Microaerophilic alkane degradation in Pseudomonas extremaustralis: a transcriptomic and physiological approach

  • Paula M. Tribelli
  • Leticia Rossi
  • Martiniano M. Ricardi
  • Maria Gomez-Lozano
  • Søren Molin
  • Laura J. Raiger IustmanEmail author
  • Nancy I. Lopez
Environmental Microbiology - Original Paper


Diesel fuel is one of the most important sources of hydrocarbon contamination worldwide. Its composition consists of a complex mixture of n-alkanes, branched alkanes and aromatic compounds. Hydrocarbon degradation in Pseudomonas species has been mostly studied under aerobic conditions; however, a dynamic spectrum of oxygen availability can be found in the environment. Pseudomonas extremaustralis, an Antarctic bacterium isolated from a pristine environment, is able to degrade diesel fuel and presents a wide microaerophilic metabolism. In this work RNA-deep sequence experiments were analyzed comparing the expression profile in aerobic and microaerophilic cultures. Interestingly, genes involved in alkane degradation, including alkB, were over-expressed in micro-aerobiosis in absence of hydrocarbon compounds. In minimal media supplemented with diesel fuel, n-alkanes degradation (C13–C19) after 7 days was observed under low oxygen conditions but not in aerobiosis. In-silico analysis of the alkB promoter zone showed a putative binding sequence for the anaerobic global regulator, Anr. Our results indicate that some diesel fuel components can be utilized as sole carbon source under microaerophilic conditions for cell maintenance or slow growth in a Pseudomonas species and this metabolism could represent an adaptive advantage in polluted environments.


Pseudomonas extremaustralis Micro-aerobiosis alkB RNA-seq Alkane degradation 



This work was partially supported by grants from UBA, CONICET, and ANPCyT. PMT, MMR, LJRI and NIL are career investigators from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina). RNA-seq experiments were performed by PMT at Dr. Molin’s Lab supported by a short term EMBO fellowship. Authors want to thank to anonymous reviewers for the helpful criticisms.

Supplementary material

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Supplementary material 1 (PDF 1013 kb)


  1. 1.
    Alonso H, Kleifeld O, Yeheskel A, Ong PC, Liu YC, Stok JE, De Voss JJ, Roujeinikova A (2014) Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB. Biochem J 460:283–293. CrossRefPubMedGoogle Scholar
  2. 2.
    Alvarez-Ortega C, Harwood CS (2007) Responses of Pseudomonas aeruginosa to low oxygen indicate that growth in the cystic fibrosis lung is by aerobic respiration. Mol Microbiol 65:153–165. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Ayub ND, Tribelli PM, López NI (2009) Polyhydroxyalkanoates are essential for maintenance of redox state in the Antarctic bacterium Pseudomonas sp. 14-3 during low temperature adaptation. Extremophiles 13:59–66. CrossRefPubMedGoogle Scholar
  4. 4.
    Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST server: rapid annotations using subsystems technology. BMC Genom 9:75. CrossRefGoogle Scholar
  5. 5.
    van Beilen JB, Funhoff EG (2007) Alkane hydroxylases involved in microbial alkane degradation. Appl Microbiol Biotechnol 74:13–21. CrossRefPubMedGoogle Scholar
  6. 6.
    van Beilen JB, Panke S, Lucchini S, Franchini AG, 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. CrossRefPubMedGoogle Scholar
  7. 7.
    Berthe-Corti L, Fetzner S (2002) Bacterial metabolism of n-alkanes and ammonia under oxic, suboxic and anoxic conditions. Acta Biotechnol 22:299–336CrossRefGoogle Scholar
  8. 8.
    Callaghan AV (2013) Enzymes involved in the anaerobic oxidation of n-alkanes: from methane to long-chain paraffins. Front Microbiol 4:89CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Chayabutra C, Ju LK (2000) Degradation of n-hexadecane and its metabolites by Pseudomonas aeruginosa under microaerobic and anaerobic denitrifying conditions. Appl Environ Microbiol 66:493–498. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Conesa A, Götz S (2008) Blast2GO: a comprehensive suite for functional analysis in plant genomics. Int J Plant Genom. Google Scholar
  11. 11.
    Dinamarca MA, Aranda-Olmedo I, Puyet A, Rojo F (2003) Expression of the Pseudomonas putida OCT plasmid alkane degradation pathway is modulated by two different global control signals: evidence from continuous cultures. J Bacteriol 185:4772–4778. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Eschbach M, Schreiber K, Trunk K, Buer J, Jahn D, Schobert M (2004) Long-term anaerobic survival of the opportunistic pathogen Pseudomonas aeruginosa via pyruvate fermentation. J Bacteriol 186:4596–4604. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Fisher R (1934) Statistical methods for research workers. Oliver and Boyd, EdinburghGoogle Scholar
  14. 14.
    Fonseca P, Moreno R, Rojo F (2011) Growth of Pseudomonas putida at low temperature: global transcriptomic and proteomic analyses. Environ Microbiol Rep 3:329–339. CrossRefPubMedGoogle Scholar
  15. 15.
    Gómez-Lozano M, Marvig RL, Molin S, Long KS (2012) Genome-wide identification of novel small RNAs in Pseudomonas aeruginosa. Environ Microbiol 14:2006–2016. CrossRefPubMedGoogle Scholar
  16. 16.
    Gunasekera TS, Striebich RC, Mueller SS, Strobel EM, Ruiz ON (2013) Transcriptional profiling suggests that multiple metabolic adaptations are required for effective proliferation of Pseudomonas aeruginosa in jet fuel. Environ Sci Technol 47:13449–13458. CrossRefPubMedGoogle Scholar
  17. 17.
    Hernández-Arranz S, Moreno R, Rojo F (2013) The translational repressor Crc controls the Pseudomonas putida benzoate and alkane catabolic pathways using a multi-tier regulation strategy. Environ Microbiol 15:227–241. CrossRefPubMedGoogle Scholar
  18. 18.
    Kok M, Oldenhuis R, Van Der Linden MPG, Raatjes P, Kingma J, Van Lelyveld PH, Witholt B (1989) The Pseudomonas oleovorans alkane hydroxylase gene. Sequence and expression. J Biol Chem 264:5435–5441PubMedGoogle Scholar
  19. 19.
    Lageveen RG, Huisman GW, Preusting H, Ketelaar P, Eggink G, Witholt B (1988) Formation of polyesters by Pseudomonas oleovorans: effect of substrates on formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates. Appl Environ Microbiol 54:2924–2932PubMedPubMedCentralGoogle Scholar
  20. 20.
    Larionov A, Krause A, Miller W (2005) A standard curve based method for relative real time PCR data processing. BMC Bioinform 6:62. CrossRefGoogle Scholar
  21. 21.
    Liu H, Liang R, Tao F, Ma C, Liu Y, Liu X, Liu J (2012) Genome sequence of Pseudomonas aeruginosa strain SJTD-1, a bacterium capable of degrading long-chain alkanes and crude oil. J Bacteriol 194:4783–4784. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Liu H, Sun W-B, Liang R-B, Huang L, Hou J-L, Liu J-H (2015) iTRAQ-based quantitative proteomic analysis of Pseudomonas aeruginosa SJTD-1: a global response to n-octadecane induced stress. J Proteom 123:14–28. CrossRefGoogle Scholar
  23. 23.
    López NI, Pettinari MJ, Stackebrandt E, Tribelli PM, Põtter M, Steinbüchel A, Méndez BS (2009) Pseudomonas extremaustralis sp. nov., a poly(3-hydroxybutyrate) producer isolated from an antarctic environment. Curr Microbiol 59:514–519. CrossRefPubMedGoogle Scholar
  24. 24.
    Mann HB, Whitney DR (1947) On a test of whether one of two random variables is stochastically larger than the other. Ann Math Stat 18(1):50–60CrossRefGoogle Scholar
  25. 25.
    Martínez-Antonio A, Collado-Vides J (2003) Identifying global regulators in transcriptional regulatory networks in bacteria. Curr Opin Microbiol 6:482–489. CrossRefPubMedGoogle Scholar
  26. 26.
    Mason OU, Hazen TC, Borglin S, Chain PS, Dubinsky EA, Fortney JL, Han J, Holman HY, Hultman J, Lamendella R, Mackelprang R, Malfatti S, Tom LM, Tringe SG, Woyke T, Zhou J, Rubin EM, Jansson JK (2012) Metagenome, metatranscriptome and single-cell sequencing reveal microbial response to deepwater horizon oil spill. ISME J 6:1715–1727. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    McClure R, Balasubramanian D, Sun Y, Bobrovskyy M, Sumby P, Genco CA, Vanderpool CK, Tjaden B (2013) Computational analysis of bacterial RNA-Seq data. Nucleic Acids Res 41:e140. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Münch R, Hiller K, Grote A, Scheer M, Klein J, Schobert M, Jahn D (2005) Virtual footprint and PRODORIC: an integrative framework for regulon prediction in prokaryotes. Bioinformatics 21:4187–4189. CrossRefPubMedGoogle Scholar
  29. 29.
    Rodriguez-R LM, Overholt WA, Hagan C, Huettel M, Kostka JE, Konstantinidis KT (2015) Microbial community successional patterns in beach sands impacted by the deepwater horizon oil spill. ISME J 9:1928–1940. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Rojo F (2005) Specificity at the end of the tunnel: understanding substrate length discrimination by the AlkB Alkane hydroxylase. J Bacteriol 187:19–22. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Rojo F (2009) Degradation of alkanes by bacteria: minireview. Environ Microbiol 11:2477–2490. CrossRefPubMedGoogle Scholar
  32. 32.
    Sawers RG (1991) Identification and molecular characterization of a transcriptional regulator from Pseudomonas aeruginosa PAO1 exhibiting structural and functional similarity to the FNR protein of Escherichia coli. Mol Microbiol 5:1469–1481CrossRefPubMedGoogle Scholar
  33. 33.
    Schirmer A, Rude MA, Li X, Popova E, Del Cardayre SB (2010) Microbial biosynthesis of alkanes. Science 329:559–562. CrossRefPubMedGoogle Scholar
  34. 34.
    Smits THM, Witholt B, van Beilen JB (2003) Functional characterization of genes involved in alkane oxidation by Pseudomonas aeruginosa. Antonie Van Leeuwenhoek 84:193–200. CrossRefPubMedGoogle Scholar
  35. 35.
    Tribelli PM, Hay AG, López NI (2013) The global anaerobic regulator Anr, is involved in cell attachment and aggregation influencing the first stages of biofilm development in Pseudomonas extremaustralis. PLoS One 8:e76685. CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Tribelli PM, Di Martino C, López NI, Raiger Iustman LJ (2012) Biofilm lifestyle enhances diesel bioremediation and biosurfactant production in the Antarctic polyhydroxyalkanoate producer Pseudomonas extremaustralis. Biodegradation 23:645–651. CrossRefPubMedGoogle Scholar
  37. 37.
    Tribelli PM, Méndez BS, López NI (2010) Oxygen-sensitive global regulator, anr, is involved in the biosynthesis of poly(3-Hydroxybutyrate) in Pseudomonas extremaustralis. J Mol Microbiol Biotechnol 19:180–188. CrossRefPubMedGoogle Scholar
  38. 38.
    Tribelli PM, Nikel PI, Oppezzo OJ, López NI (2013) Anr, the anaerobic global regulator, modulates the redox state and oxidative stress resistance in Pseudomonas extremaustralis. Microbiology 159:259–268. CrossRefPubMedGoogle Scholar
  39. 39.
    Tribelli PM, Venero ECS, Ricardi MM, Gómez-Lozano M, Raiger Iustman LJ, Molin S, López NI (2015) Novel essential role of ethanol oxidation genes at low temperature revealed by transcriptome analysis in the antarctic bacterium Pseudomonas extremaustralis. PLoS One 10:e0145353. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Trunk K, Benkert B, Quäck N, Münch R, Scheer M, Garbe J, Jänsch L, Trost M, Wehland J, Buer J, Jahn M, Schobert M, Jahn D (2010) Anaerobic adaptation in Pseudomonas aeruginosa: definition of the Anr and Dnr regulons. Environ Microbiol 12:1719–1733. CrossRefPubMedGoogle Scholar
  41. 41.
    Ugidos A, Morales G, Rial E, Williams HD, Rojo F (2008) The coordinate regulation of multiple terminal oxidases by the Pseudomonas putida ANR global regulator. Environ Microbiol 10:1690–1702. CrossRefPubMedGoogle Scholar
  42. 42.
    Wentzel A, Ellingsen TE, Kotlar HK, Zotchev SB, Throne-Holst M (2007) Bacterial metabolism of long-chain n-alkanes. Appl Microbiol Biotechnol 76:1209–1221. CrossRefPubMedGoogle Scholar
  43. 43.
    Yamada T, Letunic I, Okuda S, Kanehisa M, Bork P (2011) IPath2.0: interactive pathway explorer. Nucleic Acids Res 39:W412–W415. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2017

Authors and Affiliations

  • Paula M. Tribelli
    • 1
    • 2
  • Leticia Rossi
    • 2
  • Martiniano M. Ricardi
    • 3
  • Maria Gomez-Lozano
    • 4
  • Søren Molin
    • 4
  • Laura J. Raiger Iustman
    • 1
    • 2
    Email author
  • Nancy I. Lopez
    • 1
    • 2
  1. 1.Departamento de Química Biológica, Facultad de Ciencias Exactas y NaturalesUniversidad de Buenos AiresBuenos AiresArgentina
  2. 2.IQUIBICEN, CONICETBuenos AiresArgentina
  3. 3.Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE-CONICET), Facultad de Ciencias Exactas y NaturalesUniversidad de Buenos AiresBuenos AiresArgentina
  4. 4.Novo Nordisk Foundation Center for BiosustainabilityTechnical University of DenmarkHørsholmDenmark

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