| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 College of Life Sciences, Capital Normal University, Beijing 100037, People's Republic of China
2 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China
3 College of Life Sciences, Heibei University, Baoding 071002, People's Republic of China
Correspondence
Wei Song
songwei{at}mail.cnu.edu.cn
Zhiheng Liu
zhliu{at}sun.im.ac.cn
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
-cyclohexylundecanoic and anteiso- and iso-branched saturated fatty acids, MK-11 as the major menaquinone and DL-2,4-diaminobutyric acid in its cell-wall peptidoglycan. The DNA G+C content of strain wged11T was 66.4 mol%. Levels of similarity between the 16S rRNA gene sequence of strain wged11T and those of the type strains of other members of the genus Leifsonia ranged from 94.7 to 97.6 %. The mean level of DNA–DNA relatedness between strain wged11T and Leifsonia poae DSM 15202T, its nearest phylogenetic neighbour, was 35.3 %. Based on these findings, strain wged11T (=CGMCC 4.3491T=JCM 13908T) is proposed as the type strain of a novel species of the genus Leifsonia, Leifsonia ginsengi sp. nov.
A table comparing carbon source utilization between strain wged11T and recognized species of the genus Leifsonia is available as supplementary material in IJSEM Online.
These authors contributed equally to this work. 
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
and at the time of writing comprises eight recognized species: Leifsonia poae (Evtushenko et al., 2000), Leifsonia aquatica (Leifson, 1962), Leifsonia xyli (Davis et al., 1984), Leifsonia naganoensis, Leifsonia shinshuensis and Leifsonia cynodontis (Suzuki et al., 1999), and Leifsonia rubra and Leifsonia aurea (Reddy et al., 2003). Members of the genus Leifsonia are characterized by coryneform cell morphology, peptidoglycans based upon 2,4-diaminobutyric acid, MK-11 as the major menaquinone, phosphatidylglycerol and diphosphatidylglycerol as principal phospholipids, a high content of anteiso- and iso-branched saturated fatty acids, and a DNA G+C content in the range 66–73 mol% (Evtushenko et al., 2000). During an investigation on the diversity of endophytic bacteria of ginseng, a novel strain, designated wged11T, was isolated. Genotypic and phenotypic data indicated that this strain represents a novel species of the genus Leifsonia.
Strain wged11T was isolated on plates of nitrogen-free medium (Von Bulow & Döbereiner, 1975) that had been seeded with a tissue suspension of ginseng roots and incubated at 28 °C for 7 days. Healthy 3-year-old ginseng roots were collected from Fusong county, Jilin Province, China. The root tissue suspension was prepared according to the following procedure. Roots were separated from the soil and washed with tap water, surface-sterilized with 75 % ethanol for 3 min and 2.6 % sodium hypochlorite solution for 3–5 min, and then rinsed with sterile double distilled water (ddH2O). The surface-sterilized root mass was pulverized in a ceramic mortar and diluted with sterile ddH2O using the standard dilution plating technique. The new isolate and reference strain L. poae DSM 15202T were maintained on LB medium or corynebacterial (CB) agar (Zgurskaya et al., 1993) slants at 4 °C and as a glycerol suspension (20 %, v/v) at –20 °C. Biomass for chemotaxonomic and molecular systematic studies was prepared by growing the strains in shake flasks of CB medium (l–1: 10 g casein peptone, 5 g yeast extract, 5 g glucose and 5 g NaCl; pH 7.0) at 28 °C for 3–5 days. Cells were harvested by centrifugation, washed with ddH2O and freeze-dried before use in chemical studies.
Colony properties of the isolates were observed on CB agar. Cell morphology was examined by light microscopy and transmission electron microscopy (TEM). Presence of flagella was observed by TEM. Cell motility was examined by phase-contrast microscopy of cells from exponentially growing cultures. Acid production from carbohydrates was examined by using the modified method of Hugh & Leifson (1953), as described by Gledhill & Casida (1969). Utilization of a variety of substrates as sole carbon source was tested using a GP2 Microplate (Biolog). The ability of strain wged11T to grow in the presence of nine antibiotics was examined by placing 6-mm antibiotic discs centrally in plates of CB agar seeded with 200 µl of 3-day-old CB medium culture, and inhibition zones were observed after 3–5 days incubation at 28 °C.
Cell walls were prepared as described by Streshinskaya et al. (1979). Analysis of peptidoglycan amino acids was carried out according to the methods given by Schleifer & Kandler (1972) and Bousfield et al. (1985). Cell-wall sugars were identified according to the methods described by Maltsev et al. (1992). Cellular menaquinones were extracted and purified as described by Collins (1985) and were analysed by HPLC (Wu et al., 1989). Non-hydroxylated fatty acids were extracted, purified, methylated, identified and quantified by GC with the standard Microbial Identification System (MIDI; Sasser, 1990; Kämpfer & Kroppenstedt, 1996). The results were validated by GC-MS given that monoenoic fatty acid was found as a major component in the profile.
A loop of biomass was scraped off the agar plate, suspended in 20 µl ddH2O and lysed by boiling for 10 min and freezing for 5 min. Following centrifugation, the supernatant was used as the template for PCR with the universal primers 27F and 1492R (Lane, 1991). Automated sequencing was performed by using an ABI Big Dye Primer cycle sequencing ready reaction kit and Applied Biosystems 3730 DNA sequencer.
The program CLUSTAL_X v1.8 (Thompson et al., 1997) was used for multiple alignment of 16S rRNA gene sequences of type strains of members of the genus Leifsonia together with sequences of related species retrieved from GenBank. Three tree-making algorithms, neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Eck & Dayhoff, 1966; Fitch, 1971) and maximum-likelihood (Felsenstein & Churchill, 1996), from the PHYLIP package v3.6 were used to infer the phylogenetic evolutionary trees. The resulting unrooted tree topologies were evaluated by bootstrap analyses (Felsenstein, 1985) of the neighbour-joining method based on 1000 resamplings.
Preparation of genomic DNA was carried out according to the method of Marmur (1961). The G+C content of the DNA of strain wged11T was determined using the thermal denaturation method (Marmur & Doty, 1962) with Escherichia coli K-12 as a control. Levels of DNA–DNA relatedness between the new isolate and the reference strains were determined by using the reassociation rate method (Dong et al., 2000).
An almost complete 16S rRNA gene sequence (1485 nt) for strain wged11T was determined. Although a comparative 16S rRNA gene sequence analysis (BLAST search; http://www.ncbi.nlm.nih.gov/blast) revealed strain wged11T to be related most closely to Agreia pratensis DSM 14226T (98 %, 1267/1293 nt), strain wged11T fell within the radiation of the cluster of the genus Leifsonia in the phylogenetic tree generated with the PHYLIP package (Fig. 1). It formed a separate phyletic line and showed highest 16S rRNA gene sequence similarity to L. poae DSM 15202T (97.6 %). The 16S rRNA gene sequence similarity between strain wged11T and A. pratensis was 96.8 %, and that between strain wged11T and the type strains of other Leifsonia species, except L. aurea (94.7 %), was between 96 and 96.5 %. According to the criterion of Ludwig et al. (1998) used for genus definition (95 % 16S rRNA gene sequence similarity), strain wged11T can be classified as representing a member of the genus Leifsonia. Its phylogenetic distinctiveness suggested that it represents a taxon separate from recognized Leifsonia species.
|
and Supplementary Table S1 (available in IJSEM Online). Amino acid composition (alanine, 2,4-diaminobutyric acid, glutamic acid and glycine) of the cell-wall peptidoglycan, cell-wall sugars (mannose, galactose, glucose, rhamnose) and menaquinone profile (MK-11 as the major component, with a minor amount of MK-10 and a trace amount of MK-12) are in agreement with the description of the genus Leifsonia. The major cellular fatty acid profile of strain wged11T [-cyclohexylundecanoic acid (52.0 %), anteiso-C15 : 0 (19.6 %), anteiso-C17 : 0 (10.5 %) and iso-C16 : 0 (8.3 %); data from GC-MS], except the unusual -cyclohexylundecanoic acid (identified as C18 : 17cis in the MIDI Sherlock system), is consistent with the type strains of recognized members of the genus Leifsonia. -Cyclohexylundecanoic acid was also found as a major component of the cellular fatty acids in Curtobacterium pusillum (Kawaguchi et al., 1986); however, strain wged11T formed a robust clade with recognized members of the genus Leifsonia but not with recognized members of the genus Curtobacterium (Fig. 1
). At the same time, the mean level of DNA–DNA relatedness (35.3 %) is low enough to distinguish strain wged11T from the type strain of L. poae, its nearest phylogenetic neighbour. In addition, strain wged11T showed differences in several phenotypic characteristics from recognized species of the genus Leifsonia (see Table 1 and Supplementary Table S1 in IJSEM Online). Therefore, on the basis of the data presented, strain wged11T is considered to represent a novel species of the genus Leifsonia, for which the name Leifsonia ginsengi sp. nov. is proposed.
|
Colonies on CB agar are white to yellow, circular, convex, glistening, opaque, butyrous and 1–2 mm in diameter. Cells are Gram-positive, non-spore-forming, aerobic, straight or curved rods (length 1.2–2.5 µm, width 0.6–0.8 µm). Flagella are not found by TEM. Cell motility is not detected by phase-contrast microscopy. Cell growth occurs between 4 and 37 °C; optimum growth is at 26–30 °C. Cell growth is observed at 1 % but not at 3 % NaCl. Positive for catalase, hydrolysis of starch and indole production. Production of H2S is weak. Negative for oxidase, urease, gelatinase and the Voges–Proskauer reaction. Cells are susceptible to tetracycline (10 µg ml–1), erythromycin (15 µg ml–1), rifampicin (30 µg ml–1) and chloramphenicol (10 µg ml–1), but resistant to ampicillin (10 µg ml–1), lincomycin (15 µg ml–1), penicillin (10 U ml–1), streptomycin (10 µg ml–1) and kanamycin (15 µg ml–1). Acid is produced from fructose, mannose, glucose, arabinose, sucrose and maltose, but not from ribose. Dextrin (weak), Tween 40 (weak), D-cellobiose, 
-D-glucose, maltose, D-mannitol, D-ribose, D-sorbitol, sucrose, turanose, D-xylose, acetic acid (weak), 
-hydroxybutyric acid, 2'-deoxyadenosine, pyruvic acid methyl ester, 2,3-butanediol (weak), D-mannose, pyruvic acid, glycerol, D-fructose, adenosine and thymidine are used as sole carbon sources for energy and growth. Cell-wall peptidoglycan contains alanine, 2,4-diaminobutyric acid, glutamic acid and glycine. Cell-wall sugars are galactose, glucose, mannose and rhamnose. The major menaquinone is MK-11, with a minor amount of MK-10 and a trace amount of MK-12. The fatty acid profile comprises mainly -cyclohexylundecanoic acid (52.0 %), anteiso-C15 : 0 (19.6 %), anteiso-C17 : 0 (10.5 %) and iso-C16 : 0 (8.3 %).
The DNA G+C content of the type strain is 66.4 mol%. The type strain, wged11T (=CGMCC 4.3491T=JCM 13908T), was isolated from a tissue suspension of a surface-sterilized ginseng root.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Collins, M. D. (1985). Isoprenoid quinine analysis in classification and identification. In Chemical Methods in Bacterial Systematics, pp. 267–287. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press.
Davis, M. J., Gillaspie, A. G., Vidaver, A. K. & Harris, R. W. (1984). Clavibacter: a new genus containing some phytopathogenic coryneform bacteria, including Clavibacter xyli subsp. xyli sp. nov., subsp. nov. and Clavibacter xyli subsp. cynodontis subsp. nov., pathogens that cause ratoon stunting disease of sugarcane and bermudagrass stunting disease. Int J Syst Bacteriol 34, 107–117.
Dong, X., Xin, Y., Jian, W., Liu, X. & Ling, D. (2000). Bifidobacterium thermacidophilum sp. nov., isolated from an anerobic digester. Int J Syst Evol Microbiol 50, 119–125.[Abstract]
Eck, R. V. & Dayhoff, M. O. (1966). Atlas of Protein Sequence and Structure. Silver Springs, MD: National Biomedical Research Foundation.
Evtushenko, L. I., Dorofeeva, L. V., Subbotin, S. A., Cole, J. R. & Tiedje, J. M. (2000). Leifsonia poae gen. nov., sp. nov., isolated from nematode galls on Poa annua, and reclassification of ‘Corynebacterium aquaticum’ Leifson 1962 as Leifsonia aquatica (ex Leifson 1962) gen. nov., nom. rev., comb. nov. and Clavibacter xyli Davis et al. 1984 with two subspecies as Leifsonia xyli (Davis et al. 1984) gen. nov., comb. nov. Int J Syst Evol Microbiol 50, 371–380.[Abstract]
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Felsenstein, J. & Churchill, G. A. (1996). A Hidden Markov Model approach to variation among sites in rate of evolution. Mol Biol Evol 13, 93–104.[Abstract]
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416.[CrossRef]
Gledhill, W. E. & Casida, L. E., Jr (1969). Predominant catalase-negative soil bacteria. III. Agromyces, gen. n., microorganisms intermediary to Actinomyces and Nocardia. Appl Environ Microbiol 18, 340–349.
Hugh, R. & Leifson, E. (1953). The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria. J Bacteriol 66, 24–26.
Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005.
Kawaguchi, A., Uemura, N. & Okuda, S. (1986). Characterization of the fatty acid synthetase system of Curtobacterium pusillum. J Biochem (Tokyo) 99, 1735–1742.
Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, pp. 115–175. Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley.
Leifson, E. (1962). The bacterial flora of distilled and stored water. III. New species of the genera Corynebacterium, Flavobacterium, Spirillum and Pseudomonas. Int Bull Bacteriol Nomencl Taxon 12, 161–170.
Ludwig, W., Strunk, O., Klugbauer, S., Klugbauer, N., Weizenegger, M., Neumaier, J., Bachleitner, M. & Schleifer, K. H. (1998). Bacterial phylogeny based on comparative sequence analysis. Electrophoresis 19, 554–568.[CrossRef][Medline]
Maltsev, I. I., Kalinovskii, A. I., Zgurskaya, H. I. & Evtushenko, L. I. (1992). Tyvelose in Agromyces cell walls. Syst Appl Microbiol 15, 187–189.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118.[Medline]
Reddy, G. S. N., Prakash, J. S. S., Srinivas, R., Matsumoto, G. I. & Shivaji, S. (2003). Leifsonia rubra sp. nov. and Leifsonia aurea sp. nov., psychrophiles from a pond in Antarctica. Int J Syst Evol Microbiol 53, 977–984.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.
Streshinskaya, G. M., Naumova, I. B. & Panina, L. I. (1979). Cell wall composition of Streptomyces chrysomallus producing the antibiotic aurantin. Mikrobiologiia 48, 814–819.[Medline]
Suzuki, K. I., Suzuki, M., Sasaki, J., Park, Y. H. & Komagata, K. (1999). Leifsonia gen. nov., a genus for 2,4-diaminobutyric acid-containing actinomycetes to accommodate "Corynebacterium aquaticum" Leifson 1962 and Clavibacter xyli susp. cynodontis Davis et al. 1984. J Gen Appl Microbiol 45, 253–262.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Von Bulow, J. F. W. & Döbereiner, J. (1975). Potential for nitrogen fixation in maize genotypes in Brazil. Proc Natl Acad Sci U S A 72, 2389–2393.
Wu, C., Lu, X., Qin, M., Wang, Y. & Ruan, J. (1989). Analysis of menaquinone compound in microbial cells by HPLC. Microbiology [English translation of Microbiology (Beijing)] 16, 176–178.
Zgurskaya, H. I., Evtushenko, L. I., Akimov, V. N. & Kalakoutskii, L. V. (1993). Rathayibacter gen. nov., including the species Rathayibacter rathayi comb. nov., Rathayibacter tritici comb. nov., Rathayibacter iranicus comb. nov., and six strains from annual grasses. Int J Syst Bacteriol 43, 143–149.
This article has been cited by other articles:
|
|
 |
|
  P. Monciardini, L. Cavaletti, A. Ranghetti, P. Schumann, M. Rohde, R. Bamonte, M. Sosio, A. Mezzelani, and S. Donadio Novel members of the family Micromonosporaceae, Rugosimonospora acidiphila gen. nov., sp. nov. and Rugosimonospora africana sp. nov. Int J Syst Evol Microbiol, November 1, 2009; 59(11): 2752 - 2758. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Schumann, P. Kampfer, H.-J. Busse, L. I. Evtushenko, and for the Subcommittee on the Taxonomy of the Subord Proposed minimal standards for describing new genera and species of the suborder Micrococcineae Int J Syst Evol Microbiol, July 1, 2009; 59(7): 1823 - 1849. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. K. Pindi, K. H. Kishore, G. S. N. Reddy, and S. Shivaji Description of Leifsonia kafniensis sp. nov. and Leifsonia antarctica sp. nov. Int J Syst Evol Microbiol, June 1, 2009; 59(6): 1348 - 1352. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Qiu, X. Zhang, L. Liu, L. Sun, P. Schumann, and W. Song Bacillus beijingensis sp. nov. and Bacillus ginsengi sp. nov., isolated from ginseng root Int J Syst Evol Microbiol, April 1, 2009; 59(4): 729 - 734. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. G. Dastager, J.-C. Lee, Y.-J. Ju, D.-J. Park, and C.-J. Kim Leifsonia kribbensis sp. nov., isolated from soil Int J Syst Evol Microbiol, January 1, 2009; 59(1): 18 - 21. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. S. N. Reddy, S. R. Prabagaran, and S. Shivaji Leifsonia pindariensis sp. nov., isolated from the Pindari glacier of the Indian Himalayas, and emended description of the genus Leifsonia Int J Syst Evol Microbiol, September 1, 2008; 58(9): 2229 - 2234. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. G. Dastager, J.-C. Lee, Y.-J. Ju, D.-J. Park, and C.-J. Kim Leifsonia bigeumensis sp. nov., isolated from soil on Bigeum Island, Korea Int J Syst Evol Microbiol, August 1, 2008; 58(8): 1935 - 1938. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Vaz-Moreira, M. F. Nobre, A. C. S. Ferreira, P. Schumann, O. C. Nunes, and C. M. Manaia Humibacter albus gen. nov., sp. nov., isolated from sewage sludge compost Int J Syst Evol Microbiol, April 1, 2008; 58(4): 1014 - 1018. [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 | |
