Advertisement

Applied Microbiology and Biotechnology

, Volume 97, Issue 13, pp 5779–5791 | Cite as

Expression of genes involved in rhamnolipid synthesis in Pseudomonas aeruginosa PAO1 in a bioreactor cultivation

  • Anke SchmidbergerEmail author
  • Marius Henkel
  • Rudolf Hausmann
  • Thomas SchwartzEmail author
Biotechnological products and process engineering

Abstract

There is a growing demand for economic bioprocesses based on sustainable resources rather than petrochemical-derived substances. Particular attention has been paid to rhamnolipids—surface-active glycolipids—that are naturally produced by Pseudomonas aeruginosa. Rhamnolipids have gained increased attention over the past years due to their versatile chemical and biological properties as well as numerous biotechnological applications. However, rhamnolipid synthesis is tightly governed by a complex growth-dependent regulatory network. Quantitative comprehension of the molecular and metabolic mechanisms during bioprocesses is key to manipulating and improving rhamnolipid production capacities in P. aeruginosa. In this study, P. aeruginosa PAO1 was grown under nitrogen limitation with sunflower oil as carbon and nitrate as nitrogen source in a batch fermentation process. Gene expression was monitored using quantitative PCR over the entire time course. Until late deceleration phase, an increase in relative gene expression of the las, rhl, and pqs quorum-sensing regulons was observed. Thereafter, expression of the rhamnolipid synthesis genes, rhlA and rhlC, as well as the las regulon was downregulated. RhlR was shown to remain upregulated at the late phase of the fermentation process.

Keywords

Pseudomonas aeruginosa PAO1 Rhamnolipid Biosurfactant Gene expression Batch cultivation Quorum sensing 

Notes

Acknowledgments

This work was financed by the Baden-Württemberg Stiftung as part of the Environmental Technology Research Programme as well as the Karlsruhe Institute of Technology (KIT).

References

  1. Abdel-Mawgoud AM, Lepine F, Deziel E (2010) Rhamnolipids: diversity of structures, microbial origins and roles. Appl Microbiol Biotechnol 86(5):1323–1336CrossRefGoogle Scholar
  2. Banat IM, Makkar RS, Cameotra SS (2000) Potential commercial applications of microbial surfactants. Appl Microbiol Biotechnol 53(5):495–508CrossRefGoogle Scholar
  3. Bazire A, Diab F, Taupin L, Rodrigues S, Jebbar M, Dufour A (2009) Effects of osmotic stress on rhamnolipid synthesis and time-course production of cell-to-cell signal molecules by Pseudomonas aeruginosa. Open Microbiol J 3:128–135CrossRefGoogle Scholar
  4. Bertani I, Sevo M, Kojic M, Venturi V (2003) Role of GacA, LasI, RhlI, Ppk, PsrA, Vfr and ClpXP in the regulation of the stationary-phase sigma factor rpoS/RpoS in Pseudomonas. Arch Microbiol 180(4):264–271CrossRefGoogle Scholar
  5. Cabrera-Valladares N, Richardson AP, Olvera C, Trevino LG, Deziel E, Lepine F, Soberon-Chavez G (2006) Monorhamnolipids and 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs) production using Escherichia coli as a heterologous host. Appl Microbiol Biotechnol 73(1):187–194CrossRefGoogle Scholar
  6. Cha M, Lee N, Kim M, Lee S (2008) Heterologous production of Pseudomonas aeruginosa EMS1 biosurfactant in Pseudomonas putida. Bioresour Technol 99(7):2192–2199CrossRefGoogle Scholar
  7. Croda-Garcia G, Grosso-Becerra V, Gonzalez-Valdez A, Servin-Gonzalez L, Soberon-Chavez G (2011) Transcriptional regulation of Pseudomonas aeruginosa rhlR: role of the CRP orthologue Vfr (virulence factor regulator) and quorum-sensing regulators LasR and RhlR. Microbiology 157(Pt 9):2545–2555CrossRefGoogle Scholar
  8. de Kievit TR, Iglewski BH (2000) Bacterial quorum sensing in pathogenic relationships. Infect Immun 68(9):4839–4849CrossRefGoogle Scholar
  9. Deziel E, Lepine F, Milot S, Villemur R (2003) RhlA is required for the production of a novel biosurfactant promoting swarming motility in Pseudomonas aeruginosa: 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), the precursors of rhamnolipids. Microbiology 149(Pt 8):2005–2013CrossRefGoogle Scholar
  10. Goni R, García P, Foissac S (2009) The qPCR data statistical analysis, Integromics White PaperGoogle Scholar
  11. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8(2):R19CrossRefGoogle Scholar
  12. Henkel M, Schmidberger A, Kühnert C, Beuker J, Bernard T, Schwartz T, Syldatk C, Hausmann R (2013) Kinetic modeling of N-butyryl-homoserine lactone in a batch cultivation of Pseudomonas aeruginosa PAO1. Appl Microbiol Biot (in press)Google Scholar
  13. Heurlier K, Denervaud V, Pessi G, Reimmann C, Haas D (2003) Negative control of quorum sensing by RpoN (sigma54) in Pseudomonas aeruginosa PAO1. J Bacteriol 185(7):2227–2235CrossRefGoogle Scholar
  14. Heurlier K, Williams F, Heeb S, Dormond C, Pessi G, Singer D, Camara M, Williams P, Haas D (2004) Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1. J Bacteriol 186(10):2936–2945CrossRefGoogle Scholar
  15. Hong KW, Koh CL, Sam CK, Yin WF, Chan KG (2012) Quorum quenching revisited—from signal decays to signalling confusion. Sensors 12(4):4661–4696CrossRefGoogle Scholar
  16. Huang JJ, Han JI, Zhang LH, Leadbetter JR (2003) Utilization of acyl-homoserine lactone quorum signals for growth by a soil pseudomonad and Pseudomonas aeruginosa PAO1. Appl Environ Microbiol 69(10):5941–5949CrossRefGoogle Scholar
  17. Huang JJ, Petersen A, Whiteley M, Leadbetter JR (2006) Identification of QuiP, the product of gene PA1032, as the second acyl-homoserine lactone acylase of Pseudomonas aeruginosa PAO1. Appl Environ Microbiol 72(2):1190–1197CrossRefGoogle Scholar
  18. Jarvis FG, Johnson MJ (1949) A glycolipid produced by Pseudomonas aeruginosa. J Am Chem Soc 71(12):4124–4126CrossRefGoogle Scholar
  19. Kayama S, Murakami K, Ono T, Ushimaru M, Yamamoto A, Hirota K, Miyake Y (2009) The role of rpoS gene and quorum-sensing system in ofloxacin tolerance in Pseudomonas aeruginosa. FEMS Microbiol Lett 298(2):184–192CrossRefGoogle Scholar
  20. Lamont IL, Martin LW (2003) Identification and characterization of novel pyoverdine synthesis genes in Pseudomonas aeruginosa. Microbiology 149(Pt 4):833–842CrossRefGoogle Scholar
  21. Lee KM, Yoon MY, Park Y, Lee JH, Yoon SS (2011) Anaerobiosis-induced loss of cytotoxicity is due to inactivation of quorum sensing in Pseudomonas aeruginosa. Infect Immun 79(7):2792–2800CrossRefGoogle Scholar
  22. Lin YH, Xu JL, Hu J, Wang LH, Ong SL, Leadbetter JR, Zhang LH (2003) Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes. Mol Microbiol 47(3):849–860CrossRefGoogle Scholar
  23. Medina G, Juarez K, Diaz R, Soberon-Chavez G (2003a) Transcriptional regulation of Pseudomonas aeruginosa rhlR, encoding a quorum-sensing regulatory protein. Microbiology 149(Pt 11):3073–3081CrossRefGoogle Scholar
  24. Medina G, Juarez K, Soberon-Chavez G (2003b) The Pseudomonas aeruginosa rhlAB operon is not expressed during the logarithmic phase of growth even in the presence of its activator RhlR and the autoinducer N-butyryl-homoserine lactone. J Bacteriol 185(1):377–380CrossRefGoogle Scholar
  25. Medina G, Juarez K, Valderrama B, Soberon-Chavez G (2003c) Mechanism of Pseudomonas aeruginosa RhlR transcriptional regulation of the rhlAB promoter. J Bacteriol 185(20):5976–5983CrossRefGoogle Scholar
  26. Morici LA, Carterson AJ, Wagner VE, Frisk A, Schurr JR, Honer zu Bentrup K, Hassett DJ, Iglewski BH, Sauer K, Schurr MJ (2007) Pseudomonas aeruginosa AlgR represses the Rhl quorum-sensing system in a biofilm-specific manner. J Bacteriol 189(21):7752–7764CrossRefGoogle Scholar
  27. Müller MM, Hörmann B, Syldatk C, Hausmann R (2010) Pseudomonas aeruginosa PAO1 as a model for rhamnolipid production in bioreactor systems. Appl Microbiol Biotechnol 87(1):167–174CrossRefGoogle Scholar
  28. Müller MM, Hörmann B, Kugel M, Syldatk C, Hausmann R (2011) Evaluation of rhamnolipid production capacity of Pseudomonas aeruginosa PAO1 in comparison to the rhamnolipid over-producer strains DSM 7108 and DSM 2874. Appl Microbiol Biotechnol 89(3):585–592CrossRefGoogle Scholar
  29. Nadal Jimenez P, Koch G, Papaioannou E, Wahjudi M, Krzeslak J, Coenye T, Cool RH, Quax WJ (2010) Role of PvdQ in Pseudomonas aeruginosa virulence under iron-limiting conditions. Microbiology 156(Pt 1):49–59CrossRefGoogle Scholar
  30. Neilson JW, Zhang L, Veres-Schalnat TA, Chandler KB, Neilson CH, Crispin JD, Pemberton JE, Maier RM (2010) Cadmium effects on transcriptional expression of rhlB/rhlC genes and congener distribution of monorhamnolipid and dirhamnolipid in Pseudomonas aeruginosa IGB83. Appl Microbiol Biotechnol 88(4):953–963CrossRefGoogle Scholar
  31. Ochsner UA, Wilderman PJ, Vasil AI, Vasil ML (2002) GeneChip expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes. Mol Microbiol 45(5):1277–1287CrossRefGoogle Scholar
  32. Oinuma K, Greenberg EP (2011) Acyl-homoserine lactone binding to and stability of the orphan Pseudomonas aeruginosa quorum-sensing signal receptor QscR. J Bacteriol 193(2):421–428CrossRefGoogle Scholar
  33. Pessi G, Williams F, Hindle Z, Heurlier K, Holden MT, Camara M, Haas D, Williams P (2001) The global posttranscriptional regulator RsmA modulates production of virulence determinants and N-acylhomoserine lactones in Pseudomonas aeruginosa. J Bacteriol 183(22):6676–6683CrossRefGoogle Scholar
  34. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45CrossRefGoogle Scholar
  35. Potvin E, Sanschagrin F, Levesque RC (2008) Sigma factors in Pseudomonas aeruginosa. FEMS Microbiol Rev 32(1):38–55CrossRefGoogle Scholar
  36. Rahim R, Ochsner UA, Olvera C, Graninger M, Messner P, Lam JS, Soberon-Chavez G (2001) Cloning and functional characterization of the Pseudomonas aeruginosa rhlC gene that encodes rhamnosyltransferase 2, an enzyme responsible for di-rhamnolipid biosynthesis. Mol Microbiol 40(3):708–718CrossRefGoogle Scholar
  37. Rahman P, Dusane D, Zinjarde S, Venugopalan V, McLean R, Weber M (2010) Quorum sensing: implications on rhamnolipid biosurfactant production. Biotechnol Genet Eng Rev 27:159–184CrossRefGoogle Scholar
  38. Rampioni G, Bertani I, Zennaro E, Polticelli F, Venturi V, Leoni L (2006) The quorum-sensing negative regulator RsaL of Pseudomonas aeruginosa binds to the lasI promoter. J Bacteriol 188(2):815–819CrossRefGoogle Scholar
  39. Rampioni G, Schuster M, Greenberg EP, Bertani I, Grasso M, Venturi V, Zennaro E, Leoni L (2007) RsaL provides quorum sensing homeostasis and functions as a global regulator of gene expression in Pseudomonas aeruginosa. Mol Microbiol 66(6):1557–1565CrossRefGoogle Scholar
  40. Reis RS, Pereira AG, Neves BC, Freire DM (2011) Gene regulation of rhamnolipid production in Pseudomonas aeruginosa—a review. Bioresource Technol 102(11):6377–6384CrossRefGoogle Scholar
  41. Rosenau F, Isenhardt S, Gdynia A, Tielker D, Schmidt E, Tielen P, Schobert M, Jahn D, Wilhelm S, Jaeger KE (2010) Lipase LipC affects motility, biofilm formation and rhamnolipid production in Pseudomonas aeruginosa. FEMS Microbiol Lett 309(1):25–34Google Scholar
  42. Savli H, Karadenizli A, Kolayli F, Gundes S, Ozbek U, Vahaboglu H (2003) Expression stability of six housekeeping genes: a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J Med Microbiol 52(Pt 5):403–408CrossRefGoogle Scholar
  43. Schuster M, Greenberg EP (2006) A network of networks: quorum-sensing gene regulation in Pseudomonas aeruginosa. Int J Med Microbiol 296(2–3):73–81CrossRefGoogle Scholar
  44. Schuster M, Greenberg EP (2007) Early activation of quorum sensing in Pseudomonas aeruginosa reveals the architecture of a complex regulon. BMC Genomics 8:287CrossRefGoogle Scholar
  45. Schwartz T, Walter S, Marten SM, Kirschhofer F, Nusser M, Obst U (2007) Use of quantitative real-time RT-PCR to analyse the expression of some quorum-sensing regulated genes in Pseudomonas aeruginosa. Anal Bioanal Chem 387(2):513–521CrossRefGoogle Scholar
  46. Sio CF, Otten LG, Cool RH, Diggle SP, Braun PG, Bos R, Daykin M, Camara M, Williams P, Quax WJ (2006) Quorum quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1. Infect Immun 74(3):1673–1682CrossRefGoogle Scholar
  47. Soberon-Chavez G, Lepine F, Deziel E (2005) Production of rhamnolipids by Pseudomonas aeruginosa. Appl Microbiol Biotechnol 68(6):718–725CrossRefGoogle Scholar
  48. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK, Wu Z, Paulsen IT, Reizer J, Saier MH, Hancock RE, Lory S, Olson MV (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406(6799):959–964CrossRefGoogle Scholar
  49. Sullivan ER (1998) Molecular genetics of biosurfactant production. Curr Opin Biotechnol 9(3):263–269CrossRefGoogle Scholar
  50. Viducic D, Ono T, Murakami K, Katakami M, Susilowati H, Miyake Y (2007) rpoN gene of Pseudomonas aeruginosa alters its susceptibility to quinolones and carbapenems. Antimicrob Agents Chemother 51(4):1455–1462CrossRefGoogle Scholar
  51. Wahjudi M, Papaioannou E, Hendrawati O, van Assen AH, van Merkerk R, Cool RH, Poelarends GJ, Quax WJ (2011) PA0305 of Pseudomonas aeruginosa is a quorum quenching acylhomoserine lactone acylase belonging to the Ntn hydrolase superfamily. Microbiology 157(Pt 7):2042–2055CrossRefGoogle Scholar
  52. Waters CM, Bassler BL (2005) Quorum sensing: cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol 21:319–346CrossRefGoogle Scholar
  53. Winsor GL, Van Rossum T, Lo R, Khaira B, Whiteside MD, Hancock RE, Brinkman FS (2009) Pseudomonas genome database: facilitating user-friendly, comprehensive comparisons of microbial genomes. Nucleic Acids Res 37(Database issue):483–488CrossRefGoogle Scholar
  54. Wittgens A, Tiso T, Arndt TT, Wenk P, Hemmerich J, Müller C, Wichmann R, Kupper B, Zwick M, Wilhelm S, Hausmann R, Syldatk C, Rosenau F, Blank LM (2011) Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440. Microb Cell Fact 10:80CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Institute of Functional Interfaces, Department of Interface MicrobiologyKarlsruhe Institute of Technology (KIT)Eggenstein-LeopoldshafenGermany
  2. 2.Institute of Process Engineering in Life Sciences, Section Technical BiologyKarlsruhe Institute of Technology (KIT)KarlsruheGermany
  3. 3.Institute of Food Science and Biotechnology, Bioprocess EngineeringUniversity of HohenheimStuttgartGermany

Personalised recommendations