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1 Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan
2 Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan
3 Laboratory of Electron Microscopy, School of Dentistry, Hokkaido University, Kita-ku, Sapporo 060-8586, Japan
Correspondence
Isao Yumoto
i.yumoto{at}aist.go.jp
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, Orn–D-Glu. A phylogenetic analysis based on 16S rRNA gene sequence data indicated that strain IDR2-2T is a member of the genus Alkalibacterium. DNA–DNA hybridization revealed low relatedness (less than 25 %) between the isolate and two phylogenetically related strains, Alkalibacterium olivapovliticus and Marinilactibacillus psychrotolerans. On the basis of phenotypic characteristics, phylogenetic data and DNA–DNA relatedness data, the isolate merits classification as a novel species, for which the name Alkalibacterium psychrotolerans sp. nov. is proposed. The type strain is IDR2-2T (=JCM 12281T=NCIMB 13981T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain IDR2-2T is AB125938.
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; Krulwich et al., 2001; Yumoto, 2002, 2003). Although considered to be extremophiles, alkaliphilic micro-organisms are distributed not only in unique places such as alkaline soda lakes (Duckworth et al., 1996) but also in ordinary soils or faeces (Horikoshi, 1991). In this respect, alkaliphilic micro-organisms are different from other extremophiles. This might due to the fact that niches for alkaliphiles are relatively small, e.g. the intestines of insects (high pH), and are distributed throughout natural ordinary environments (Thongaram et al., 2003). Alkaliphilic bacteria can be either facultative or obligate (Krulwich & Guffanti, 1989). Bacteria of the former type can grow at neutral pH, whereas the latter cannot. Numerous alkaliphilic micro-organisms have been isolated, including members of the Archaea and Bacteria. Both Gram-positive and Gram-negative alkaliphilic bacteria have been isolated. Alkaliphilic bacteria generally consist of not only aerobic or facultatively anaerobic micro-organisms but also obligate anaerobes; they are divided into psychroalkaliphiles, mesoalkaliphiles, thermoalkaliphiles and haloalkaliphiles (Pikuta et al., 2000; Yumoto et al., 2001, 2002; Belduz et al., 2003).
Traditionally, indigo blue dye has been produced from a Japanese indigo plant. The indigo leaves are air-dried and then appropriately wetted to increase aerobic microbial oxidization activity. The product is fermented under alkaline conditions (pH values above 10), at which point the original insoluble oxidized form of indigo is converted, by the micro-organisms, to the soluble reduced form. The use of fermentation to produce indigo declined around 1960 because of difficulties in the production and maintenance processes; since then, the dye has been produced by a chemical process. Recently, traditional methods have been re-evaluated, because they produce better products, in terms of colour, than do the recently invented methods. Indigo-reducing bacteria have been isolated by Takahara & Tanabe (1960) and Padden et al. (1999) and identified as Bacillus sp. and Clostridium isatidis, respectively.
In this study, a halotolerant, psychrotolerant, obligately alkaliphilic micro-organism that can reduce indigo at high pH values was isolated. Phenotypic characterization, phylogenetic analysis based on 16S rRNA gene sequences and DNA–DNA hybridization showed that the isolate merits classification as a novel Alkalibacterium species.
Fermented polygonum indigo (Polygonum tinctorium Lour.) samples were obtained from Date, Hokkaido, Japan (42° 42' N, 140° 42' E). Fermentation liquor (5 ml) was inoculated into 100 ml 0·01 % indigo-containing peptone/yeast extract/alkaline buffer (PYA) broth consisting of 8 g peptone (Kyokuto), 3 g yeast extract (Merck), 1 g K2HPO4, 3·5 mg EDTA, 3 mg ZnSO4.7H2O, 10 mg FeSO4.7H2O, 2 mg MnSO4.nH2O, 1 mg CuSO4.5H2O, 2 mg Co(NO3)2.6H2O and 1 mg H3BO3 in 1 l NaHCO3/Na2CO3 buffer (100 mM in deionized water; pH 10) at 27 °C. After checking for microbial reduction, the culture was transferred to another 100 ml fresh 0·01 % indigo-containing PYA broth. This enrichment process was continued five times using the same medium. The enrichment culture thus obtained was inoculated on a reinforced clostridial agar (RCA; Sigma) plate containing 100 mM NaHCO3/Na2CO3 buffer at pH 10 (alkali-RCA) and incubated in an argon gas exchange jar. After 48 h cultivation, single colonies were picked up and re-isolated five times using alkali-RCA plates. The purified colonies obtained were inoculated into 0·01 % indigo-containing PYA broth to check the ability of the isolate to reduce indigo. Cells for chemotaxonomic analysis were harvested in the late-exponential phase after cultivation with reciprocal shaking (130 strokes min–1) at 27 °C in reinforced clostridial broth (RCB; Sigma) containing 100 mM NaHCO3/Na2CO3 (alkali-RCB). Alkali solution for media used in this study was sterilized separately by autoclaving. Alkalibacterium olivapovliticus NCIMB 13710T and Marinilactibacillus psychrotolerans NCIMB 13873T were used as reference strains for DNA–DNA hybridization. These micro-organisms were cultivated using alkali-RCB by the same method.
For phenotypic characterization, alkali-RCA or alkali-RCB was used as the basal medium. The culture was incubated at 27 °C for 2 weeks and experiments were performed three times for reproducibility of results. Acid production from carbohydrates was determined according to the method of Hugh & Leifson (1953), using thymol blue, instead of bromothymol blue, at pH 10. Growth experiments at pH 7–10 were performed using PYA medium containing 100 mM NaH2PO4/Na2HPO4 buffer (pH 7–8), 100 mM NaHCO3/Na2CO3 buffer (pH 9–10) or 100 mM Na2CO3/NaOH buffer (pH 11–12·3). Anaerobic growth was tested in PYA broth (pH 10) by replacing air with argon gas. Other physiological and biochemical characteristics were examined according to the methods of Yumoto et al. (1998) and as described by Barrow & Feltham (1993). The aerobic growth rate, at pH 10, of the isolate in the temperature range 10–40 °C was determined using a temperature-gradient incubator (Bio-Photorecorder, type TN-2612; Advantec).
For observation of negatively stained cells by transmission electron microscopy, cells were grown on alkali-RCA; preparation and observation were performed as described previously (Yumoto et al., 2002).
Whole-cell fatty acid analysis and isoprenoid quinone determination were performed as described previously (Yumoto et al., 2001). Peptidoglycan and its hydrolysate were prepared by the method of Okada et al. (1992). The amino acid composition was determined using a Hitachi L-8500A automated amino acid analyser.
Fermentation products from D-glucose were analysed by HPLC with a TSKgel Oapak (Tosoh) column (7·8 mmx30 cmx2). The optical purity of L-lactate in the spent medium was determined by HPLC with a TSKgel ENANRIO L1 (Tosoh) column (4·6 mmx25 cm).
Bacterial DNA was prepared according to the method of Marmur (1961). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984). The level of DNA–DNA relatedness was determined fluorometrically by the method of Ezaki et al. (1989), using photobiotin-labelled DNA probes and black microplates.
The 16S rRNA gene sequence corresponding to positions 27–1519 in the 16S rRNA gene sequence of Escherichia coli (Brosius et al., 1978) was amplified by a PCR with primers 20F (AGTTTGATCATGGCTCA) and 1541R (AAGGAGGTGATCCAGCC). The PCR product (approx. 1·5 kb) was sequenced directly by the dideoxynucleotide chain-termination method using a DNA sequencer (PRISM 3100; Applied Biosystems). Multiple alignments of the sequences were performed and the nucleotide substitution rate (Knuc value) was calculated. A phylogenetic tree was constructed by the neighbour-joining method (Kimura, 1980; Saitou & Nei, 1987) using the CLUSTAL W program (Thompson et al., 1994). Similarity values for sequences were calculated using the GENETYX computer program (Software Development).
One strain was isolated using RCA (pH 10). The colonies of strain IDR2-2T on an alkali-RCA plate were circular, convex and pale white; IDR2-2T cells were Gram-positive, peritrichously flagellated rods measuring 0·4–0·9x0·7–3·1 µm and did not produce spores (Fig. 1).
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The end-products of glucose utilization were determined. Lactate was produced with a yield of 80–88 % relative to the amount of added D-glucose (0·5 %). The optical purity of the L-lactate was found to be 52 %, indicating that the bacterium produced DL-lactate.
GLC analysis revealed that the fatty acids of IDR2-2T are C10 : 0 (0·7 %), C14 : 0 (10·9 %), C16 : 0 (37·7 %), C16 : 17c (2·2 %), C16 : 19c (34·7 %), C18 : 0 (1·5 %) and C18 : 19c (10·3 %). The isolate contained a large amount of unsaturated fatty acids. The fatty acid composition of IDR2-2T was similar to that of A. olivapovliticus rather than that of M. psychrotolerans.
Isoprenoid quinones could not be detected by HPLC analysis of the concentrated extracted fraction. The purified peptidoglycan of IDR2-2T contained glutamic acid, alanine and ornithine in a molar ratio of 1 : 1·2 : 0·8. From the results, it was suggested that the strain possessed peptidoglycan of the A4 type (Orn–D-Glu). The amino acid composition in the peptidoglycan is similar to that of M. psychrotolerans NCIMB 13873T rather than that of A. olivapovliticus NCIMB 13710T.
A 1492-base 16S rRNA gene sequence of IDR2-2T was compared with those of strains belonging to other phylogenetically related taxa. The phylogenetic tree constructed using the neighbour-joining method (Fig. 2) and 16S rRNA gene sequence similarities with 12 related strains (data not shown) showed that IDR2-2T is a member of the genus Alkalibacterium. The values for similarity to the related taxa were less than 97 %. IDR2-2T was placed in a related phylogenetic position with respect to A. olivapovliticus (Ntougias & Russell, 2001) and M. psychrotolerans (Ishikawa et al., 2003), which are both alkaliphiles. The highest similarities, in terms of 16S rRNA gene sequence, for IDR2-2T were with the obligate alkaliphile A. olivapovliticus (96·9 %) and the facultative alkaliphile M. psychrotolerans (94·6 %). IDR2-2T exhibited higher levels of 16S rRNA gene sequence similarity with the obligate alkaliphile than with the facultative alkaliphile. These results demonstrate that the obligately alkaliphilic nature of IDR2-2T is consistent with the phylogenetic placement of this isolate.
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According to the results of 16S rRNA gene sequence analysis, IDR2-2T is closely related to A. olivapovliticus and M. psychrotolerans. DNA–DNA hybridization indicates that IDR2-2T is different from A. olivapovliticus (24·3 % DNA–DNA relatedness) and M. psychrotolerans (7·6 % DNA–DNA relatedness).
IDR2-2T differed phenotypically and chemotaxonomically from its phylogenetically close relatives A. olivapovliticus and M. psychrotolerans, as shown in Table 1. The strain could be distinguished from other strains belonging to other phylogenetically related genera: it is distinguished from the genus Alloiococcus by cellular morphology, motility, metabolism of glucose, catalase reaction and the DNA G+C content (the genus Alloiococcus is catalase-positive, comprises non-motile cocci, does not ferment glucose and the DNA G+C content is 44–45 mol%); from the genus Dolosigranulum by cellular morphology and the peptidoglycan type (the genus Dolosigranulum has non-motile, ovoid cells and has lysine at position 3 in the peptidoglycan); and from the genus Carnobacterium by peptidoglycan type and in the main enantiomer of lactate produced (the genus Carnobacterium has peptidoglycan of the A1
type, direct cross-linkage and produces mainly L-lactate from D-glucose).
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Description of Alkalibacterium psychrotolerans sp. nov.
Alkalibacterium psychrotolerans (psy.chro.to'le.rans. Gr. adj. psychros cold; L. part. adj. tolerans tolerating; N.L. neut. part. adj. psychrotolerans tolerating cold environments).
Cells are Gram-positive, peritrichously flagellated straight rods (0·4–0·9x0·7–3·1 µm) and do not produce spores. Grows equally well aerobically and anaerobically. Colonies are circular, convex and pale white. Catalase and oxidase reactions are negative. The species grows at pH 9–12 but not at pH 7–8; optimum growth is at pH 9·5–10·5 at 27 °C. It grows in 0–17 % (w/v) NaCl, with 2–12 % (w/v) as the optimum concentration. It grows at temperatures between 5 and 45 °C, the optimum growth temperature being around 34 °C. It does not hydrolyse starch or gelatin. Ferments D-arabinose, D-xylose, D-glucose and maltose. Does not ferment melibiose, raffinose, myo-inositol, mannitol, erythritol, sorbitol, xylitol, adonitol, dulcitol, sucrose, galactose, inulin or rhamnose. DL-lactic acid is the major end-product from D-glucose. No quinones can be detected. The peptidoglycan type is A4, Orn–D-Glu. The major cellular fatty acids are C14 : 0, C16 : 0, C16 : 19c and C18 : 19c. The DNA G+C content is 40·6 mol%.
The type strain, strain IDR2-2T (=JCM 12281T=NCIMB 13981T), was isolated from polygonum indigo (Polygonum tinctorium Lour.) fermentation liquor obtained from Date, Hokkaido, Japan.
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Belduz, A. O., Dulger, S. & Demirbag, Z. (2003). Anoxybacillus gonensis sp. nov., a moderately thermophilic, xylose-utilizing, endospore-forming bacterium. Int J Syst Evol Microbiol 53, 1315–1320.
Brosius, J., Palmer, J. L., Kennedy, J. P. & Noller, H. F. (1978). Complete nucleotide sequence of 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci U S A 75, 4801–4805.
Duckworth, A. W., Grant, W. D., Jones, B. E. & Steenbergen, R. (1996). Phylogenetic diversity of soda lake alkaliphiles. FEMS Microbiol Lett 19, 181–191.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization 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.
Horikoshi, K. (1991). Microorganisms in Alkaline Environments. Weinheim: VCH.
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.
Ishikawa, M., Nakajima, K., Yanagi, M., Yamamoto, Y. & Yamasato, K. (2003). Marinilactibacillus 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 Microbiol 53, 711–720.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Krulwich, T. A. & Guffanti, A. A. (1989). Alkalophilic bacteria. Annu Rev Microbiol 43, 435–463.[CrossRef][Medline]
Krulwich, T. A., Ito, M. & Guffanti, A. A. (2001). The Na+-dependence of alkaliphily in Bacillus. Biochim Biophys Acta 1505, 158–168.[Medline]
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3, 208–218.
Ntougias, S. & Russell, N. J. (2001). Alkalibacterium olivoapovliticus gen. nov., sp. nov., a new obligately alkaliphilic bacterium isolated from edible-olive wash waters. Int J Syst Evol Microbiol 51, 1161–1170.[Abstract]
Okada, S., Uchimura, T. & Kozaki, M. (1992). Laboratory Manual for Lactic Acid Bacteria. Tokyo: Asakura-shoten (in Japanese).
Padden, A. N., Dillon, V. M., Edmonds, J., Collins, M. D., Alvarez, N. & John, P. (1999). An indigo-reducing moderate thermophile from a woad vat, Clostridium isatidis sp. nov. Int J Syst Bacteriol 49, 1025–1031.
Pikuta, E., Lysenko, A., Chuvilskaya, N., Mendrock, U., Hippe, H., Suzina, N., Nikitin, D., Osipov, G. & Laurinavichius, K. (2000). Anoxybacillus pushchinensis gen. nov., sp. nov., a novel anaerobic, alkaliphilic, moderately thermophilic bacterium from manure, and description of Anoxybacillus flavithermus comb. nov. Int J Syst Evol Microbiol 50, 2109–2117.[Abstract]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Takahara, Y. & Tanabe, O. (1960). Studies on the reduction of indigo in industrial fermentation vat (VII). J Ferment Technol 38, 329–331.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Thongaram, T., Kosono, S., Ohkuma, M., Hongoh, Y., Kitada, M., Yoshinaka, T., Trakulnaleamsai, S., Noparatnaraporn, N. & Kudo, T. (2003). Gut of higher termites as a niche for alkaliphiles as shown by culture-based and culture-independent studies. Microb Environ 18, 152–159.[CrossRef]
Yumoto, I. (2002). Bioenergetics of alkaliphilic Bacillus spp. J Biosci Bioeng 93, 342–353.
Yumoto, I. (2003). Electron transport system in alkaliphilic Bacillus spp. Recent Res Devel Bacteriol 1, 131–149.
Yumoto, I., Yamazaki, K., Sawabe, T., Nakano, K., Kawasaki, K., Ezura, Y. & Shinano, H. (1998). Bacillus horti sp. nov., a new Gram-negative alkaliphilic bacillus. Int J Syst Bacteriol 48, 565–571.
Yumoto, I., Yamazaki, K., Hishinuma, M., Nodasaka, Y., Suemori, A., Nakajima, K., Inoue, N. & Kawasaki, K. (2001). Pseudomonas alcaliphila sp. nov., a novel facultatively psychrophilic alkaliphile isolated from seawater. Int J Syst Evol Microbiol 51, 349–355.[Abstract]
Yumoto, I., Nakamura, A., Iwata, H., Kojima, K., Kusumoto, K., Nodasaka, Y. & Matsuyama, H. (2002). Dietzia psychralcaliphila sp. nov., a novel, facultatively psychrophilic alkaliphile that grows on hydrocarbons. Int J Syst Evol Microbiol 52, 85–90.[Abstract]
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