| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Environmental and Molecular Microbiology Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, South Korea
2 College of Bio & Food Technology, Dalian Polytechnic University, Qinggong-yuan No. 1, Ganjingzi-qu, Dalian 116034, P. R. China
Correspondence
Wan-Taek Im
wandra{at}kaist.ac.kr
Feng-Xie Jin
fxjin{at}dlili.edu.cn
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
7c/9t/12t, C19 : 0 cyclo 8c and C18 : 0. The G+C content of the genomic DNA of strain Ko04T was 67.8 mol%. The level of DNA–DNA relatedness with K. adipata Chj404T was 15 %. The results of the genotypic analyses in combination with chemotaxonomic and physiological data demonstrated that strain Ko04T represents a novel species within the genus Kaistia, for which the name Kaistia granuli sp. nov. is proposed. The type strain is Ko04T (=KCTC 12575T=LMG 23410T).
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
, and at present comprises only one species, Kaistia adipata (Im et al., 2004). It was isolated from a sediment sample collected from an industrial stream. K. adipata was characterized as a Gram-negative, strictly aerobic, chemo-organotrophic, non-motile rod to coccus-shaped bacterium. Q-10 was the predominant quinone and C18 : 17c/9t/12t was the major fatty acid when K. adipata was grown on trypticase soy agar (TSA) for 24 h but, when grown on TSA for 48 h, C19 : 0 cyclo 8c was the major fatty acid. According to the 16S rRNA gene sequence, the genus Kaistia belongs to the order Rhizobiales of the class Alphaproteobacteria.
During the course of studies on the culturable aerobic bacterial community in granules used to treat wastewater of brewing factories in Korea, a large number of novel bacterial strains were isolated (Im et al., 2003). Some of these strains have already been classified as representing novel species (An et al., 2006; Aslam et al., 2005; Bae et al., 2005; Kim et al., 2005; La et al., 2005). In this study, we have characterized one of these isolates, strain Ko04T. Phenotypic, chemotaxonomic and phylogenetic analyses established the affiliation of this isolate to the genus Kaistia. The data obtained in this study suggest that the isolate represents a novel species of this genus.
For the isolation of aerobic bacteria, brownish-black granules (about 2 mm in diameter) from an upflow anaerobic sludge blanket reactor used to treat brewery wastewater, which had been operated anaerobically for 2 years, were homogenized by using an Ace Homogenizer (Nihonseiki Co.). The suspension was spread on R2A agar plates (Scharlau) after being serially diluted with 50 mM phosphate buffer (pH 7.0). The plates were incubated at 30 °C for 2 weeks. Single colonies on the plates were purified by being transferred onto new plates that were incubated again under the same conditions. The purified colonies were tentatively identified by partial sequences of the 16S rRNA gene. Strain Ko04T was one of the isolates that appeared on the plates under aerobic conditions. The strain was routinely cultured on R2A agar at 30 °C and maintained as a glycerol suspension (20 %, w/v) at –70 °C.
Cell morphology and motility were observed under a Nikon light microscope (x1000 magnification) using cells grown for 2 days at 30 °C on R2A agar. The Gram reaction was determined by using the non-staining method as described by Buck (1982). Catalase activity was determined by bubble production in 3 % (v/v) H2O2 and oxidase activity was determined using 1 % (w/v) tetramethyl-p-phenylenediamine. Growth at different temperatures (4, 15, 18, 25, 30, 37, 42 and 45 °C) and various pH values (pH 5.0–10.0 at intervals of 0.5 pH units) was assessed after 5 days incubation. Salt tolerance was tested on R2A agar supplemented with 1–10 % (w/v) NaCl after 5 days incubation. Growth on nutrient agar, TSA and MacConkey agar was also evaluated, at 30 °C. Utilization of various substrates as sole carbon sources was investigated, together with some physiological characteristics, using API ID 32 GN, API 20NE and API ZYM galleries according to the instructions of the manufacturer (bioMérieux). Anaerobic growth was tested in serum bottles by addition of thioglycolate (1 g l–1) to R2A broth and replacement of the upper air layer with nitrogen gas. Tests for the degradation of DNA [DNase agar (Scharlau), by flooding plates with 1 M HCl], casein, chitin, starch (Atlas 1993), lipid (Kouker & Jaeger, 1987), xylan and cellulose (Ten et al., 2004) were performed and evaluated after 10 days.
For phylogenetic analysis of strain Ko04T, DNA was extracted using a genomic DNA extraction kit (Solgent) and PCR of the 16S rRNA gene and sequencing of the purified PCR product were carried out according to Kim et al. (2005). Full sequences of the 16S rRNA gene were compiled using SeqMan software (DNASTAR). The 16S rRNA gene sequences of related taxa were obtained from GenBank. Multiple alignments were performed by using the program CLUSTAL_X (Thompson et al., 1997). Gaps were edited in the program BioEdit (Hall, 1999). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). Phylogenetic trees were constructed by using the neighbour-joining method (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) using the program MEGA 3 (Kumar et al., 2004), with bootstrap values based on 1000 replications (Felsenstein, 1985).
The length of the almost-complete 16S rRNA gene sequence of strain Ko04T was 1429 bp (bp 18–1512 with respect to the Escherichia coli numbering system). Sequence similarity calculations after neighbour-joining analysis indicated that the closest relative of strain Ko04T was K. adipata Chj404T (97.5 %). Lower sequence similarities (92.5 %) were found with other recognized species of Rhizobiales. The relationship between strain Ko04T and members of the order Rhizobiales was also evident in the phylogenetic tree (Fig. 1). Strain Ko04T and K. adipata Chj404T formed a monophyletic clade supported by a high bootstrap value (100 %), which was supported by the two types of tree-making methods employed.
|
and enzymically degraded into nucleosides. The DNA G+C content was determined as described by Mesbah et al. (1989) using reversed-phase HPLC. Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), evaporated under vacuum conditions and re-extracted in n-hexane/water (1 : 1, v/v). The crude n-hexane/quinone solution was purified using Sep-Pak Vac cartridges silica (Waters) and was subsequently analysed by HPLC as described by Hiraishi et al. (1996). Cellular fatty acid profiles were determined for strain Ko04T, grown under different conditions. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification system (MIDI). The fatty acids were analysed using a gas chromatograph (Hewlett Packard 6890) and were identified using the Microbial Identification Software package (Sasser, 1990).
The G+C content of the genomic DNA of strain Ko04T was 67.8 mol%. Q-10 was the predominant respiratory ubiquinone. As shown in Table 1, the major fatty acids of strain Ko04T grown on TSA for 24 h were summed feature 7 (C18 : 17c/9t/12t; 38.1 %), C19 : 0 cyclo 8c (35.9 %) and C18 : 0 (15.7 %). The profile of the whole-cell fatty acid composition changed with time. However, the total amount of C19 : 0 cyclo 8c and C18 : 17c/9t/12t was similar. Thus we could infer that C18 : 17c/9t/12t was transformed to C19 : 0 cyclo 8c when cells became older. Comparison of the fatty acid profiles of strain Ko04T showed that they were similar to those of K. adipata Chj404T analysed previously, although there were differences in the proportions of some fatty acids. DNA–DNA hybridization experiments were performed between strain Ko04T and K. adipata Chj404T using the method described by Ezaki et al. (1989) with photobiotin-labelled DNA probes and micro-dilution wells.
|
.
|
) and in this case DNA–DNA hybridizations needed to be performed. A DNA–DNA relatedness of 70 % is the generally accepted limit for species delineation (Wayne et al., 1987). Strain Ko04T exhibited 15 % DNA–DNA relatedness with the type strain of K. adipata. This level is sufficiently low to permit the classification of strain Ko04T as representing a distinct species (Wayne et al., 1987). On the basis of the morphological, physiological and chemotaxonomic characteristics, together with data from the 16S rRNA gene sequence comparisons described above, strain Ko04T should be placed within a novel species, for which we propose the name Kaistia granuli sp. nov.
Description of Kaistia granuli sp. nov.
Kaistia granuli (gra.nu'li. L. gen. n. granuli of a small grain, pertaining to a granule, from which the type strain was isolated).
Cells are Gram-negative, chemo-organotrophic, strictly aerobic, non-spore-forming, rod-shaped and 0.4–0.6 µm in width and 0.9–1.5 µm in length when grown for 2 days at 30 °C on R2A agar. After 2 days incubation on R2A agar, colonies are ivory-pigmented, round and raised with a greasy surface, and 1.0–3.0 mm in diameter. Growth occurs at 18–30 °C, but not at 4 °C or above 37 °C. Growth occurs on TSA, R2A, nutrient agar and MacConkey agar. Grows at pH 5.0–8.0, with optimum growth at pH 6.5–7.0. Tolerates 2 % (w/v) NaCl. Substrate utilization, enzyme activity and other physiological characteristics are given in Table 2
. Q-10 is the predominant ubiquinone, and C18 : 17c/9t/12t isomer, C19 : 0 cyclo 8c and C18 : 0 are the major cellular fatty acids. The DNA G+C content of the type strain is 67.8 mol% (as determined by HPLC).
The type strain, Ko04T (=KCTC 12575T=LMG 23410T), was isolated from granules used in a wastewater-treatment plant in Gongju, South Korea.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Aslam, Z., Im, W.-T., Kim, M.-K. & Lee, S.-T. (2005). Flavobacterium granuli sp. nov., isolated from granules used in a wastewater treatment plant. Int J Syst Evol Microbiol 55, 747–751.
Atlas, R. M. (1993). Handbook of Microbiological Media. Edited by L. C. Parks. Boca Raton, FL: CRC Press.
Bae, H.-S., Im, W.-T. & Lee, S.-T. (2005). Lysobacter concretionis sp. nov., isolated from anaerobic granules in an upflow anaerobic sludge blanket reactor. Int J Syst Evol Microbiol 55, 1155–1161.
Buck, J. D. (1982). Nonstaining (KOH) method for determination of gram reactions of marine bacteria. Appl Environ Microbiol 44, 992–993.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416.[Abstract]
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.
Hiraishi, A., Ueda, Y., Ishihara, J. & Mori, T. (1996). Comparative lipoquinone analysis of influent sewage and activated sludge by high-performance liquid chromatography and photodiode array detection. J Gen Appl Microbiol 42, 457–469.[CrossRef]
Im, W.-T., Kang, M.-S., Park, H.-Y., Kim, M.-K. & Lee, S.-T. (2003). Culturable bacterial strain's diversity of environmental samples. In Proceedings of the International Meeting of the Federation of Korean Microbiological Societies, abstract B4023, pp. 165. Seoul: Federation of Korean Microbiological Societies.
Im, W.-T., Yokota, A., Kim, M.-K. & Lee, S.-T. (2004). Kaistia adipata gen. nov., sp. nov., a novel 
-proteobacterium. J Gen Appl Microbiol 50, 249–254.[CrossRef][Medline]
Kim, M.-K., Im, W.-T., Ohta, H., Lee, M. & Lee, S.-T. (2005). Sphingopyxis granuli sp. nov., a 
-glucosidase producing bacterium in the family Sphingomonadaceae in -4 subclass of the Proteobacteria. J Microbiol 43, 152–157.[Medline]
Kimura, M. (1983). The Neutral Theory of Molecular Evolution. Cambridge: Cambridge University Press.
Kouker, G. & Jaeger, K.-E. (1987). Specific and sensitive plate assay for bacterial lipase. Appl Environ Microbiol 53, 211–213.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5, 150–163.
La, H.-J., Im, W.-T., Ten, L. N., Kang, M.-S., Shin, D.-Y. & Lee, S.-T. (2005). Paracoccus koreensis sp. nov., isolated from anaerobic granules in an upflow anaerobic sludge blanket (UASB) reactor. Int J Syst Evol Microbiol 55, 1657–1660.
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.
Moore, D. D. & Dowhan, D. (1995). Preparation and analysis of DNA. In Current Protocols in Molecular Biology, pp. 2–11. Edited by F. W. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York: Wiley.
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.
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.
Ten, L. N., Im, W.-T., Kim, M.-K., Kang, M.-S. & Lee, S.-T. (2004). Development of a plate technique for screening of polysaccharide-degrading microorganisms by using a mixture of insoluble chromogenic substrates. J Microbiol Methods 56, 375–382.[CrossRef][Medline]
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.
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & 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.
This article has been cited by other articles:
|
|
 |
|
  H.-Y. Weon, C.-M. Lee, S.-B. Hong, B.-Y. Kim, S.-H. Yoo, S.-W. Kwon, and S.-J. Go Kaistia soli sp. nov., isolated from a wetland in Korea Int J Syst Evol Microbiol, July 1, 2008; 58(7): 1522 - 1524. [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 | |
