Molecular Biology

, 42:217 | Cite as

alkB homologs in thermophilic bacteria of the genus Geobacillus

  • T. P. Tourova
  • T. N. Nazina
  • E. M. Mikhailova
  • T. A. Rodionova
  • A. N. Ekimov
  • A. V. Mashukova
  • A. B. Poltaraus
Genomics. Transcriptomics. Proteomics


Screening for alkane hydroxylase genes (alkB) was performed in thermophilic aerobic bacteria of the genus Geobacillus. Total DNAs were isolated from the biomass of 11 strains grown on a mixture of saturated C10–C20 hydrocarbons. Fragments of alkB genes were amplified by PCR with degenerate oligonucleotide primers, and the PCR products were cloned and sequenced. For the first time, a set of alkB gene homologs was detected in the genomes of thermophilic bacteria. The strains each contained three to six homologs, of which only two were common for all of the strains. Phylogenetic analysis of the nucleotide sequences and the deduced amino acid sequences showed that six of the variants revealed in Geobacillus were closely related to alkB4, alkB3, and alkB2, found in Rhodococcus erythropolis strains NRRL B-16531 and Q15. All variants of alkB sequences were unique. Analysis of the GC composition showed that the Geobacillus alkB homologs are closer to Rhodococcus than to Geobacillus chromosomal DNA. It was assumed that the alkB genes were introduced in the Geobacillus genome via interspecific horizontal transfer and that Rhodococcus or other representatives of Actinobacteria served as donors. Analysis of the codon usage in the fragments of alkB genes confirmed the suggestion that the pool of these genes is common to the majority of Gram-positive and certain Gram-negative bacteria. The formation of a set of several alkB homologs in a genome of a particular microorganism may result from free gene exchange within this pool.

Key words

alkB genes homologs thermophilic bacteria Geobacillus horizontal gene transfer 


  1. 1.
    van Beilen J.B., Li Z., Duetz W.A., Smits T.H.M., Witholt B. 2003. Diversity of alkane hydroxylase systems in the environment. Oil Gas Sci. Technol. 58, 427–440.CrossRefGoogle Scholar
  2. 2.
    Smits T.H., Rothlisberger M., Witholt B., van Beilen J.B. 1999. Molecular screening for alkane hydroxylase genes in Gram-negative and Gram-positive strains. Environ. Microbiol. 1, 307–317.PubMedCrossRefGoogle Scholar
  3. 3.
    Smits T.H., Balada S.B., Witholt B., van Beilen J.B. 2002. Functional analysis of alkane hydroxylases from Gram-negative and Gram-positive bacteria. J. Bacteriol. 184, 1733–1742.PubMedCrossRefGoogle Scholar
  4. 4.
    Andreoni V., Bernasconi S., Colombo M., van Beilen J.B., Cavalca L. 2000. Detection of genes for alkane and naphthalene catabolism in Rhodococcus sp. strain 1BN. Environ. Microbiol. 2, 572–577.PubMedCrossRefGoogle Scholar
  5. 5.
    Vomberg A., Klinner U. 2000. Distribution of alkB genes within n-alkane-degrading bacteria. J. Appl. Microbiol. 89, 339–348.PubMedCrossRefGoogle Scholar
  6. 6.
    van Beilen J.B., Smits T.H., Whyte L.G., et al. 2002. Alkane hydroxylase homologues in Gram-positive strains. Environ. Microbiol. 4, 676–682.PubMedCrossRefGoogle Scholar
  7. 7.
    Whyte L.G., Smits T.H., Labbe D., Witholt B., Greer C.W., van Beilen J.B. 2002. Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531. Appl. Environ. Microbiol. 68, 5933–5942.PubMedCrossRefGoogle Scholar
  8. 8.
    Hara A., Baik S.-H., Syutsubo K., Misawa N., Smits T.H.M., van Beilen J.B., Harayama S. 2004. Cloning and functional analysis of alkB genes in Alcanivorax borkumensis SK2. Environ. Microbiol. 6, 191–197.PubMedCrossRefGoogle Scholar
  9. 9.
    Marchant R., Sharkey F.H., Banat I.M., Rahman T.J., Perfumo A. 2006. The degradation of n-hexadecane in soil by thermophilic geobacilli. FEMS Microbiol. Ecol. 56, 44–54.PubMedCrossRefGoogle Scholar
  10. 10.
    Sharkey F.H., Banat I.M., Marchant R. 2004. A rapid and effective method of extracting fully intact RNA from thermophilic geobacilli that is suitable for gene expression analysis. Extremophiles. 8, 73–77.PubMedCrossRefGoogle Scholar
  11. 11.
    Nazina T.N., Tourova T.P., Poltaraus A.B., et al. 2001. 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. 51, 433–446.PubMedGoogle Scholar
  12. 12.
    Onstott T.C., Phelps T.J., Colwell F.S., et al. 1998. Observations pertaining to the origin and ecology of microorganisms recovered from deep subsurface Taylorsville Basin, Virginia. Geomicrobiol. J. 15, 353–385.CrossRefGoogle Scholar
  13. 13.
    Hao R., Lu A., Wang G. 2004. Crude-oil-degrading thermophilic bacterium isolated from an oil field. Can. J. Microbiol. 50, 175–182.PubMedCrossRefGoogle Scholar
  14. 14.
    Nazina T.N., Sokolova D.Sh., Grigoryan A.A., et al. 2005. 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. 28, 43–53.PubMedCrossRefGoogle Scholar
  15. 15.
    Nazina T.N., Sokolova D.Sh., Shestakova N.M., et al. 2005. The phylogenetic diversity of aerobic organotrophic bacteria from the Dagang high-temperature oil field. Mikrobiologiya. 74, 343–351.Google Scholar
  16. 16.
    Kato T., Haruki M., Imanaka T., Morikawa M., Kanaya S. 2001. Isolation and characterization of long-chain-alkane degrading Bacillus thermoleovorans from deep subterranean petroleum reservoirs. J. Biosci. Bioeng. 91, 64–70.PubMedCrossRefGoogle Scholar
  17. 17.
    Wang L., Tang Y., Wang S., et al. 2006. Isolation and characterization of a novel thermophilic Bacillus strain degrading long-chain n-alkanes. Extremophiles. 10, 347–356.PubMedCrossRefGoogle Scholar
  18. 18.
    Mutzel A., Reinscheid U.M., Antranikian G., Müller R. 1996. Isolation and characterization of a thermophilic bacillus strain that degrades phenol and cresols as sole carbon source at 70°C. Appl. Microbiol. Biotechnol. 46, 593–596.CrossRefGoogle Scholar
  19. 19.
    Annweiler E., Richnow H.H., Antranikian G., et al. 2000. Naphthalene degradation and incorporation of naphthalene-derived carbon into biomass by the thermophilic Bacillus thermoleovorans. Appl. Environ. Microbiol. 66, 518–523.PubMedCrossRefGoogle Scholar
  20. 20.
    Feitkenhauer H., Schnicke S., Müller R., Märkl H. 2003. Kinetic parameters of continuous cultures of Bacillus thermoleovorans sp. A2 degrading phenol at 65°C. J. Biotechnol. 103, 129–135.PubMedCrossRefGoogle Scholar
  21. 21.
    Meintanis C., Chalkou K.I., Kormas K.A., Karagouni A.D. 2006. Biodegradation of crude oil by thermophilic bacteria isolated from a volcano island. Biodegradation. 17, 3–9.CrossRefGoogle Scholar
  22. 22.
    Feng L., Wang W., Cheng J., et al. 2007. Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir. Proc. Natl. Acad. Sci. USA. 104, 5602–5607.PubMedCrossRefGoogle Scholar
  23. 23.
    Thompson J.D., Higgins D.G., Gibson T.J. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 9, 3251–3270.Google Scholar
  24. 24.
    Saitou N., Nei M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.PubMedGoogle Scholar
  25. 25.
    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.PubMedGoogle Scholar
  26. 26.
    Shanklin J., Whittle E., Fox B.G. 1994. Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase. Biochemistry. 33, 12787–12794.PubMedCrossRefGoogle Scholar
  27. 27.
    van Beilen J.B., Smits T.H.M., Roos F.F., Brunner T., Balada S.B., Röthlisberger M., Witholt B. 2005. Identification of an amino acid position that determines the substrate range of integral membrane akane hydroxylases. J. Bacteriol. 187, 85–91.PubMedCrossRefGoogle Scholar
  28. 28.
    Medigue C., Rouxel T., Vigier P., Henaut A., Danchin A. 1991. Evidence for horizontal gene transfer in Escherichia coli speciation. J. Mol. Biol. 222, 851–856.PubMedCrossRefGoogle Scholar
  29. 29.
    Dauga C. 2002. Evolution of the gyrB gene and the molecular phylogeny of Enterobacteriaceae: A model molecule for molecular systematic studies. Int. J. Syst. Evol. Microbiol. 52, 531–547.PubMedGoogle Scholar
  30. 30.
    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.PubMedCrossRefGoogle Scholar
  31. 31.
    Zeigler D.R. 2005. Application of a recN sequence similarity analysis to the identification of species within the bacterial genus Geobacillus. Int. J. Syst. Evol. Microbiol. 55, 1171–1179.PubMedCrossRefGoogle Scholar
  32. 32.
    Musto H., Romero H., Rodriguez-Maseda H. 1998. Heterogeneity in codon usage in the flatworm Schistosoma mansoni. J. Mol. Evol. 46, 159–167.PubMedCrossRefGoogle Scholar
  33. 33.
    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.PubMedCrossRefGoogle Scholar
  34. 34.
    van Beilen J.B., Marin M., Smits T.H.M., Röthlisberger M., Franchini A., Witholt B., Rojo F. 2004. Characterization of two alkane hydroxylase genes from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis. Environ. Microbiol. 6, 264–273.PubMedCrossRefGoogle Scholar
  35. 35.
    Kok M., Oldenhuis R., van der Linden M.P.G., Raatjes P., Kingma J., van Lelyveld P.H., Witholt B. 1989. The Pseudomonas oleovorans alkane hydroxylase gene. Sequence and expression. J. Biol. Chem. 264, 5435–5441.PubMedGoogle Scholar
  36. 36.
    Takami H., Takaki Y., Chee G.-J., Nishi S., Shimamura S., Suzuki H., Matsui S., Uchiyama I. 2004. Thermoadaptation trait revealed by the genome sequence of thermophilic Geobacillus kaustophilus. Nucleic Acids Res. 32, 6292–6303.PubMedCrossRefGoogle Scholar
  37. 37.
    Chakrabarty A.M., Chou G., Gunsalus I.C. 1973. Genetic regulation of octane dissimulation plasmid in Pseudomonas. Proc. Natl. Acad. Sci. USA. 70, 1137–1140.PubMedCrossRefGoogle Scholar
  38. 38.
    van Beilen J.B., Panke S., Lucchini S., Franchini A.G., Röthlisberger M., Witholt B. 2001. Analysis of Pseudomonas putida alkane degradation gene clusters and flanking insertion sequences: Evolution and regulation of the Alk genes. Microbiology. 147, 1621–1630.PubMedGoogle Scholar
  39. 39.
    Marin M.M., Smits T.H.M., van Beilen J.B., Rojo F. 2001. The alkane hydroxylase gene of Burkholderia cepacia RR10 is under catabolite repression control. J. Bacteriol. 183, 4202–4209.PubMedCrossRefGoogle Scholar

Copyright information

© MAIK Nauka 2008

Authors and Affiliations

  • T. P. Tourova
    • 1
  • T. N. Nazina
    • 1
  • E. M. Mikhailova
    • 1
  • T. A. Rodionova
    • 2
  • A. N. Ekimov
    • 2
  • A. V. Mashukova
    • 3
  • A. B. Poltaraus
    • 2
  1. 1.Winogradsky Institute of MicrobiologyRussian Academy of SciencesMoscowRussia
  2. 2.Engelhardt Institute of Molecular BiologyRussian Academy of SciencesMoscowRussia
  3. 3.Faculty of Bioengineering and BioinformaticsMoscow State UniversityMoscowRussia

Personalised recommendations