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Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, P. R. China
Correspondence
Shuang-Jiang Liu
shuangjiang{at}hotmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of strain 3-pT is AF523824.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). This facultative nature of phototrophic purple non-sulfur bacteria has led to their widespread distribution in rivers, lakes, oceans and soil. Phototrophic bacteria play important roles in the bioconversion of organic matters and the biocycling of elements such as carbon, nitrogen and sulfur in these environments. Phototrophic purple non-sulfur bacteria are also commonly isolated from wastewater treatment plants (Hiraishi & Ueda, 1994 and references cited therein). Because of their diverse metabolisms, they have been exploited in various wastewater treatment systems.
Spiral-shaped phototrophic purple non-sulfur bacteria were included in the genus Rhodospirillum (Trüper & Imhoff, 1989) before the proposal of the species Rhodocista centenaria (Kawasaki et al., 1992) and Rhodospira trueperi (Pfennig et al., 1997). As more phototrophic bacterial strains were isolated and more information on the phenotypes and genotypes (particularly 16S rDNA sequences) of these and other previously described strains became available, it was evident that the original description of the genus Rhodospirillum needed to be revised (Kawasaki et al., 1993). The establishment of the genus Rhodocista was based mainly on the results of 16S rDNA sequences and also on its unique cyst-producing morphology and cellular quinone (Q-9). Until now, R. centenaria has been the only species of the genus Rhodocista and the type strain, ATCC 43720T, was isolated from hot springs in Wyoming, USA (Favinger et al., 1989). In this paper, we describe an isolate from the Beijing municipal wastewater treatment plant, P. R. China. The isolate was characterized as a mesophilic, phototrophic bacterium that produces cysts and possesses Q-9 as the major quinone. Phylogenetic and phenotypic characterization of this isolate indicated that it is a novel species of the genus Rhodocista, for which the name Rhodocista pekingensis is proposed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Media used in this study were modified from the AT medium described by Imhoff & Trüper (1992). ATB medium was made with butyrate as the sole carbon source. ATY medium was prepared by addition of 0·05 % yeast extract and removal of sodium hydrogen carbonate from AT medium. ATYP medium was created by addition of 0·03 % peptone to ATY medium. Agar plates were prepared by addition of 1·5 % agar to the medium. Soft agar was made by reducing the amount of agar to 0·7 %.
Routine cultivation was done anaerobically in screw-capped tubes or bottles filled with medium. Incubation was conducted at 34–41 °C under incandescent illumination of 1000–2000 lx.
Isolation.
Samples of activated sludge were obtained from the municipal wastewater treatment plant located in Beijing, China. Samples were diluted and inoculated into soft-agar tubes, which were then incubated anaerobically in the light. After 1 week cultivation, the pink-reddish colonies were picked and streaked onto agar plates. The plates were placed in anaerobic jars, which were filled with argon and incubated under illumination. This was repeated three to five times to ensure purity of the culture.
Microscopy.
Morphology and ultrastructure of cells grown under illumination were observed by optical microscopy (BH-2; Olympus) and by scanning and transmission electron microscopy (S-570 and H-600, respectively; Hitachi).
Absorption spectrum.
Cells grown under illumination were harvested by centrifugation and resuspended in 60 % sucrose solution. This suspension was used for determination of the absorption spectrum of intact cells. The absorption spectrum was recorded with spectrophotometers HP1050 (Hewlett Packard) for 300–820 nm, and Hitachi model 557 for 800–1200 nm.
Physiological characterization.
Tests for photoassimilation of organic substrates were performed in screw-capped tubes that contained ATY medium, as described by Imhoff & Trüper (1989).
DNA base composition and DNA–DNA hybridization.
Genomic DNA was extracted and purified according to the method of Marmur (1961), except for the addition of proteinase K in the SDS-treatment step. DNA base composition was determined by thermal denaturation (Marmur & Doty, 1962). DNA–DNA hybridization was carried out as described by Tindall et al. (1984) with a minor modification: DNA fragments were labelled with 
-32P, according to the instructions provided with the Nick Translation kit (Boehringer Mannheim).
Phylogenetic analysis.
A fragment of 16S rDNA was amplified and sequenced by using the MicroSeq 16S rDNA Gene kit and an ABI Prism 377 DNA Sequencer (both from Applied Biosystems) and primers 27f (5'-GAGAGTTTGATCCTGGCTCAG-3') and 1541r (5'-AAGGAGGTGATCCAGCC-3'). Sequence alignment and analysis of 16S rRNA gene similarity were performed with the CLUSTAL W program (Thompson et al., 1994) and the SEQBOOT, DNADIST, NEIGHBOR and CONSENSE programs of the PHYLIP package, version 3.5c (Felsenstein, 1993).
Quinone determination.
Quinones were determined as described by Collins (1985) and Wu et al. (1989). The bacterial pellet was lyophilized and treated with chloroform/methanol (2 : 1, v/v). The quinones were separated on a 254 GF silica plate (Tsingdao Marine Chemical Corporation, China) with n-hexane/ethyl ether (34 : 6, v/v) as the developing solvent. The number of isoprene units and the relative percentage of components of quinones were determined by HPLC (HP1074; Hewlett Packard), using a Bondapak C18 column (3·9x300 mm; Waters). Rhodocista centenaria ATCC 43720T and quinone 10 (Q-10; commercial standard sample) were used as controls.
Nucleotide sequence accession numbers.
The 16S rRNA gene sequences used for construction of the phylogenetic tree were from the Ribosomal Database Project (Maidak et al., 2001). The GenBank accession number for the 16S rDNA sequence of strain 3-pT is AF523824.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The cells were motile by means of a single, polar flagellum (Fig. 1, top). Electron microscopy revealed that phototrophically grown cells formed intracytoplasmic membranes of the lamellar type, which were parallel to the cytoplasmic membrane. Cysts were produced under aerobic incubation in the dark, when grown with butyrate as the sole carbon source (Fig. 1, bottom). Unlike Rhodocista centenaria, no R-body was observed in cells of strain 3-pT.
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Growth, physiological and biochemical characteristics
Strain 3-pT was facultatively anaerobic and heterotrophic. When cultivated anaerobically in the light, strain 3-pT grew at 25–44 °C, with an optimum between 31 and 42 °C; this strain differs from Rhodocista centenaria in that the latter has an optimal growth temperature of 39–45 °C. The pH range for growth was 6·5–8·0, with an optimum of 7·0. No growth was observed in medium with 3 % NaCl. Thiamin and vitamin B12 were required for growth. Yeast extract stimulated growth significantly. Pyruvate, lactate, acetate, glutamate, aspartate and succinate supported growth. Malate, citrate, tartrate, arginine, ethanol, glycerol, mannitol, gluconate, benzoate, formate, glucose and sucrose did not support growth. Molecular hydrogen (H2), but not sulfide or thiosulfate, was used as an electron donor. Ammonium salts and glutamate were used as nitrogen sources. Sulfate was used as a sulfur source for growth.
The phenotypic differences between strain 3-pT and Rhodocista centenaria are listed in Table 1. Briefly, strain 3-pT assimilated succinate, needed thiamin for growth, had an optimal growth temperature between 31 and 42 °C and no R-bodies were seen in cells, whereas Rhodocista centenaria did not assimilate succinate, needed biotin rather than thiamin for growth, had an optimal growth temperature of 39–45 °C and R-bodies did occur in cells.
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-subclass of the Proteobacteria. Among the phototrophic bacteria, strain 3-pT was most closely related to Rhodocista centenaria (97 % similarity). A phylogenetic tree was constructed on the basis of a corrected evolutionary distances matrix (Fig. 2). Strain 3-pT formed a close cluster with the only species of the genus Rhodocista (Rhodocista centenaria) and two species of the genus Azospirillum (Azospirillum irakense and Azospirillum amazonense).
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Cells are vibrioid or spiral, 0·6–0·8 µm wide and 0·8–1·5 µm long. Motile by means of a single polar flagellum. Gram-negative -Proteobacterium. Phototrophically grown cells contain intracytoplasmic lamellar membranes that are parallel to the cytoplasmic membrane. Thiamin and vitamin B12 are required for growth and 0·05 % yeast extract stimulates growth. Anaerobically phototrophic or aerobically chemoheterotrophic. Pyruvate, lactate, acetate and succinate support growth. Malate, citrate, tartrate, arginine, ethanol, glycerol, gluconate, benzoate, formate, glucose, sucrose and mannitol do not support growth. Molecular hydrogen (H2), but not sulfide or thiosulfate, is used as an electron donor. Cells contain Q-9. DNA G+C content is 68·8 mol%.
The type strain, 3-pT (=AS 1.2194T =JCM 11669T), was isolated from a municipal wastewater treatment plant.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Favinger, J., Stadtwald, R. & Gest, H. (1989). Rhodospirillum centenum, sp. nov., a thermotolerant cyst-forming anoxygenic photosynthetic bacterium. Antonie van Leeuwenhoek 55, 291–296.[CrossRef][Medline]
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.5c. Department of Genetics, University of Washington, Seattle, USA.
Hiraishi, A. & Ueda, Y. (1994). Rhodoplanes gen. nov., a new genus of phototrophic bacteria including Rhodopseudomonas rosea as Rhodoplanes roseus comb. nov. and Rhodoplanes elegans sp. nov. Int J Syst Bacteriol 44, 665–673.
Imhoff, J. F. & Trüper, H. G. (1989). Purple nonsulfur bacteria. In Bergey's Manual of Systematic Bacteriology, vol. 3, pp. 1658–1682. Edited by J. T. Staley, M. P. Bryant, N. Pfennig & J. G. Holt. Baltimore: Williams & Wilkins.
Imhoff, J. F. & Trüper, H. G. (1992). The genus Rhodospirillum and related genera. In The Prokaryotes, 2nd edn, vol. 1, pp. 2141–2155. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K. H. Schleifer. Heidelberg: Springer.
Kawasaki, H., Hoshino, Y., Kuraiski, Y. & Yamasato, K. (1992). Rhodocista centenaria gen. nov., sp. nov., a cyst-forming anoxygenic photosynthetic bacterium and its phylogenetic position in the Proteobacteria alpha group. J Gen Appl Microbiol 38, 541–551.
Kawasaki, H., Hoshino, Y. & Yamasato, K. (1993). Phylogenetic diversity of phototrophic purple non-sulfur bacteria in the Proteobacteria group. FEMS Microbiol Lett 112, 61–66.[CrossRef]
Maidak, B. L., Cole, J. R., Lilburn, T. G. & 7 other authors (2001). The RDP-II (Ribosomal Database Project). Nucleic Acids Res 29, 173–174.
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]
Pfennig, N., Lünsdorf, H., Süling, J. & Imhoff, J. F. (1997). Rhodospira trueperi, gen. nov., sp. nov., a new phototrophic Proteobacterium of the alpha group. Arch Microbiol 168, 39–45.[CrossRef][Medline]
Stadtwald-Demchick, R., Turner, F. R. & Gest, H. (1990). Physiological properties of the thermotolerant photosynthetic bacterium, Rhodospirillum centenum. FEMS Microbiol Lett 67, 139–144.[CrossRef]
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.
Tindall, B. J., Ross, H. N. M. & Grant, W. D. (1984). Natronobacterium gen. nov. and Natronococcus gen. nov., two new genera of haloalkaliphilic archaebacteria. Syst Appl Microbiol 5, 41–57.
Trüper, H. G. & Imhoff, J. F. (1989). Genus Rhodospirillum. In Bergey's Manual of Systematic Bacteriology, vol. 3, pp. 1662–1666. Edited by J. T. Staley, M. P. Bryant, N. Pfennig & J. G. Holt. Baltimore: Williams & Wilkins.
Wu, C., Lu, X., Qin, M. & Ruan, J. (1989). Analysis of quinone in cells of microorganisms by HPLC method. Microbiology (China) 16, 176–178.
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