Abstract
Ability to degrade crude oil n-alkanes was revealed in new strains of thermophilic bacilli isolated from petroleum reservoirs and a hot spring: Geobacillus toebii В-1024, Geobacillus sp. 1017, and Aeribacillus pallidus 8m3. The strains utilized С10–С30 n-alkanes (В-1024), С10, C11, and С13–С19,22 n-alkanes (1017), and C11–C29 n-alkanes (8m3). In all three strains, PCR amplification with specific degenerate oligonucleotide primers revealed the alkB gene encoding rubredoxin-dependent alkane monooxygenase. In strains В-1024 and 1017, fragments of the genes homologous to the ladA gene determining flavin-dependent alkane monooxygenase were also amplified. Nucleotide sequences of these genes were practically identical to those of the genes ladAαB23, ladAβB23, and ladB B23, which were revealed previously in Geobacillus thermoleovorans strain B23. For the latter strain, activity of respective enzymes in the oxidation of long-chain n-alkanes has been shown. Thus, simultaneous presence of the alkB and ladA genes coding alkane monooxygenases responsible for oxidation of medium-chain and long-chain n-alkanes in thermophilic bacilli was revealed for the first time.
Similar content being viewed by others
References
Adkins, J.P., Cornell, L.A., and Tanner, R.S., Microbial composition of carbonate petroleum reservoir fluids, Geomicrobiol. J., 1992, vol. 10, pp. 87–97.
Alonso-Gutierrez, J., Teramoto, M., Yamazoe, A., Harayama, S., Figueras, A., and Novoa, B., Alkanedegrading properties of Dietzia sp. H0B, a key player in the Prestige oil spill biodegradation (NW Spain), J. Appl. Microbiol., 2011, vol. 111, pp. 800–810.
Boonmak, C., Takahashi, Y., and Morikawa, M., Cloning and expression of three ladA-type alkane monooxygenase genes from an extremely thermophilic alkane-degrading bacterium Geobacillus thermoleovorans B23, Extremophiles, 2014, vol. 18, pp. 515–523.
Borzenkov, I.A., Milekhina, E.I., Gotoeva, M.T., Rozanova, E.P., and Belyaev, S.S., The properties of hydrocarbon-oxidizing bacteria isolated from the oilfields of Tatarstan, Western Siberia, and Vietnam, Microbiology (Moscow), vol. 75, no. 1, pp. 66–72.
Bryanskaya, A.V., Rozanov, A.S., Logacheva, M.D., Kotenko, A.V., and Peltek, S.E., Draft genome sequence of Geobacillus icigianus strain G1w1T isolated from hot springs in the Valley of Geysers, Kamchatka (Russian Federation), Genome Announc., 2014, vol. 2, no. 5. e01098–14. doi 10.1128/genomeA.01098-14
Bryanskaya, A.V., Rozanov, A.S., Slynko, N.M., Shekhovtsov, S.V., and Peltek, S.E., Geobacillus icigianus sp. nov., a thermophilic bacterium isolated from a hot spring, Int. J. Syst. Evol. Microbiol., 2015, vol. 65, pp. 864–869.
Coorevits, A., Dinsdale, A.N., Halket, G., Lebbe, L., Vos, P., Landschoot, A., and Logan, N.A., Taxonomic revision of the genus Geobacillus: emendation of Geobacillus, G. stearothermophilus, G. jurassicus, G. toebii, G. thermodenitrificans and G. thermoglucosidans (nom. corrig., formerly ‘thermoglucosidasius’); transfer of Bacillus thermantarcticus to the genus as G. thermantarcticus comb. nov.; proposal of Caldibacillus debilis gen. nov., comb. nov.; transfer of G. tepidamans to Anoxybacillus as A. tepidamans comb. nov.; and proposal of Anoxybacillus caldiproteolyticus sp. nov., Int. J. Syst. Evol. Microbiol., 2012, vol. 62, pp. 1470–1485.
Feng, L., Wang, W., Cheng, J., Ren, Y., Zhao, G., Gao, C., Tang, Y., Liu, X., Han, W., Peng, X, Liu, R., and Wang, L., Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir, Proc. Natl. Acad. Sci. U. S. A., 2007, vol. 104, pp. 5602–5607.
Filippidou, S., Jaussi, M., Junier, T., Wunderlin, T., Roussel-Delif, L., Jeanneret, N., Vieth-Hillebrand, A., Vetter, A., Regenspurg, S., Johnson, S.L., McMurry, K., Gleasner, C.D., Lo, C.-C., Li, P., Vuyisich, M., Chain, P.S., and Junier, P., Genome sequence of Anoxybacillus geothermalis strain GSsed3, a novel thermophilic endospore-forming species, Genome Announc., 2015, vol. 3, no. 3. e00575–15.
Funhoff, E.G., Bauer, U., Garcia-Rubio, I., Witholt, B., and van Beilen, J.B., CYP153A6, a soluble P450 oxygenase catalyzing terminal-alkane hydroxylation, J. Bacteriol., 2006, vol. 188, pp. 5220–5227.
Korshunova, A.V., Tourova, T.P., Shestakova, N.M., Mikhailova, E.M., Poltaraus, A.B., and Nazina, T.N., Detection and transcription of n-alkane biodegradation genes (alkB) in the genome of a hydrocarbon-oxidizing bacterium Geobacillus subterraneus K, Microbiology (Moscow), 2011, vol. 80, no. 5, pp. 682–691.
Li, L., Liu, X., Yang, W., Xu, F., Wang, W., Feng, L., Bartlam, M., Wang, L., and Rao, Z., Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unveiling the long-chain alkane hydroxylase, J. Mol. Biol., 2008, vol. 376, no. 2, pp. 453–465.
Liu, C., Wang, W., Wu, Y., Zhou, Z., Lai, Q., and Shao, Z., Multiple alkane hydroxylase systems in a marine alkane degrader, Alcanivorax dieselolei B-5, Environ. Microbiol., 2011, vol. 13, no. 5, pp. 1168–1178.
Liu, Y., Zhou, T., Zhang, J., Xu, L., Zhang, Z., Shen, Q., and Shen, B., Molecular characterization of the alkB gene in the thermophilic Geobacillus sp. strain MH-1, Res. Microbiol., 2009, vol. 160, no. 8, pp. 560–566.
Lo Piccolo, L., De Pasquale, C., Fodale, R., Puglia, A.M., and Quatrini, P., Involvement of an alkane hydroxylase system of Gordonia sp. strain SoCg in degradation of solid n-alkanes, Appl. Environ. Microbiol., 2011, vol. 77, no. 4, pp. 1204–1213.
Marchant, R. and Banat, I.M., The Genus Geobacillus and hydrocarbon utilization, in Handbook of Hydrocarbon and Lipid Microbiology, Timmis, K.N., Ed., Berlin: Springer, 2010, pp. 1887–1896.
Marchant, R., Sharkey, F.H., Banat, I.M., Rahman, T.J., and Perfumo, A., The degradation of n-hexadecane in soil by thermophilic geobacilli, FEMS Microbiol. Ecol., 2006, vol. 56, pp. 44–54.
Miñana-Galbis, D., Pinzón, D.L., Lorén, J.G., Manresa, A., and Oliart-Ros, R.M., Reclassification of Geobacillus pallidus (Scholz et al. 1988) Banat et al. 2004 as Aeribacillus pallidus gen. nov., comb. nov., Int. J. Syst. Evol. Microbiol., 2010, vol. 60, pp. 1600–1604.
Nazina, T.N., Shumkova, E.S., Sokolova, D.Sh., Babich, T.L., Zhurina, M.V., Xue, Y.-F., Osipov, G.A., Poltaraus, A.B., and Tourova, T.P., Identification of hydrocarbon-oxidizing Dietzia bacteria from petroleum reservoirs based on phenotypic properties and analysis of the 16S rRNA and gyrB genes, Microbiology (Moscow), 2015, vol. 84, no. 3, pp. 377–388.
Nazina, T.N., Sokolova, D.Sh., Grigoryan, A.A., Shestakova, N.M., Mikhailova, E.M., Poltaraus, A.B., Tourova, T.P., Lysenko, A.M., Osipov, G.A., and Belyaev, S.S., Geobacillus jurassicus sp. nov., a new thermophilic bacterium isolated from a high-temperature petroleum reservoir, and the validation of the Geobacillus species, Syst. Appl. Microbiol., 2005, vol. 28, pp. 43–53.
Nazina, T.N., Tourova, T.P., Poltaraus, A.B., Novikova, E.V., Grigoryan, A.A., Ivanova, A.E., Lysenko, A.M., Petrunyaka, V.V., Osipov, G.A., Belyaev, S.S., and Ivanov, M.V., Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans, Int. J. Syst. Evol. Microbiol., 2001, vol. 51, pp. 433–446.
Nie, Y., Chi, C-Q., Fang, H., Liang, J.-L., Lu, S.-L., Lai, G.-L., Tang, Y.-Q., and Wu, X.-L., Diverse alkane hydroxylase genes in microorganisms and environments, Sci. Rep., 2014, vol. 4. Rep. 4968. doi 10.1038/srep04968
Nie, Y., Fang, H., Li, Y., Chi, C.-Q., Tang, Y.-Q., and Wu, X.-L., The genome of the moderate halophile Amycolicicoccus subflavus DQS3-9A1T reveals four alkane hydroxylation systems and provides some clues on the genetic basis for its adaptation to a petroleum environment, PLoS One, 2013, vol. 8. e70986.
Poltaraus, A.B., Sokolova, D.S., Grouzdev, D.S., Ivanov, T.M., Malakho S.G., Korshunova, A.V., Rozanov, A.S., Tourova, T.P., and Nazina, T.N., Draft genome sequence of Aeribacillus pallidus strain 8m3,a thermophilic hydrocarbon-oxidizing bacterium isolated from the Dagang oil field (China), Genome Announc., 2016, vol. 4, no. 3. e00500–16. doi 10.1128/genomeA.00500-16
Sharkey, F.H., Banat, I.M., and Marchant, R., A rapid and effective method of extracting fully intact RNA from thermophilic geobacilli that is suitable for gene expression analysis, Extremophiles, 2004, vol. 8, pp. 73–77.
Shestakova, N.M., Korshunova, A.V., Mikhailova, E.M., Sokolova, D.Sh., Tourova, T.P., Belyaev, S.S., Poltaraus, A.B., and Nazina, T.N., Characterization of the aerobic hydrocarbon-oxidizing enrichments from a hightemperature petroleum reservoir by comparative analysis of DNA- and RNA-derived clone libraries, Microbiology (Moscow), 2011, vol. 80, no. 1, pp. 63–73.
Throne-Holst, M., Wentzel, A., Ellingsen, T., Kotlar, H., and Zotchev, S., Identification of novel genes involved in long-chain n-alkane degradation by Acinetobacter sp. strain DSM17874, Appl. Environ. Microbiol., 2007, vol. 73, pp. 3327–3332.
Tourova, T.P., Nazina, T.N., Mikhailova, E.M., Rodionova, T.A., Ekimov, A.N., Mashukova, A.V., and Poltaraus, A.B., alkB homologs in thermophilic bacteria of the genus Geobacillus, Mol. Biol., 2008, vol. 42, no. 2, pp. 217–226.
Tourova, T.P., Korshunova, A.V., Mikhailova, E.M., Sokolova, D.S., Poltaraus, A.B., and Nazina, T.N., Application of gyrB and parE sequence similarity analyses for differentiation of species within the genus Geobacillus, Microbiology (Moscow), 2010, vol. 79, no. 3, pp. 356–369.
van Beilen, J.B., Funhoff, E.G., van Loon, A., Just, A., Kaysser, L., Bouza, M., Holtackers, R., Röthlisberger, M., Li, Z., and Witholt, B., Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases, Appl. Environ. Microbiol., 2006, vol. 72, no. 1, pp. 59–65.
Van de Peer, Y. and De Wachter, R., TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment, Comput. Appl. Biosci., 1994, vol. 10, pp. 569–570.
Wang, L., Wang, W., Lai, Q., and Shao, Z., Gene diversity of CYP153A and AlkB alkane hydroxylases in oil-degrading bacteria isolated from the Atlantic Ocean, Environ. Microbiol., 2010, vol. 12, no. 5, pp. 1230–1242.
Whyte, L.G., Smits, T.H., Labbe, D., Witholt, B., Greer, C.W., and van Beilen, J.B., Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531, Appl. Environ. Microbiol., 2002, vol. 68, pp. 5933–5942.
Author information
Authors and Affiliations
Corresponding author
Additional information
Original Russian Text © T.P. Tourova, D.Sh. Sokolova, E.M. Semenova, E.S. Shumkova, A.V. Korshunova, T.L. Babich, A.B. Poltaraus, T.N. Nazina, 2016, published in Mikrobiologiya, 2016, Vol. 85, No. 6, pp. 676–692.
Rights and permissions
About this article
Cite this article
Tourova, T.P., Sokolova, D.S., Semenova, E.M. et al. Detection of n-alkane biodegradation genes alkB and ladA in thermophilic hydrocarbon-oxidizing bacteria of the genera Aeribacillus and Geobacillus . Microbiology 85, 693–707 (2016). https://doi.org/10.1134/S0026261716060199
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0026261716060199