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1 Winogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-let Octyabrya 7/2, 117811 Moscow, Russia
2 Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands
3 Bioengineering Center, Russian Academy of Sciences, Moscow, Russia
4 Department of Microbiology, Faculty of Biology, Moscow State University, Moscow, Russia
Correspondence
Dimitry Yu. Sorokin
soroc{at}inmi.host.ru or
D.Y.Sorokin{at}tnw.tudelft.nl
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and cbb gene sequences of strain HL 5T are DQ390450 and DQ390451–DQ390453, respectively.
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TOPABSTRACTMAIN TEXTREFERENCES |
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), this is mostly true of the brines. In the sediments, which are separated from the brine by a thick salt crust, the microbial communities are more complex and include halophilic eubacteria of different physiology. In particular, halophilic sulfate-reducing bacteria can be active in these environments up to saturating salt concentrations (Ollivier et al., 1994; Sørensen et al., 2004). The latter can be a condition for development of their counterpart, halophilic sulfur-oxidizing bacteria (SOB). Among the chemolithotrophic bacteria, SOB have a good chance to adapt to extreme conditions, such as high salt, owing to the high energy yield available during complete oxidation of sulfide/thiosulfate to sulfate (Oren, 1999).
The first and so far only neutrophilic, halophilic SOB able to grow at very high salt concentrations, up to 4 M NaCl, was discovered 15 years ago in an Australian hypersaline lake (Wood & Kelly, 1991) and is currently known as Halothiobacillus halophilus (Kelly & Wood, 2000). The organism belongs to the high-salt-tolerant moderate halophiles, with an optimum salt concentration for growth around 1 M. Since that time, no attempts have been made to peer more thoroughly into the diversity of halophilic SOB in hypersaline habitats. Our current research on haloalkaliphilic SOB inhabiting soda lakes, however, has demonstrated the widespread potential of SOB to grow at very high concentrations of sodium carbonates (Sorokin & Kuenen, 2005). This prompted us to start analogous research on the diversity of SOB in hypersaline chloride–sulfate inland lakes and salterns with neutral pH.
The Kulunda Steppe (Altai, Russia) harbours numerous salt lakes with a total salt content from 10 to 38 %, a pH range of 7.0–8.5 and with Na+, Cl– and 
as the dominant ions in the brines. Samples were obtained from the top 10 cm of sediment with overlying brine water from 20 lakes and were grouped into three categories according to the salinity.
Halophilic SOB were enriched and isolated in pure culture using the following mineral base medium (g l–1): NaCl, 120; K2HPO4, 1.5; (NH4)2SO4, 0.5. The pH was adjusted to 7.2. After sterilization, the medium was supplemented with 2 mM MgCl2.6H2O, 1 ml trace metal solution l–1 (Pfennig & Lippert, 1966) and 20–50 mM NaHCO3 (from a 1 M filter-sterilized solution). The final pH was 7.5–7.8 and the incubation temperature was 25 °C. Two types of enrichments were performed. Enrichments with thiosulfate as substrate (20 mM) were incubated in sealed 100 ml serum bottles with 10 ml medium under fully aerobic conditions (gas phase, air; rotary shaker at 150 r.p.m.) and under microaerophilic conditions with 10 ml medium in 250 ml bottles at 2 % O2 (static). The second type of enrichment was based on gradient cultivation (Nelson & Jannasch, 1983) in 50 ml glass cylinders, whereby sulfide, as substrate, was diffusing upward from the bottom 2 % (w/v) agar layer (10 ml, 0.1 mmol Na2S) overlaid by 30 ml of the above-mentioned mineral medium containing one-tenth of the amount of trace elements and 0.2 % (w/v) agarose. Oxygen was diffusing downward from the 10 ml headspace. SOB usually developed within a thin layer approximately 1 cm below the surface of the top medium layer. The isolation strategy included several 1 : 100 transfers to stabilize the cultures, followed by serial dilutions and plating onto solid medium with the same composition (for thiosulfate enrichments). The plates were incubated either in plastic bags (aerobic) or in closed jars under 2 % O2 (microaerophilic). For sulfide-gradient cultures, dilution series were performed in 20 ml stoppered tubes with the proportions of different layers similar to that used in the cylinders. Growth was monitored by measuring the OD600 (in case of intermediate sulfur formation, it was removed by centrifugation at 3000 r.p.m. for 1 min) and by monitoring thiosulfate consumption and pH. The pH was maintained above pH 7 by periodic addition of NaHCO3. Sulfur compounds (thiosulfate, sulfide, tetrathionate and elemental sulfur), protein concentration and respiration rates of washed cells were measured as described previously (Sorokin et al., 2001). Phase-contrast photomicrographs were obtained using a Zeiss Axioplan Imaging 2 microscope. For electron microscopy, cells were fixed with glutaraldehyde (final, 3 % v/v) and positively contrasted with 1 % (w/v) uranyl acetate. The isolation of DNA and subsequent determination of the G+C content of the DNA and DNA–DNA hybridization were performed by the thermal denaturation/reassociation technique (Marmur, 1961; De Ley et al., 1970).
The 16S rRNA genes were selectively amplified using general bacterial primers and the PCR products were purified from low-melting-temperature agarose using a Wizard PCR Prep kit (Promega) according to the manufacturer's instructions. To amplify bacterial genes of RuBisCO form I and form II large subunit (cbbL and cbbM, respectively), specially designed and previously tested primer pairs were employed (Spiridonova et al., 2004). Purified PCR fragments were cloned into the pGEM-T vector (Promega) using competent cells of Escherichia coli DH5
for transformation. Plasmid DNA was extracted and purified using the Wizard MiniPrep kit (Promega) according to the manufacturer's recommendations. Clones containing appropriately sized inserts were sequenced from universal M13 forward and reverse primers. Sequencing was performed using a Big Dye Terminator version 3.1 sequencing reaction kit with an ABI 3730 DNA automatic sequencer (Applied Biosystems).
Preliminary phylogenetic analysis of the new sequences was done with the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/blast/). Nucleotide and inferred amino acid sequences were aligned with sequences from GenBank using CLUSTAL W. Phylogenetic trees were reconstructed using the neighbour-joining algorithm within the TREECONW program package (Van de Peer & De Wachter, 1994).
Relative synonymous codon usage (RSCU) values of the RuBisCO genes were calculated using CodonW software (http://www.molbiol.ox.ac.uk/cu). To investigate major trends in codon usage in different species, CodonW was used to carry out a correspondence analysis. This resulted in a point in the codon space for each species, the positions of which sometimes suggested codon usage bias.
Aerobic enrichments with thiosulfate at 2 M NaCl resulted in rapid growth of rod-shaped, motile SOB. From these cultures, colonies of the rod-shaped SOB could be easily obtained on mineral plates with thiosulfate and two pure cultures were identified by 16S rRNA gene sequencing as members of the genus Halothiobacillus (data not shown). However, in enrichments with thiosulfate under micro-oxic conditions and, especially in sulfide–oxygen gradient cultures, small and extremely motile vibrios developed more rapidly than halothiobacilli. Unfortunately, they did not readily form colonies on plates. As a consequence, the colony approach always resulted in the dominance of halothiobacilli, even if they were minor components in the liquid cultures. Only after several dilution series, when the vibrio phenotype became highly enriched, did tiny, sulfur-containing colonies appear on thiosulfate plates under micro-oxic conditions after 2–3 weeks incubation. Two pure cultures were obtained from single colonies originating from thiosulfate enrichments (lake salinity ranges 100–140 and 200–240 g l–1) and one culture after serial dilutions of a< sulfide-gradient enrichment (lake salinity range 100–140 g l–1). The strains were designated HL 5T, HL 8 (isolated with thiosulfate) and HLgr 1 (isolated from sulfide gradient culture). Genetic comparison (DNA–DNA hybridization) demonstrated a high level of relatedness (85–95 %) between the isolates. Therefore, subsequent experiments were performed with the representative strain HL 5T.
All three isolates were small vibrios, highly motile with a single polar flagellum (Fig. 1). Despite the fact that they outcompeted halothiobacilli under micro-oxic conditions, growth of pure cultures was not inhibited under fully aerobic conditions with thiosulfate provided that the pH was kept above 6.5 by the addition of NaHCO3. In that case, all thiosulfate was oxidized to sulfate. At lower pH, thiosulfate was partially converted to elemental sulfur. Polythionates (tetra- and trithionate) were not detected among the products. Cells of strain HL 5T, grown with thiosulfate, were capable of oxidation of sulfide, thiosulfate and elemental sulfur (the latter supplied as a saturated solution in acetone) at maximum rates of 950, 440 and 90 nmol O2 (mg protein)–1 min–1, respectively. Strain HL 5T grew with thiosulfate at NaCl concentrations between 0.5 and 3.5 M with an optimum at 1.5 M, which qualifies this bacterium as an extremely halotolerant moderate halophile. At 2 M NaCl with thiosulfate as substrate, the bacterium grew at temperatures from 20 to 43 °C with an optimum at 30 °C (lower temperatures were not tested). Thiosulfate-oxidizing activity in washed cells was maximal at 0.75–1.5 M NaCl. At salt concentrations below 0.5 M, the cells lysed, turning into spheroplasts. Growth with thiosulfate at 2 M NaCl was possible within the pH range 6.5–8.5, with an optimum at pH 7.5–7.8. Under optimal growth conditions (1.5 M NaCl, pH 7.5, 30 °C), the experimentally measured specific growth rate and growth yield on thiosulfate were 0.25 h–1 and 3.5 mg protein mmol–1, respectively.
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). Currently, this group includes eight species with validly published names, many uncharacterized isolates from various marine habitats (Brinkhoff et al., 2005) and a single hydrogen-utilizing bacterium, Hydrogenovibrio marinus (Nishihara et al., 1991). The level of 16S rRNA gene sequence similarity between strain HL 5T and other species of this cluster ranged from 93.7 to 96.2 %, whereas the similarity with species of the Thiomicrospira pelophila cluster (including the members of the genus Thioalkalimicrobium) did not exceed 93 %.
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), Thiomicrospira crunogena and Thiomicrospira kuenenii (Tourova et al., 2006). However, the G+C content in the genome of all three halophilic isolates was 56.1–57.1 mol%, which is 5–15 mol% higher than in the Thiomicrospira crunogena group (39.2–51.7 mol%). DNA–DNA hybridization of the new isolates with Thiomicrospira sp. strain SL 1, the strain with the highest G+C content within the Thiomicrospira crunogena lineage (51.7 mol%) which originated from Solar Lake, Sinai, Egypt (Brinkhoff & Muyzer, 1997), and with Thiomicrospira crunogena DSM 12353T was below 20 %. Nevertheless, analysis of both 16S rRNA gene sequences (Fig. 2a
) and translated amino acid sequences of the RuBisCO genes (Fig. 2b) firmly placed strain HL 5T into the Thiomicrospira crunogena group.
The G+C content of the cbb genes revealed a strong linear correlation with the genomic G+C content, which is common (Muto & Osawa, 1987). The large differences in the G+C content of the total DNA between the new isolates and other members of the Thiomicrospira crunogena cluster were not an artefact, since the obtained fragments of RuBisCO genes from strain HL 5T also had a similarly high G+C content. The total G+C content for the cbb genes of this strain was 54.1–58.7 mol%, compared with 45.1–48.9 mol% for the corresponding genes of phylogenetically related species. Comparison of codon usage in the RuBisCO genes between strain HL 5T and Thiomicrospira species using RSCU data identified the major trends (Fig. 3): axis 2 is associated with the G+C3 content (in the third position of codons) (Musto et al., 1998) and axis 1 shows the frequency of codons ending in C or U versus A or G (Fennoy & Bailey-Serres, 1993). The codon usage of all Thiomicrospira species was typical of A/T-biased micro-organisms, in which codons with an A or T in the third position are used preferentially (Ohtaka & Ishikawa, 1993). In contrast, the codon usage of strain HL 5T was typical of G/C-biased bacteria. Based on codon usage, the closest neighbours of the strain HL 5T were (halo)alkaliphilic sulfur-oxidizing gammaproteobacteria such as Thioalkalispira microaerophila and Halothiobacillus neapolitanus, with a similar G+C content of their genomic DNA (56.0 mol%) and the two types of RuBisCO genes (54.9–55.5 mol%).
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Cells are Gram-negative vibrios (0.3–0.5x1–2 µm), highly motile due to a single polar flagellum. Colonies on thiosulfate agar at 1–2 M NaCl reach a maximum diameter of 1 mm and are convex, circular and silver–white (because of sulfur accumulation). Strictly aerobic, with a tendency for microaerophilism. Grows chemolithoautotrophically with thiosulfate and sulfide, oxidizing them to sulfate with elemental sulfur as an intermediate. Does not produce or oxidize polythionates. Autotrophic growth with thiosulfate is possible within the range of 0.5–3.5 M NaCl (optimum 1.5 M) and at pH 6.5–8.5 (optimum pH 7.5–7.8). Inorganic carbon is assimilated through the Calvin cycle. Possesses three different RuBisCO genes. Differs from the other Thiomicrospira species by its high salt tolerance and much higher G+C content of genomic DNA. Represented by three closely related strains isolated from hypersaline inland lakes in south-western Siberia (Altai, Russia) with DNA G+C contents of between 56.1 and 57.1 mol%.
The type strain is HL 5T (=DSM 15072T=UNIQEM U 221T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Brinkhoff, T., Kuever, J., Muyzer, G. & Jannasch, H. W. (2005). Genus VI. Thiomicrospira Kuenen and Veldkamp 1972, 253AL. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 2, part B, The Gammaproteobacteria, pp. 193–199. Edited by D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer.
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
Fennoy, S. L. & Bailey-Serres, J. (1993). Synonymous codon usage in Zea mays L. nuclear genes is varied by levels of C- and G-ending codons. Nucleic Acids Res 21, 5294–5300.
Kelly, D. P. & Wood, A. P. (2000). Reclassification of some species of Thiobacillus to the newly designated genera Acidithiobacillus gen. nov., Halothiobacillus gen. nov. and Thermithiobacillus gen. nov. Int J Syst Evol Microbiol 50, 511–516.[Abstract]
Knittel, K., Kuever, J., Meyerdierks, A., Meinke, R., Amann, R. & Brinkhoff, T. (2005). Thiomicrospira arctica sp. nov. and Thiomicrospira psychrophila sp. nov., psychrophilic, obligately chemolithoautotrophic, sulfur-oxidizing bacteria isolated from marine Arctic sediments. Int J Syst Evol Microbiol 55, 781–786.
Marmur, J. (1961). A procedure for isolation of DNA from microorganisms. J Mol Biol 3, 208–214.
Musto, H., Romero, H. & Rodriguez-Maseda, H. (1998). Heterogeneity in codon usage in the flatworm Schistosoma mansoni. J Mol Evol 46, 159–167.[CrossRef][Medline]
Muto, A. & Osawa, S. (1987). The guanine and cytosine content of genomic DNA and bacterial evolution. Proc Natl Acad Sci U S A 84, 166–169.
Nelson, D. C. & Jannasch, H. W. (1983). Chemolithoautotrophic growth of a marine Beggiatoa in sulfide-gradient cultures. Arch Microbiol 136, 262–269.[CrossRef]
Nishihara, H., Igarashi, Y. & Kodama, T. (1991). Hydrogenovibrio marinus gen. nov., sp. nov., a marine obligately chemolithoautotrophic hydrogen-oxidizing bacterium. Int J Syst Bacteriol 41, 130–133.
Ohtaka, C. & Ishikawa, H. (1993). Accumulation of adenine and thymine in a groE-homologous operon of an intracellular symbiont. J Mol Evol 36, 121–126.[CrossRef][Medline]
Ollivier, B., Caumette, P., Garcia, J.-L. & Mah, R. A. (1994). Anaerobic bacteria from hypersaline environments. Microbiol Rev 58, 27–38.
Oren, A. (1999). Bioenergetic aspects of halophilism. Microbiol Mol Biol Rev 63, 334–348.
Oren, A. (2002). Halophilic Microorganisms and their Environments. Dordrecht: Kluwer.
Pfennig, N. & Lippert, K. D. (1966). Über das Vitamin B12-bedürfnis phototropher Schwefel bacterien. Arch Microbiol 55, 245–256 (in German).
Sørensen, K. B., Canfield, D. E. & Oren, A. (2004). Salinity responses of benthic microbial communities in a solar saltern (Eilat, Israel). Appl Environ Microbiol 70, 1608–1616.
Sorokin, D. Yu. & Kuenen, J. G. (2005). Haloalkaliphilic sulfur-oxidizing bacteria in soda lakes. FEMS Microbiol Rev 29, 685–702.[CrossRef][Medline]
Sorokin, D. Yu., Lysenko, A. M., Mityushina, L. L., Tourova, T. P., Jones, B. E., Rainey, F. A., Robertson, L. A. & Kuenen, G. J. (2001). Thioalkalimicrobium aerophilum gen. nov., sp. nov. and Thioalkalimicrobium sibericum sp. nov., and Thioalkalivibrio versutus gen. nov., sp. nov., Thioalkalivibrio nitratis sp. nov. and Thioalkalivibrio denitrificans sp. nov., novel obligately alkaliphilic and obligately chemolithoautotrophic sulfur-oxidizing bacteria from soda lakes. Int J Syst Evol Microbiol 51, 565–580.[Abstract]
Spiridonova, E. M., Berg, I. A., Kolganova, T. V., Ivanovskii, R. N., Kuznetsov, B. B. & Tourova, T. P. (2004). An oligonucleotide primer system for amplification of the ribulose-1,5-bisphosphate carboxylase/oxygenase genes of bacteria of various taxonomic groups. Microbiology (English translation of Mikrobiologiia) 73, 377–387.
Takai, K., Hirayama, H., Nakagawa, T., Suzuki, Y., Nealson, K. H. & Horikoshi, K. (2004). Thiomicrospira thermophila sp. nov., a novel microaerobic, thermotolerant, sulfur-oxidizing chemolithomixotroph isolated from a deep-sea hydrothermal fumarole in the TOTO caldera, Mariana Arc, Western Pacific. Int J Syst Evol Microbiol 54, 2325–2333.
Tourova, T. P., Spiridonova, E. M., Berg, I. A., Kuznetsov, B. B. & Sorokin, D. Yu. (2006). Occurrence, phylogeny and evolution of ribulose-1,5-bisphosphate carboxylase/oxygenase genes in obligately chemolithoautotrophic sulfur-oxidizing bacteria of the genera Thiomicrospira and Thioalkalimicrobium. Microbiology 152, 2159–2169.
Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570.
Wood, A. P. & Kelly, D. P. (1991). Isolation and characterisation of Thiobacillus halophilus sp. nov., a sulfur-oxidising autotrophic eubacterium from a Western Australian hypersaline lake. Arch Microbiol 156, 277–280.[CrossRef]
Yoshizawa, Y., Toyoda, K., Arai, H., Ishii, M. & Igarashi, Y. (2004). CO2-responsive expression and gene organization of three ribulose-1,5-bisphosphate carboxylase/oxygenase enzymes and carboxysomes in Hydrogenovibrio marinus strain MH-110. J Bacteriol 186, 5685–5691.
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