to the formula of the Difco medium and NaCl concentrations were varied (0, 0.5 and 1.0–7.0 %, w/v, at intervals of 1.0 %). The pH range for growth was determined in nutrient broth (NB; Difco) that was adjusted to various pH values (pH 4.5–10.5 at intervals of 0.5 pH units) prior to sterilization by adding directly HCl or Na2CO3. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on NA and on NA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Catalase and oxidase activities and hydrolysis of casein, gelatin, hypoxanthine, starch, Tweens 20, 40, 60 and 80, tyrosine, urea and xanthine were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin and nitrate reduction were studied as described by Lanyi (1987). Utilization of substrates as sole carbon and energy sources was tested according to the method of Kämpfer et al. (1991). Susceptibility to antibiotics was tested on NA plates using discs containing the following antibiotics: polymyxin B (100 U), streptomycin (50 µg), penicillin G (20 U), chloramphenicol (100 µg), ampicillin (10 µg), cephalothin (30 µg), gentamicin (30 µg), novobiocin (5 µg), tetracycline (30 µg), kanamycin (30 µg), lincomycin (15 µg), oleandomycin (15 µg), neomycin (30 µg) and carbenicillin (100 µg). Enzyme activities and other physiological properties were tested by using the API ZYM and API 20E systems (bioMérieux).
Cell biomass of strain DS-52T for DNA extraction and for cell-wall, isoprenoid quinone and polar lipid analyses was obtained from cultures grown in NB at 25 °C. Cell mass of N. multipartita DSM 44233T for cell-wall and polar lipid analyses was obtained by cultivation in trypticase soy broth (Difco) at 28 °C. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996), with the exception that RNase T1 was used in combination with RNase A to minimize contamination with RNA. The 16S rRNA gene was amplified by PCR using two universal primers as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with a modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. The isomer type of the diamino acid in the cell-wall peptidoglycan was analysed by using TLC according to the method described by Komagata & Suzuki (1987). Whole-cell sugars were determined as described by Komagata & Suzuki (1987). Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed by using reversed-phase HPLC and a YMC ODS-A (250x4.6 mm) column. Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) and identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987). For fatty acid analysis, cell mass of strain DS-52T was harvested from NA plates after incubation for 10 days at 25 °C, and cell mass of N. multipartita DSM 44233T was harvested from NA plates after incubation for 10 days at 28 °C. The fatty acids were extracted and fatty acid methyl esters were prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990).
Cells of strain DS-52T were Gram-positive, non-spore-forming cocci (0.6–1.2 µm in diameter). Morphological, cultural, physiological and biochemical characteristics of strain DS-52T are given in the genus and species descriptions or are shown in Table 1. The almost complete 16S rRNA gene sequence of strain DS-52T, comprising 1475 nucleotides (approximately 96 % of the Escherichia coli 16S rRNA gene sequence), was determined in this study. In the neighbour-joining tree based on 16S rRNA gene sequences, strain DS-52T joined the phylogenetic lineage of N. multipartita at a bootstrap resampling value of 100 % and this cluster was independent from the clade comprising other members of the suborder Frankineae (Fig. 1). These topologies were also found in the trees generated with the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain DS-52T exhibited 16S rRNA gene sequence similarity values of 96.5 % to N. multipartita JCM 9543T and of 92.0–93.9 % to other members of the suborder Frankineae used in the phylogenetic analysis. Strain DS-52T had meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan. The whole-cell sugars detected in strain DS-52T were galactose, rhamnose, xylose and mannose, whereas N. multipartita DSM 44233T was found to have glucose, rhamnose, xylose and mannose as whole-cell sugars. Strain DS-52T contained similar amounts of MK-8(H4) and MK-9(H4) as the predominant menaquinones. Strain DS-52T had a cellular fatty acid profile that contained large amounts of branched and straight-chain fatty acids; the major components (>10 % of total fatty acids) were anteiso-C15 : 0, iso-C15 : 0 and C17 : 0 (Table 2). Major polar lipids detected in strain DS-52T and N. multipartita DSM 44233T were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidyldimethylethanolamine. The DNA G+C content of strain DS-52T was 72.6 mol%.
|
|
|
). Cell morphology, peptidoglycan diamino acid type and major polar lipids of strain DS-52T were the same as those of N. multipartita (Yoshimi et al., 1996; Table 1). However, strain DS-52T showed distinct differences from N. multipartita in content of the predominant menaquinones and fatty acid profile. N. multipartita contains MK-8(H4), at a peak area ratio of 97 %, as the predominant menaquinone (Yoshimi et al., 1996); a similar result was also obtained in this study. Strain DS-52T showed notable differences from N. multipartita in the proportions of anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 0 and C17 : 0 and in the absence of unsaturated and 10-methyl fatty acids (Table 2). There was also a slight difference in the whole-cell sugars between strain DS-52T and N. multipartita (Table 1). Based on the data presented here, it is proposed that strain DS-52T represents a novel species in a new genus, Humicoccus flavidus gen. nov., sp. nov.
Description of Humicoccus gen. nov.
Humicoccus (Hu.mi.coc'cus. L. n. humus the soil; Gr. n. kokkos a grain or berry; N.L. masc. n. Humicoccus coccus isolated from soil).
Cells are Gram-positive, non-spore-forming cocci. Strictly aerobic. The cell-wall peptidoglycan contains meso-diaminopimelic acid. The predominant menaquinones are MK-8(H4) and MK-9(H4). The cellular fatty acid profile consists of straight-chain and branched fatty acids. The type species is Humicoccus flavidus.
Description of Humicoccus flavidus sp. nov.
Humicoccus flavidus (fla'vi.dus. L. masc. adj. flavidus pale yellow).
Has the following properties in addition to those given for the genus. Cells are 0.6–1.2 µm in diameter. Colonies are circular, convex, smooth, glistening, light-yellow in colour and 1.0–1.8 mm in diameter after incubation for 10 days at 25 °C on NA. Growth occurs at 4 and 32 °C, but not at 33 °C. The optimal pH for growth is 6.0–7.0; growth occurs at pH 5.0 and 8.5, but not at pH 4.5 or 9.0. Growth occurs in the absence of NaCl; growth does not occur in the presence of greater than 5 % (w/v) NaCl. Growth does not occur under anaerobic conditions on NA or on NA supplemented with nitrate. Gelatin is hydrolysed, but Tweens 20, 40, 60 and 80 are not. H2S and indole are not produced. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. In assays with the API ZYM system, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, 
-glucosidase and 
-glucosidase are present, but alkaline phosphatase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. D-Cellobiose, D-trehalose, L-arabinose and salicin are utilized as sole carbon and energy sources, but benzoate, citrate and formate are not. The whole-cell sugars are galactose, mannose, xylose and rhamnose. The major polar lipids are diphosphatidylglycerol, phosphatidylethanolamine and phosphatidyldimethylethanolamine. The major fatty acids (>10 % of total fatty acids) are anteiso-C15 : 0, iso-C15 : 0 and C17 : 0. The DNA G+C content is 72.6 mol% (HPLC). Other phenotypic properties are given in Table 1.
The type strain, DS-52T (=KCTC 19127T=CIP 108919T), was isolated from soil.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Kämpfer, P., Steiof, M. & Dott, W. (1991). Microbiological characterization of a fuel-oil contaminated site including numerical identification of heterotrophic water and soil bacteria. Microb Ecol 21, 227–251.
Komagata, K. & Suzuki, K.-I. (1987). Lipid and cell wall analysis in bacterial systematics. Methods Microbiol 19, 161–207.
Lanyi, B. (1987). Classical and rapid identification methods for medically important bacteria. Methods Microbiol 19, 1–67.
Minnikin, D. E., O'Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.[CrossRef]
Sasser, M. (1990). Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids. Newark, DE: MIDI.
Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. (1997). Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol 47, 479–491.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reverse-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Tao, T.-S., Yue, Y.-Y., Chen, W.-X. & Chen, W.-F. (2004). Proposal of Nakamurella gen. nov. as a substitute for the bacterial genus Microsphaera Yoshimi et al. 1996 and Nakamurellaceae fam. nov. as a substitute for the illegitimate bacterial family Microsphaeraceae Rainey et al. 1997. Int J Syst Evol Microbiol 54, 999–1000.
Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Goodfellow, M. & Park, Y.-H. (1996). Identification of Saccharomonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505.
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rRNA gene sequences. Int J Syst Bacteriol 48, 187–194.
Yoon, J.-H., Kim, I.-G., Shin, D.-Y., Kang, K. H. & Park, Y.-H. (2003). Microbulbifer salipaludis sp. nov., a moderate halophile isolated from a Korean salt marsh. Int J Syst Evol Microbiol 53, 53–57.
Yoshimi, Y., Hiraishi, A. & Nakamura, K. (1996). Isolation and characterization of Microsphaera multipartita gen. nov., sp. nov., a polysaccharide-accumulating Gram-positive bacterium from activated sludge. Int J Syst Bacteriol 46, 519–525.
This article has been cited by other articles:
|
|
 |
|
  M. R. Carlsohn, I. Groth, H.-P. Saluz, P. Schumann, and E. Stackebrandt Fodinicola feengrottensis gen. nov., sp. nov., an actinomycete isolated from a medieval mine Int J Syst Evol Microbiol, July 1, 2008; 58(7): 1529 - 1536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. D. Lee, S. K. Park, Y.-W. Yun, and D. W. Lee Saxeibacter lacteus gen. nov., sp. nov., an actinobacterium isolated from rock Int J Syst Evol Microbiol, April 1, 2008; 58(4): 906 - 909. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
