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1 Laboratoire d'Ecologie Moléculaire, EA 3525, IBEAS, Université de Pau et des Pays de l'Adour, BP1155, F-64013 Pau Cedex, France
2 Institut für Meereskunde an der Universität Kiel, Abteilung Marine Mikrobiologie, Düsternbrooker Weg 20, D-24105 Kiel, Germany
3 Institute of Microbiology, Russian Academy of Sciences, pr. 60-letiya Oktyabrya 7 k. 2, 117312 Moscow, Russia
Correspondence
Pierre Caumette
pierre.caumette{at}univ-pau.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of strain 5811T is Y12301.
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), and the more recently described Thiocapsa litoralis (Puchkova et al., 2000). In contrast, two species that were previously described as members of the genus Thiocapsa are now recognized as members of separate genera, i.e. Thiococcus pfennigii and Thiohalocapsa halophila (Imhoff et al., 1998).
During ecological investigations in the brackish coastal lagoon ‘Etang du Prevost’ (French Mediterranean coast), a particular purple–red, okenone-containing strain of Thiocapsa (strain 5811T) was isolated (Caumette, 1986) and characterized according to its phenotypic characteristics (Caumette et al., 1985). At that time, it was concluded that it would be premature to consider strain 5811T as a novel species solely on the basis of differences in pigment composition. Subsequently, other okenone-containing Thiocapsa strains (strains 5812, BS-1 and BM-3) were isolated from different environments (Prevost Lagoon, Romanian coast of the Black Sea and Russian coast of the White Sea, respectively). In this paper, on the basis of cytological, biochemical, physiological and genetic properties, it is proposed that these four strains should be classified as members of a novel species, Thiocapsa marina sp. nov., with strain 5811T (=DSM 5653T=ATCC 43172T) as the type strain.
Strains 5811T and 5812 were isolated from Prevost Lagoon (French Mediterranean coast) during red-water periods in July 1977 and 1979 (Caumette, 1986). During the time of sampling, the water was anoxic, contained high sulfide concentrations (up to 3–4 mM) and had a salinity of 2·0–3·5 %. Strain BS-1 was isolated from hydrogen sulfide-containing sediment in the Black Sea near the coast of Romania, at a depth of 30 m. Strain BM-3 was isolated from a microbial mat located in a natural, shallow, supralittoral beach, which was only periodically covered by sea water, on the Russian coast of the White Sea. For isolation of strains, the culture medium, prepared according to Pfennig & Trüper (1992), contained [(l distilled water)–1]: KH2PO4, 0·3 g; NH4Cl, 0·5 g; CaCl2.2H2O, 0·05 g; MgCl2.6H2O, 1 g; MgSO4.7H2O, 0·5 g; NaCl, 20 g; trace element solution SL12 (Overmann et al., 1992), 1 ml; vitamin B12, 0·02 mg; NaHCO3, 1·5 g; and Na2S.9H2O, 0·5 g. The final pH was adjusted to 7·2–7·4. Pure cultures were obtained as described by Guyoneaud et al. (1997). Microscopic observations, absorption spectra of living cells and acetone extracts were realized according to Caumette et al. (1985) and Caumette (1986). Growth tests for substrate utilization, optimum NaCl concentration, pH and temperature and light intensity requirements were performed according to Guyoneaud et al. (1997). Semi-aerobic growth, vitamin requirements, capacity for assimilatory sulfate reduction and capacity for dinitrogen fixation were tested according to Guyoneaud et al. (1997).
Individual cells of the four purple–red strains were non-motile cocci, 1·5–3·0 µm in diameter. They were similar morphologically to Thiocapsa roseopersicina and Thiocapsa litoralis, but differed from Thiocapsa pendens and Thiocapsa rosea by their lack of gas vesicles. The four strains contained bacteriochlorophyll a and okenone as main pigments. In the near-infrared region of the spectra, the major peak of bacteriochlorophyll a was 825–830 nm, whereas the usual 800 nm peak was very low, appearing as a shoulder on the major peak (Fig. 1b). The broad peak at 520–525 nm is characteristic of the presence of okenone as the main carotenoid. In comparison, spirilloxanthin-containing Thiocapsa roseopersicina has two major peaks in the near-infrared region around 800 and 860 nm (Fig. 1a). Chemical carotenoid analysis of strains 5811T, 5812 and BM-3 confirmed okenone as the main carotenoid (Caumette et al., 1985; Sidorova et al., 1998). However, results revealed the presence of three carotenoid series in these strains. In addition to okenone, which is also the major carotenoid, spirilloxanthin and the ketocarotenoids found in Rhodopila globiformis were also present. No modification in carotenoid composition was detected when growth occurred under different culture conditions. This peculiar pigment composition is the most obvious phenotypic characteristic that distinguishes these four purple–red strains from other Thiocapsa species.
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DNA was isolated by the method of Marmur (1961). The DNA base composition was determined according to Owen et al. (1969). DNA–DNA hybridizations were performed according to standard procedures (De Ley et al., 1970). Cell material for 16S rDNA sequencing was taken from 1–2 ml well-grown liquid cultures. DNA was extracted and purified by using a QIAamp DNA extraction kit (Qiagen). PCR amplification and 16S rDNA sequencing were done as described previously (Imhoff et al., 1998; Imhoff & Pfennig, 2001). Sequences were aligned by using the program CLUSTAL_W (Thompson et al., 1994) and corrected manually. A distance matrix was calculated on the basis of the F84-algorithm according to Kishino & Hasegawa (1989), with the program DNADIST within the PHYLIP package (Felsenstein, 1989). The program FITCH (Fitch & Margoliash, 1967) in the PHYLIP package fitted a tree to the evolutionary distances. Phylogenetic trees calculated by maximum-likelihood were constructed by the program DNAML (Felsenstein & Churchill, 1996) of the PHYLIP package. Parameter G (global rearrangement) was invoked and J (jumble) was set to 10 in all calculations (see PHYLIP documentation). Phylogenetic trees based on distance matrices and maximum-likelihood calculations were bootstrapped with 100 resamplings.
The DNA G+C contents of the four isolates were 62·7, 62·9, 62·9 and 63·2 mol% for strains 5811T, 5812, BS-1 and BM-3, respectively; these values are lower than those of other Thiocapsa species. Caumette et al. (1985) already suggested that the purple–red strains 5811T and 5812 may be considered as subspecies or forma specialis of Thiocapsa roseopersicina. These authors considered analysis of the genetic relatedness between purple–red and rose-red strains of Thiocapsa roseopersicina to be crucial for a definite assignment of the purple–red strains. More recently, taxonomic rearrangement of the genera Thiocapsa and Amoebobacter on the basis of 16S rDNA sequence analysis (Guyoneaud et al., 1998) revealed that both strains 5811T and 5812 were not related closely to the type strains of Thiocapsa roseopersicina, Thiocapsa rosea or Thiocapsa pendens. Comparative 16S rDNA sequence analysis revealed a common ancestor between the four novel strains and the type strains of recognized Thiocapsa species (Fig. 2). No close relationship was found between the novel isolates and Thiohalocapsa halophila, which also represents a coccoid, purple sulfur bacterium from marine and saline environments and contains okenone as the main carotenoid (Caumette et al., 1991). Within the genus Thiocapsa, all four strains formed a tight cluster, with 16S rDNA sequence similarities of >99 %. This suggests that these strains could be recognized as representatives of a novel species of the genus Thiocapsa.
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). Thiohalocapsa halophila (formerly Thiocapsa halophila) was already found to be well-separated from representatives of the genus Thiocapsa (Guyoneaud et al., 1998). DNA–DNA hybridization studies (Table 1) indicate that there is no close relationship between Thiohalocapsa halophila DSM 6210T and the purple–red strain 5811T or the type strain of the species Thiocapsa roseopersicina, with only 14 and 10 % DNA–DNA hybridization, respectively. These results confirm those of 16S rDNA sequence analysis, which have already led to the transfer of this halophilic species to a novel genus, Thiohalocapsa (Imhoff et al., 1998). Concerning the relationship between the four purple–red Thiocapsa strains described in this study and the type species Thiocapsa roseopersicina, the results of DNA–DNA hybridization clearly separate the four purple–red strains from Thiocapsa roseopersicina (DNA relatedness values of 41–47 %). Moreover, the four purple–red strains formed a close cluster, with DNA similarities within the group of 70–87 %. On the basis of DNA–DNA hybridization studies, Brenner (1973) considered similarity values of 70 % and higher as representative of strains of the same species. Moreover, Johnson (1973) considered that different species from the same genus could have 20–60 % DNA similarity.
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, the presence of strong phenotypic coherency among strains should always be the deciding factor when grouping strains into one species. In the case of the four purple–red strains described in this paper, the presence of carotenoids of the okenone series, which is a feature of strong ecological relevance, together with the results of both 16S rDNA sequence analysis and DNA–DNA hybridization, suggest strongly that all four strains should be considered as members of a novel Thiocapsa species (Table 2). Thiocapsa marina sp. nov. is proposed as the name for this novel species and strain 5811T (=DSM 5653T=ATCC 43172T) is the type strain.
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Individual cells are spherical and 1·5–3·0 µm in diameter; cells occur singly or in pairs, tetrads or small aggregates. Division is by binary fission. Gram-negative. Non-motile. Cell suspension is purple–red. Photosynthetic membrane system is of the vesicular type. Contains bacteriochlorophyll a as photosynthetic pigment and okenone as main carotenoid. Phototrophic growth is observed under anoxic conditions in light; chemolithotrophic and chemoorganotrophic growth is seen under micro-oxic conditions in the dark. Electron donors for phototrophic growth are H2, sulfide, elemental sulfur and thiosulfate. Globules of elemental sulfur are stored in cells as an intermediary product when grown photolithotrophically with reduced sulfur compounds. In the presence of sulfide and bicarbonate, the following compounds are photoassimilated: formate, acetate, propionate, lactate, pyruvate, malate, succinate, fumarate, glycerol, fructose, peptone and Casamino acids. Chemolithotrophic growth occurs with sulfide and thiosulfate; chemoorganotrophic growth occurs with acetate and pyruvate. Not capable of assimilatory sulfate reduction. No vitamins are required. Optimal pH for growth is 7·2–7·4 and optimal temperature is 30–35 °C. Grows in 0–8 % NaCl (optimum, 1–2 %). DNA G+C content of the type strain is 62·7 mol%.
The type strain is 5811T (=DSM 5653T=ATCC 43172T), which was isolated from Prevost Lagoon on the French Mediterranean coast. Isolated from anoxic sediments and microbial mats exposed to light in coastal brackish to marine lagoons.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Caumette, P. (1986). Phototrophic sulphur bacteria and sulphate-reducing bacteria causing red waters in a shallow brackish coastal lagoon (Prévost Lagoon, France). FEMS Microbiol Ecol 38, 113–124.
Caumette, P., Schmidt, K., Biebl, H. & Pfennig, N. (1985). Characterization of a Thiocapsa strain containing okenone as major carotenoid. Syst Appl Microbiol 6, 132–136.
Caumette, P., Baulaigue, R. & Matheron, R. (1991). Thiocapsa halophila sp. nov., a new halophilic phototrophic purple sulfur bacterium. Arch Microbiol 155, 170–176.[CrossRef]
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Guyoneaud, R., Matheron, R., Liesack, W., Imhoff, J. F. & Caumette, P. (1997). Thiorhodococcus minus, gen. nov., sp. nov., a new purple sulfur bacterium isolated from coastal lagoon sediments. Arch Microbiol 168, 16–23.[CrossRef][Medline]
Guyoneaud, R., Süling, J., Petri, R., Matheron, R., Caumette, P., Pfennig, N. & Imhoff, J. F. (1998). Taxonomic rearrangements of the genera Thiocapsa and Amoebobacter on the basis of 16S rDNA sequence analyses, and description of Thiolamprovum gen. nov. Int J Syst Bacteriol 48, 957–964.
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Johnson, J. L. (1973). Use of nucleic-acid homologies in the taxonomy of anaerobic bacteria. Int J Syst Bacteriol 23, 308–315.
Kishino, H. & Hasegawa, M. (1989). Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea. J Mol Evol 29, 170–179.[CrossRef][Medline]
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
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Puchkova, N. N., Imhoff, J. F. & Gorlenko, V. M. (2000). Thiocapsa litoralis sp. nov., a new purple sulfur bacterium from microbial mats from the White Sea. Int J Syst Evol Microbiol 50, 1441–1447.[Abstract]
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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.
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