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1 Leibniz Institut für Meereswissenschaften IFM-GEOMAR, Düsternbrooker Weg 20, D-24105 Kiel, Germany
2 Winogradsky Institute of Microbiology, Russian Academy of Sciences, pr. 60-letiya Oktyabrya 7, k. 2, Moscow, 117312, Russia
Correspondence
Johannes F. Imhoff
jimhoff{at}ifm-geomar.de
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). Due to the transient situation in estuaries between freshwater and marine salt concentrations, both marine and freshwater species of phototrophic bacteria can be found (Gorlenko et al., 1985; Puchkova et al., 2000).
Purple sulfur bacteria belong to the Gammaproteobacteria and comprise the two families Chromatiaceae and Ectothiorhodospiraceae (Imhoff, 1984). These families differ in respect to the deposition of sulfur globules formed during the oxidation of reduced sulfur compounds: members of the Chromatiaceae deposit the globules intracellularly, whereas representatives of the Ectothiorhodospiraceae deposit them extracellularly. Phenotypic characteristics like cell shape or pigment composition were traditional criteria to distinguish bacterial species (Winogradsky, 1888; Pfennig & Trüper, 1974). Today, the analysis of 16S rRNA gene nucleotide sequences is an additional important tool for species differentiation (Imhoff & Süling, 1996; Imhoff et al., 1998a, b). It is particularly valuable if combined with phenotypic properties to allow a detailed characterization of new bacterial isolates.
Strain WST was isolated from material taken from a microbial mat in an estuary of the Nilma river, on the White Sea coast of north-western Russia. In this area, regions of shallow supralittoral beach, which are covered only periodically by brackish water, exhibit the development of natural microbial mats. In addition to cells similar in morphology to strain WST, the purple sulfur bacteria Thiocapsa roseopersicina, Thiocapsa purpurea, Thiocapsa litoralis, Thiorhodovibrio winogradskyi and Allochromatium vinosum and the green sulfur bacterium Prosthecochloris aestuarii were found in these microbial mats (Gorlenko et al., 1985; Puchkova et al., 2000).
The 16S rRNA gene sequence of strain WST exhibited clear affiliation to the genus Thiorhodococcus. The genus Thiorhodococcus, with the type species Thiorhodococcus minor (Guyoneaud et al., 1997; Imhoff et al., 1998b), is one of the genera belonging to the family Chromatiaceae. Important morphological features such as coccoid cells are common to the genera Thiorhodococcus, Thiocystis, Thiocapsa, Thiococcus, Thiohalocapsa, Thiolamprovum, Lamprocystis, Thioalkalicoccus and Thioflavicoccus (Guyoneaud et al., 1997; Imhoff, 2001; Imhoff & Pfennig, 2001; Bryantseva et al., 2000). Both the sequence distance and a number of differences in phenotypic properties differentiate the novel bacterium from known Thiorhodococcus species, which consequently necessitates the description of a novel species.
For isolation of strain WST, cultivation of the pure culture and for growth experiments, Pfennig's medium was used (Pfennig & Trüper, 1992) (l–1): 0.34 g KH2PO4, 0.34 g NH4Cl, 0.5 g MgSO4.7H2O, 0.05 g CaCl2.2H2O, 0.34 g KCl, 1 ml SL A (Imhoff, 1992), 20 µg vitamin B12, 1.5 g NaHCO3, 0.4 g Na2S.7–9H2O, 0.5 g Na2S3O3.5H2O, 15 g NaCl, 2.5 g MgCl2.6H2O. The pH was adjusted to 7.5.
Pure cultures were obtained by repeated application of the deep-agar dilution method (Pfennig & Trüper, 1992). Agar tubes were incubated at 30 °C under a light–dark cycle (16 h 500 lx light, 8 h dark) using tungsten lamps. Purity of the isolate was checked by both microscopy and growth tests in deep-agar or liquid media supplemented with 5 mM acetate and incubated in the dark. Pure cultures were grown in 100 ml screw-capped bottles filled with synthetic medium, incubated at 2000 lx (42 µmol quanta m–2 s–1) at 25 °C. Repeated addition of neutralized sulfide solution was used to obtain high cell yields (Siefert & Pfennig, 1984). Stock cultures were stored at 5 °C in the dark. Growth was followed photometrically by measuring optical density at 650 nm (UV/VIS spectrophotometer Lambda 2; Perkin Elmer).
Microscopic observations of cells of strain WST were done using a phase-contrast microscope (Axiophot; Zeiss). The fine structure of the cells was studied by electron microscopy after fixation of a cell pellet by the method of Ryter & Kellenberger (1958) and ultrathin sectioning of the cells. Observations were made with a JEOL 100 electron microscope.
The absorption spectrum of the living cells was measured after suspension of a cell pellet in 50 % glycerol using a UV/VIS spectrophotometer Lambda 2 (Perkin Elmer).
Growth tests were performed using Pfennig's medium described above aliquotted into 20 ml screw-capped tubes. According to the test conditions, the pH and the salt concentration were varied. Different salt concentrations were obtained using a concentrated salt solution containing (l–1) 294 g NaCl and 47 g MgCl2.6H2O (N. Pfennig, personal communication). The optimal pH was determined first and experiments to determine the optimal salt concentration were performed at optimal pH. Additional tests were performed at optimal pH and salt concentration. For nutritional experiments, several electron donors and carbon sources were tested, according to the recommended standards for the description of novel species (Imhoff & Caumette, 2004), with the final concentrations indicated in Table 1. The tubes were inoculated with a volume of 5 % preculture and incubated at 25 °C and 2000 lx (42 µmol quanta s–1 m–2) for 5 days. For each experiment, three serial repetitions were carried out. Bacterial growth was measured as OD650 as described above. The measurements were performed using sterile Pfennig's medium as a blank and reference sample. Bacterial growth in standard Pfennig's medium incubated under exactly the same conditions was used as control.
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DNA from pure cultures was extracted using the QIAamp DNA Mini kit (Qiagen). The 16S rRNA gene was amplified using eubacterial primers 5'-27F (5'-AGTTTGATCCTGGCTCAG-3') and 3'-1492R (5'-GGTTACCTTGTTACGACTT-3') and puReTaq Ready-To-Go PCR beads (Amersham Biosciences). The QIAquick PCR purification kit (Qiagen) was used to purify the PCR products. Sequence data were obtained using the method of Sanger et al. (1977). Automated sequence determination was performed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The complete sequence was assembled from several fragments by using the software SeqMan II 4.03 (DNASTAR) (Swindell & Plasterer, 1997).
For phylogenetic classification, alignments including various sequences from databases were created with the aid of the software program CLUSTAL X 1.83 (Thompson et al., 1997). PHYLIP version 3.63 (Felsenstein, 2004) was used to create a distance matrix (based on the maximum-likelihood algorithm) and a phylogenetic tree was constructed with PHYML. For the determination of DNA base composition, the DNA was isolated by applying the method of Marmur (1961). The DNA base composition was determined according to Owen et al. (1969).
Under optimal growth conditions in the medium described above, single cells of strain WST exhibit a coccoid morphology. During binary fission, diplococci are formed. The cells are motile, have a mean cell diameter of 1.85 µm and do not contain gas vesicles (Fig. 1). Around individual cells, slight slime production is visible. In phases of stationary growth, cells form microcolonies of irregular shape (Fig. 1). When growing with sulfide and thiosulfate as photosynthetic electron donors, the cells contain sulfur globules stored inside the cell (Figs 1 and 2a). Electron microscopy of thin sections revealed the presence of an intracellular membrane system of the vesicular type and a cell wall typical of Gram-negative bacteria (Fig. 2). The external layer of the cell wall exhibits a multilayered structure (Fig. 2b).
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), which indicates the presence of bacteriochlorophyll a and carotenoids of the rhodopinal series.
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). The pH optimum is 7.0–7.5 and the pH tolerance is between 7.0 and 8.5. Growth experiments concerning the salt concentration revealed slightly decreased growth of strain WST at concentrations higher then 2 % NaCl and in strongly decreased growth with more than 3 % NaCl. No growth occurred at 5 % NaCl. Without salt, strain WST exhibited no growth, but growth increased very steeply at 0.1 % NaCl (Fig. 4b). Thus, the salt optimum of strain WST lies between 0.5 and 2.0 %, while the tolerance range can be defined as 0.1–3.0 %. Growth optima for temperature and light were found at 25–30 °C and 2000 lx (42 µmol quanta s–1 m–2).
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Analysis of the 16S rRNA gene sequence was used to reveal the phylogenetic placement of strain WST among other species of the family Chromatiaceae. The data clearly show that strain WST belongs to the genus Thiorhodococcus (Fig. 5). The highest sequence similarities found were to Trc. minor CE2203T (97.3 %) and ‘Thiorhodococcus drewsii’ DSM 15006 (96.1 %). The base composition of purified DNA of strain WST is 61.8 mol% G+C.
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). Consequently, the taxonomy of phototrophic bacteria has been carefully revised by combining information from gene sequences and selected phenotypic characteristics as diagnostic properties (Imhoff & Süling, 1996; Imhoff et al., 1998a, b; Imhoff, 2003; Guyoneaud et al., 1998). By combining genetic and phenotypic features, clear differentiation of the novel bacterial species described in this communication from closely related species was achieved. Morphological properties such as cell shape and size, the absence of gas vesicles and motility of the new isolate are in accordance with properties of the genus Thiorhodococcus as well as Thiocystis. Genetically, strain WST is affiliated to the genus Thiorhodococcus. Experimental studies revealed significant phenotypic differences from related Thiorhodococcus species (Table 1
) as well as clear separation by 16S rRNA gene sequence similarities from both Trc. minor (97.3 % similarity to the type strain) and ‘Trc. drewsii’ (96.1 % similarity to the proposed type strain). Both Trc. minor and ‘Trc. drewsii’ contain spirilloxanthin as main carotenoid, while rhodopinal is the major component in strain WST. With respect to physiological properties, slight differences can be seen in pH and salt requirements. Compared with other species of this genus, the salt optimum of strain WST is shifted slightly towards lower salt concentrations. This property is probably connected with the isolation of the novel strain from microbial mats developing in brackish, shallow supralittoral zones of the White Sea. On the other hand, the pH requirement of strain WST exhibits an affinity to slightly more alkaline conditions (pH 7.5 and 8.5). In terms of substrate utilization, again similarities and differences can be seen. Common to all members of the genus Thiorhodococcus is the utilization of acetate, pyruvate, succinate, malate and fumarate as carbon substrates. Glucose is used exclusively by strain WST. An outstanding and unique characteristic of strain WST is good growth with mannitol.
Description of Thiorhodococcus mannitoliphagus sp. nov.
Thiorhodococcus mannitoliphagus (mann.i.to'li.pha'gus. N.L. n. mannitolum mannitol; Gr. v. phagein to eat; N.L. masc. adj. mannitoliphagus consuming mannitol).
Cells are coccoid with mean cell diameter of 1.85 µm. During binary fission, diplococci are formed. Cells are Gram-negative, motile by flagella and do not contain gas vesicles. In the stationary growth phase, cells form irregular microcolonies. Colour of cell suspensions is purple–violet. Photosynthetic membrane system is of the vesicular type. Photosynthetic pigments are bacteriochlorophyll a and carotenoids of the rhodopinal series. Phototrophic growth occurs under anoxic conditions in the light. No growth occurs under microaerobic conditions in the light or under chemotrophic conditions in the dark. Vitamin B12 is required as a growth factor. Electron donors used for photolithoautotrophic growth are sulfide, thiosulfate, sulfite and elemental sulfur. Globules of elemental sulfur, which are formed during photolithoautotrophic growth with sulfide and thiosulfate, are stored transiently inside the cells and are oxidized further to sulfate. In the presence of carbonate and a reduced sulfur source (sulfide and/or thiosulfate), oxoglutarate, fructose, acetate, lactate, pyruvate, malate, peptone, Casamino acids, yeast extract, propionate, succinate, fumarate, glucose and mannitol are photoassimilated. The utilization of mannitol results in an outstanding increase in growth. Conditions for optimal growth are 25–30 °C, 2000 lx (42 µmol m–2 s–1), pH 7.0–7.5 and concentrations of 0.5–2 % NaCl. The DNA base composition of the type strain is 61.8 mol% G+C.
The type strain, WST (=ATCC BAA-1228T=VKM B-2393T), was isolated from microbial mat communities of an estuary of the White Sea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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