Abstract
The Black Sea is the largest meromictic basin, in the bottom sediments of which a powerful biogenic process of sulfide production occurs. The goal of the present work was to obtain data on phylogenetic diversity of the sulfur cycle microorganisms (sulfate-reducing and sulfur-oxidizing bacteria) in the Black Sea coastal gas-saturated bottom sediments. The samples were collected in the Chersonesus (Blue) Bay near Sevastopol from whitish bacterial mats of sulfurettes, and from the upper layer of the nearby seabed. Using DNA isolated from the native samples and obtained enrichment cultures, PCR analysis was performed with oligonucleotide primers specific to the fragments of the 16S rRNA genes of the main subgroups of sulfatereducing bacteria (SRB) and to the fragments of the dsrB gene (both reductive and oxidative types), encoding the β-subunit of dissimilatory (bi)sulfite reductase, the key enzyme in the sulfur cycle, inherent in both sulfate- reducing and sulfur-oxidizing microorganisms. The presence of 16S rRNA gene fragments specific to the genera Desulfobacterium, Desulfobacter, Desulfococcus–Desulfonema–Desulfosarcina, and Desulfovibrio–Desulfomicrobium was detected in the DNA samples isolated from coastal bottom bacterial mats. Usage of denaturing gradient gel electrophoresis (DGGE) with subsequent sequencing of reamplified dsrB gene fragments revealed that according to deduced amino acid sequences encoded by the dsrB gene (reductive type), SRB from the coastal gas-saturated bottom sediments of the Black Sea had the highest homology (92−99%) with the dsrB gene of cultured SRB belonging to the genera Desulfovibrio, Desulfatitalea, Desulfobacter, and Desulfobacterium, as well as with uncultured SRB strains from various marine habitats, such as bottom sediments of the Northern and Japanese seas. Deduced amino acid sequences encoded by the oxidative dsrB gene had the highest homology (90−99%) with the relevant sequences of the genera Thiocapsa, Thiobaca, Thioflavicoccus, and Thiorhodococcus.
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References
Amouroux, D., Roberts, G., Rapsomanikis, S., and Andreae, M.O., Biogenic gas (CH4, N2O, DMS) emission to the atmosphere from nearshore and shelf waters of the north-western Black Sea, Estuar. Coast. Shelf Sci., 2002, vol. 54, pp. 575–587.
Bagwell, C.E., Formolo, M., Ye, Q., Yeager, C.M., Lyons, T.W., and Zhang, C.L., Direct analysis of sulfatereducing bacterial communities in gas hydrate-impacted marine sediments by PCR-DGGE, J. Basic Microbiol., 2009, vol. 49, pp. 87–92.
Blazejak, A. and Schippers, A., Real-time PCR quantification and diversity analysis of the functional genes aprA and dsrA of sulfate-reducing prokaryotes in marine sediments of the Peru continental margin and the Black Sea, Front. Microbiol., 2011, vol. 2, pp. 253–263.
Boetius, A., Ravenschlag, K., Schubert, C.J., Rickert, D., Widdel, F., Gieseke, A., Amann, R., Jorgensen, B.B., Witte, U., and Pfannkuche, O., A marine microbial consortium apparently mediating anaerobic oxidation of methane, Nature, 2000, vol. 407, pp. 623–627.
Bryukhanov, A.L., Korneeva, V.A., Kanapatskii, T.A., Zakharova, E.E., Men’ko, E.V., Rusanov, I.I., and Pimenov, N.V., Investigation of the sulfate-reducing bacterial community in the aerobic water and chemocline zone of the Black Sea by the FISH technique, Microbiology (Moscow), 2011, vol. 80, pp. 108–116.
Daly, K., Sharp, R.J., and McCarthy, A.J., Development of oligonucleotide probes and PCR primers for detecting phylogenetic subgroups of sulfate-reducing bacteria, Microbiology (UK), 2000, vol. 146, pp. 1693–1705.
Dimitrov, L., Contribution to atmospheric methane by natural gas seepages on the Bulgarian continental shelf, Cont. Shelf Res., 2002, vol. 22, pp. 2429–2442.
Egorov, V.N., Pimenov, N.V., Malakhova, T.V., Kanapatskii, T.A., Artemov, Yu.G., and Malakhova, L.V., Biogeochemical characteristics of methane distribution in the water and bottom sediments at gas seepage sites in the Sevastopol bays, Mor. Ekol. Zh., 2012, vol 11, pp. 41–52.
Geets, J., Borremans, B., Diels, L., Springael, D., Vangronsveld, J., van der Lelie, D., and Vanbroekhoven, K., DsrB gene-based DGGE for community and diversity surveys of sulfate-reducing bacteria, J. Microbiol. Methods, 2006, vol. 66, pp. 194–205.
Hoehler, T.M., Alperin, M.J., Albert, D.B., and Martens, C.S., Field and laboratory studies of methane oxidation in an anoxic marine sediment: evidence for a methanogen-sulfate reducer consortium, Global Geochem. Cycles, 1994, vol. 8, pp. 451–463.
Hovland, M., Judd, A.G., and Burke, R.A., Jr., The global flux of methane from shallow submarine sediments, Chemosphere, 1993, vol. 26, pp. 559–578.
Ivanov, M.V., Pimenov, N.V., Rusanov, I.I., and Lein, A.Y., Microbial processes of the methane cycle at the north-western shelf of the Black Sea, Estuar. Coast. Shelf. Sci., 2002, vol. 54, pp. 589–599.
Jessen, G.L., Lichtschlag, A., Struck, U., and Boetius, A., Distribution and composition of thiotrophic mats in the hypoxic zone of the Black Sea (150–170 m water depth, Crimea margin), Front. Microbiol., 2016, vol. 7, pp. 1011–1024.
Jørgensen, B.B., Bang, M., and Blackburn, T.H., Anaerobic mineralization in marine sediments from the Baltic Sea–North Sea transition, Mar. Ecol. Prog. Ser., 1990, vol. 59, pp. 39–54.
Judd, A.G., Natural seabed gas seeps as sources of atmospheric methane, Environ. Geol., 2004, vol. 46, pp. 988–996.
Kleindienst, S., Ramette, A., Amann, R., and Knittel, K., Distribution and in situ abundance of sulfate-reducing bacteria in diverse marine hydrocarbon seep sediments, Environ. Microbiol., 2012, vol. 14, pp. 2689–2710.
Kumar, S., Stecher, G., and Tamura, K., MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets, Mol. Biol. Evol., 2016, vol. 33, pp. 1870–1874.
Leloup, J., Loy, A., Knab, N.J., Borowski, C., Wagner, M., and Jørgensen, B.B., Diversity and abundance of sulfatereducing microorganisms in the sulfate and methane zones of a marine sediment, Black Sea, Environ. Microbiol., 2007, vol. 9, pp. 131–142.
Lenk, S., Arnds, J., Zerjatke, K., Musat, N., Amann, R., and Mussmann, M., Novel groups of Gammaproteobacteria catalyse sulfur oxidation and carbon fixation in a coastal, intertidal sediment, Environ. Microbiol., 2011, vol. 13, pp. 758–774.
Lever, M.A., Rouxel, O., Alt, J.C., Shimizu, N., Ono, S., Coggon, R.M., Shanks, W.C., Lapham, L., Elvert, M., Prieto-Mollar, X., Hinrichs, K.-U., Inagaki, F., and Teske, A., Evidence for microbial carbon and sulfur cycling in deeply buried ridge flank basalt, Science, 2013, vol. 339, pp. 1305–1308.
Loy, A., Duller, S., Baranyi, C., Mussmann, M., Ott, J., Sharon, I., Béjà, O., Le Paslier, D., Dahl, C., and Wagner, M., Reverse dissimilatory sulfite reductase as phylogenetic marker for a subgroup of sulfur-oxidizing prokaryotes, Environ. Microbiol., 2009, vol. 11, pp. 289–299.
Lysenko, V. and Shik, N., Modern processes of carbonate formation associated with hydrocarbon degassing in the Laspi Bay (Southern Crimean coast), Prostr. Vremya, 2013, vol. 2, pp. 151–157.
Malakhova, T.V., Kanapatskii, T.A., Egorov, V.N., Malakhova, L.V., Artemov, Yu.G., Evtushenko, D.B., Gulin, S.B., and Pimenov, N.V., Microbial processes and genesis of methane gas jets in the coastal areas of the Crimean Peninsula, Microbiology (Moscow), 2015, vol. 84, pp. 838–845.
Marchesi, J.R., Sato, T., Weightman, A.J., Martin, T.A., Fry, J.C., Hiom, S.J., Dymock, D., and Wade, W.G., Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA, Appl. Environ. Microbiol., 1998, vol. 64, pp. 795–799.
Michaelis, W., Seifert, R., Nauhaus, K., Treude, T., Thiel, V., Blumenberg, M., Knittel, K., Gieseke, A., Peterknecht, K., Pape, T., Boetius, A., Amann, R., Jørgensen, B. B., Widdel, F., Peckmann, J., Pimenov, N.V., and Gulin, M.B., Microbial reefs in the Black Sea fueled by anaerobic oxidation of methane, Science, 2002, vol. 297, pp. 1013–1015.
Müller, A.L., Kjeldsen, K.U., Rattei, T., Pester, M., and Loy, A., Phylogenetic and environmental diversity of DsrAB-type dissimilatory (bi)sulfite reductases, ISME J., 2015, vol. 9, pp. 1152–1165.
Muyzer, G., de Waal, E.C., and Uitterlinden, A.G., Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA, Appl. Environ. Microbiol., 1993, vol. 59, pp. 695–700.
Neretin, L.N., Abed, R.M., Schippers, A., Schubert, C.J., Kohls, K., and Kuypers, M.M., Inorganic carbon fixation by sulfate-reducing bacteria in the Black Sea water column, Environ. Microbiol., 2007, vol. 9, pp. 3019–3024.
Pester, M., Bittner, N., Deevong, P., Wagner, M., and Loy, A., A “rare biosphere” microorganism contributes to sulfate reduction in a peatland, ISME J., 2010, vol. 4, pp. 1591–1602.
Pimenov, N.V. and Ivanova, A.E., Anaerobic methane oxidation and sulfate reduction in bacterial mats of coral-like carbonate structures in the Black Sea, Microbiology (Moscow), 2005, vol. 74, pp. 362–370.
Pimenov, N.V., Egorov, V.N., Kanapatskii, T.A., Malakhova, T.V., Artemov, Ju.G., Sigalevich, P.A., and Malakhova, L.V., Sulfate reduction and microbial processes of the methane cycle in the sediments of the Sevastopol Bay, Microbiology (Moscow), 2013, vol. 82, pp. 618–627.
Schippers, A., Kock, D., Höft, C., Köweker, G., and Siegert, M., Quantification of microbial communities in subsurface marine sediments of the Black Sea and off Namibia, Front. Microbiol., 2012, vol. 3, pp. 16–26.
Tkeshelashvili, G.I., Egorov, V.N., Mestvirishvili, Sh.A., Parkhaladze, G.Sh., Gulin, M.B., Gulin, S.B., and Artemov, Yu.G., Methane emissions from the Black Sea bottom in the mouth zone of the Supsa River at the coast of Georgia, Geochem. Int., 1997, vol. 35, pp. 284–288.
Trüper, H.G. and Schlegel, H.G., Sulfur metabolism in Thiorhodaceae. I. Quantitative measurements in growing cells of Cromatium okenii, Antonie van Leeuwenhoek. J. Microbiol. Serol., 1964, vol. 30, pp. 225–238.
Vetriani, C., Tran, H.V., and Kerkhof, L.J., Fingerprinting microbial assemblages from the oxic/anoxic chemocline of the Black Sea, Appl. Environ. Microbiol., 2003, vol. 69, pp. 6481–6488.
Wrede, C., Heller, C., Reitner, J., and Hoppert, M., Correlative light/electron microscopy for the investigation of microbial mats from Black Sea Cold Seeps, J. Microbiol. Methods, 2008, vol. 73, pp. 85–91.
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Original Russian Text © A.L. Bryukhanov, M.A. Vlasova, T.V. Malakhova, A.A. Perevalova, N.V. Pimenov, 2018, published in Mikrobiologiya, 2018, Vol. 87, No. 3.
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Bryukhanov, A.L., Vlasova, M.A., Malakhova, T.V. et al. Phylogenetic Diversity of the Sulfur Cycle Bacteria in the Bottom Sediments of the Chersonesus Bay. Microbiology 87, 372–381 (2018). https://doi.org/10.1134/S0026261718030025
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DOI: https://doi.org/10.1134/S0026261718030025