Applied Microbiology and Biotechnology

, Volume 100, Issue 23, pp 9995–10004 | Cite as

Pseudomonas aeruginosa ATCC 9027 is a non-virulent strain suitable for mono-rhamnolipids production

  • María-Victoria Grosso-Becerra
  • Abigail González-Valdez
  • María-Jessica Granados-Martínez
  • Estefanía Morales
  • Luis Servín-González
  • José-Luis Méndez
  • Gabriela Delgado
  • Rosario Morales-Espinosa
  • Gabriel-Yaxal Ponce-Soto
  • Miguel Cocotl-Yañez
  • Gloria Soberón-ChávezEmail author
Applied genetics and molecular biotechnology


Rhamnolipids produced by Pseudomonas aeruginosa are biosurfactants with a high biotechnological potential, but their extensive commercialization is limited by the potential virulence of P. aeruginosa and by restrictions in producing these surfactants in heterologous hosts. In this work, we report the characterization of P. aeruginosa strain ATCC 9027 in terms of its genome-sequence, virulence, antibiotic resistance, and its ability to produce mono-rhamnolipids when carrying plasmids with different cloned genes from the type strain PAO1. The genes that were expressed from the plasmids are those coding for enzymes involved in the synthesis of this biosurfactant (rhlA and rhlB), as well as the gene that codes for the RhlR transcriptional regulator. We confirm that strain ATCC 9027 forms part of the PA7 clade, but contrary to strain PA7, it is sensitive to antibiotics and is completely avirulent in a mouse model. We also report that strain ATCC 9027 mono-rhamnolipid synthesis is limited by the expression of the rhlAB-R operon. Thus, this strain carrying the rhlAB-R operon produces similar rhamnolipids levels as PAO1 strain. We determined that strain ATCC 9027 with rhlAB-R operon was not virulent to mice. These results show that strain ATCC 9027, expressing PAO1 rhlAB-R operon, has a high biotechnological potential for industrial mono-rhamnolipid production.


Metabolic engineering Biosurfactant production Pseudomonas aeruginosa virulence Quorum-sensing response 



We acknowledge the support in providing the radioactive material and the use of laboratory facilities of Guadalupe Espín of the Instituto de Biotecnología, Universidad Nacional Autónoma de México.

Compliance with ethical standards


This work was supported in part by grant PAPIIT IN200416 (DGAPA-UNAM) and from a grant from Fundación Miguel Alemán for the project “Análisis genómico de cepas de Pseudomonas aeruginosa que presentan una respuesta de detección de quórum atípica.”

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. In particular, all mouse studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Committee for the Update of the Guide for the Care and Use of Laboratory Animals and Institute for Laboratory Animal Research, Washington, DC, 2011) and the Comité para el Cuidado y Uso de Animales de Laboratorio (CCUAL) and were approved by the ethics committee of Instituto de Investigaciones Biomédicas—UNAM (approval No. ID201 09/02-2010).

Supplementary material

253_2016_7789_MOESM1_ESM.pdf (5.2 mb)
ESM 1 (PDF 5298 kb)


  1. Abdel-Maugoud AM, Lépine F, Déziel E (2010) Rhamnolipids diversity, structures, microbial origin and roles. Appl Microbiol Biotechnol 86:1323–1336CrossRefGoogle Scholar
  2. Abdel-Maugoud AM, Hausmann R, Lépine F, Muller MM, Déziel E (2011) Rhamnolipids: detection, analysis, biosynthesis, genetic regulation and bioengineering of production. In: Soberón-Chávez G (ed) Biosurfactants: from genes to applications, Microbiol Monographs, vol 20. Springer, Berlin Heilderberg, pp. 13–55CrossRefGoogle Scholar
  3. Abdel-Maugoud AM, Lépine F, Déziel E (2014) A stereospecific pathway diverts β-oxidation intermediates to the biosynthesis of rhamnolipid biosurfactants. Chem Biol 21(1):156–164CrossRefGoogle Scholar
  4. Aguirre-Ramírez M, Medina G, González-Valdez A, Grosso-Becerra V, Soberón-Chávez G (2012) Pseudomonas aeruginosa rmlBDAC operon, encoding dTDP-L-rhamnose biosynthetic enzymes, is regulated by the quorum-sensing transcriptional regulator RhlR and the alternative sigma S factor. Microbiol-UK 158:908–916CrossRefGoogle Scholar
  5. Angiuoli SV, Salzberg SL (2011) Mugsy: fast multiple alignment of closely related whole genomes. Bioinformatics 27(3):334–342CrossRefPubMedGoogle Scholar
  6. Arino S, Marchal R, Vandecasteele JP (1996) Identification and production of a rhamnolipidic biosurfactant by a Pseudomonas species. Appl Microbiol Biotechnol 45:162–168CrossRefGoogle Scholar
  7. 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-Genomics 9:75CrossRefPubMedPubMedCentralGoogle Scholar
  8. Beatson SA, Whitchurch CB, Sargent JL, Levesque RC, Mattick JS (2002) Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa. J Bacteriol 184:3605–3613CrossRefPubMedPubMedCentralGoogle Scholar
  9. Boukerb AM, Marti R, Cournoyer B (2015) Genome sequences of three strains of the Pseudomonas aeruginosa PA7 clade. Genom Announc 3:e01366–e01315. doi: 10.1128/genomeA.01366-15 CrossRefGoogle Scholar
  10. Boukerb AM, Decor A, Ribun S, Tabaroni R, Rousset A, Commin L, Buff S, Doléans-Jordheim A, Vidal S, Varrot A, Inverty A, Cournoyer B (2016) Genomic rearrangements and functional diversification of lecA and lecB lectin-coding regions impacting the efficacy of glycomimetics directed against Pseudomonas aeruginosa. Front Microbiol 7:811. doi: 10.3389/fmicb.2016.00811 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Cabrera-Valladares N, Richardson A-P, Olvera C, Treviño LG, Déziel E, Lépine F, Soberón-Chávez G (2006) Mono-rhamnolipid and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) production using Escherichia coli as a heterologous host. Appl Microbiol Biotechnol 73:187–194CrossRefPubMedGoogle Scholar
  12. Cha M, Lee N, Kim M, Kim M, Lee S (2008) Heterologous production of Pseudomonas aeruginosa EMS1 biosurfactant in Pseudomonas putida. Bioresource Technol 99:2192–2199CrossRefGoogle Scholar
  13. Chandrasekaran EV, Bemiller JN (1980) Constituent analyses of glycosaminoglycans. Methods Carbohydr Chem 8:89–96Google Scholar
  14. Choi MH, Xu J, Gutierrez M, Yoo T, Cho YH, Yoon SC (2011) Metabolic relationship between polyhydroxyalkanoic acid and rhamnolipid synthesis in Pseudomonas aeruginosa: comparative 13C NMR analysis of the products in wild type and mutants. J Biotechnol 151:30–42CrossRefPubMedGoogle Scholar
  15. Croda-García G, Grosso-Becerra V, González A, Servín-González L, Soberón-Chávez G (2011) Transcriptional regulation of Pseudomonas aeruginosa rhlR: role of the Crp-ortholog Vfr (virulence factor regulator) and quorum-sensing regulators LasR and RhlR. Microbiol-UK 157:2545–2555CrossRefGoogle Scholar
  16. Deziel E, Lépine F, Milot F, Villemur R (2000) Mass spectrometry monitoring of rhamnolipids from a growing culture of Pseudomonas aeruginosa strain 57RP. Biochem Biophys Acta 1485:145–152PubMedGoogle Scholar
  17. Diggle SP, Fletcher MP, Cámara M, Williams P (2011) Detection of 2-alkyl-4-quinolone using biosensors. In: Rumbaugh KP (ed) Quorum sensing: methods and protocols, methods in molecular biology, vol 692CrossRefGoogle Scholar
  18. Essar DW, Eberly L, Crawford IP (1990) Evolutionary differences in chromosomal locations of four early genes of tryptophan pathway in fluorescent Pseudomonas: DNA sequences and characterization of Pseudomonas putida trpE and trpGDC. J Bacteriol 172:867–883CrossRefPubMedPubMedCentralGoogle Scholar
  19. Gellatly SL, Hancock REW (2013) Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. Pathog Dis 67:159–173CrossRefPubMedGoogle Scholar
  20. Giardine B, Riemer C, Hardison RC, Burhans R, Elnitski L, Shah P, Zhang Y, Blankenberg D, Albert I, Taylor J, Miller W, Kent WJ, Nekrutenko A (2005) Galaxy: a platform for interactive large-scale genome analysis. Genome Res 15(10):1451–1455CrossRefPubMedPubMedCentralGoogle Scholar
  21. Grosso-Becerra MV, Santos-Medellín C, González-Valdez A, Méndez JL, Delgado G, Morales-Espinosa R, Servín-González L, Alcaraz LD, Soberón-Chávez G (2014a) Pseudomonas aeruginosa clinical and environmental isolates constitute a single population with high phenotypic diversity. BMC-Genomics 15:318CrossRefPubMedPubMedCentralGoogle Scholar
  22. Grosso-Becerra MV, Croda-García G, Merino E, Servín-González L, Mojica-Espinosa R, Soberón-Chávez G (2014b) Regulation of Pseudomonas aeruginosa virulence factors by two novel RNA thermometers. Proc Natl Acad Sci U S A 111:15562–15567CrossRefPubMedPubMedCentralGoogle Scholar
  23. Holloway BW (1955) Genetic recombination in Pseudomonas aeruginosa. J Gen Microbiol 13:572–581PubMedGoogle Scholar
  24. Laemmli UK (1990) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277(5259):680–685Google Scholar
  25. Law JL, Slepecky RA (1961) Assay of poly-β-hydroxybutyric acid. J Bacteriol 82:33–36PubMedPubMedCentralGoogle Scholar
  26. Lee DG, Urbach JM, Wu G, Liberati NT, Feinbaum RL, Miyata S, Diggins LT, He J, Déziel E, Friedman L, Li L, Grills G, Montgomery K, Kucherlapati R, Rahme LG, Ausubel FM (2006) Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial. Genome Biol 7:R90CrossRefPubMedPubMedCentralGoogle Scholar
  27. Madison LL, Huisman GW (1999) Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev 63:21–53PubMedPubMedCentralGoogle Scholar
  28. Mai-Prochnow A, Bradbury M, Ostrikov K, Murphy AB (2015) Pseudomonas aeruginosa biofilm response and resistance to cold atmospheric pressure plasma is linked to the redox active molecule phenazine. PLoS One 10(6):e0130373CrossRefPubMedPubMedCentralGoogle Scholar
  29. Medina G, Juárez K, Valderrama B, Soberón-Chávez G (2003) Mechanism of Pseudomonas aeruginosa RhlR transcriptional regulation of the rhlAB promoter. J Bacteriol 185:5976–5983CrossRefPubMedPubMedCentralGoogle Scholar
  30. Miller J (1972) In: Experiments in molecular genetics. Published by Cold Spring Harbor Laboratory (Cold Spring Harbor Laboratory, NY), pp 352–355Google Scholar
  31. Morales-Espinosa R, Soberón-Chávez G, Delgado-Sapién G, Sandner-Miranda L, Mendez J, Gonzalez-Valencia G, Cravioto A (2012) Genetic and phenotypic characterization of a Pseudomonas aeruginosa population with high frequency of genomic islands. PLoS One 7(5):e37459CrossRefPubMedPubMedCentralGoogle Scholar
  32. Müller MM, Hörmann B, Syldark C, Hausmann R (2010) Pseudomonas aeruginosa PAO1 as a model for rhamnolipids production in bioreactor systems. Appl Microbiol Biotechnol 87:167–174CrossRefPubMedGoogle Scholar
  33. Ochsner UA, Fietcher A, Reiser J (1994) Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis. J Biol Chem 269:19787–19795PubMedGoogle Scholar
  34. Pearson JP, Pesci EC, Iglewski BH (1997) Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis. J Bacteriol 179:5756–5767CrossRefPubMedPubMedCentralGoogle Scholar
  35. Rahim R, Ochsner U, Olvera C, Graninger M, Messner P, Lam JS, Soberón-Chávez 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 40:708–718CrossRefPubMedGoogle Scholar
  36. Rahman PK, Randhawa KK (2015) Editorial: Microbiotechnology based surfactants and their applications. Frontiers Microbiol 1 6:1344Google Scholar
  37. Roy PH, Tetu SG, Larouche A, Elbourne L, Tremblay S, Ren Q, Dodson R, Harkins D, Shay R, Watkins K, Mahamoud Y, Paulsen IT (2010) Complete genome sequence of the multiresistant taxonomic outlier Pseudomonas aeruginosa PA7. PLoS One 5:e8842CrossRefPubMedPubMedCentralGoogle Scholar
  38. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Lab Press, Cold Spring Harbor, NYGoogle Scholar
  39. Soberón-Chávez G, Aguirre-Ramírez M, Sánchez R (2005a) The Pseudomonas aeruginosa RhlA enzyme is not only involved in rhamnolipid, but also in polyhydroxyalkanoate production. J Ind Microbiol Biotechnol 32:675–677CrossRefPubMedGoogle Scholar
  40. Soberón-Chávez G, Lépine F, Déziel E (2005b) Production of rhamnolipids by Pseudomonas aeruginosa. Appl Microbiol Biotechnol 68:718–725CrossRefPubMedGoogle Scholar
  41. Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9):1312–1313CrossRefPubMedPubMedCentralGoogle Scholar
  42. 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 406:959–964CrossRefPubMedGoogle Scholar
  43. Toribio J, Escalante AE, Soberón-Chávez G (2011) Production of rhamnolipids in bacteria other than Pseudomonas aeruginosa. European J Lipid Sci Technol 112:1082–1087CrossRefGoogle Scholar
  44. Wade DS, Calfee WM, Rocha ER, Ling EA, Engstrom E, Coleman JF, Pesci EC (2005) Regulation of Pseudomonas quinolone signal synthesis in Pseudomonas aeruginosa. J Bacteriol 187:4372–4380CrossRefPubMedPubMedCentralGoogle Scholar
  45. West SEH, Schweizer HP, Dall C, Sample AK, Runyen-Janeck LJ (1994) Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148:81–86CrossRefPubMedGoogle Scholar
  46. Williams P, Cámara M (2009) Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and multifunctional signal molecules. Curr Opin Microbiol 12:82–191CrossRefGoogle Scholar
  47. Wittgens A, Tiso T, Arndt TT, Wenk P, Hemmerick J, Müller C, Wichmann R, Küpper 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 Factories 10:80CrossRefGoogle Scholar
  48. Zhang Y, Miller RM (1992) Enhanced octadecane dispersion and biodegradation by a Pseudomonas rhamnolipid surfactant (biosurfactant). Appl Environ Microbiol 58:3276–3282PubMedPubMedCentralGoogle Scholar
  49. Zhu K, Rock CO (2008) RhlA converts β-hydroxyacyl-acyl carrier protein intermediates in fatty acid synthesis to the β-hydroxydecanoyl-β-hydroxydecanoate component of rhamnolipids in Pseudomonas aeruginosa. J Bacteriol 190:3147–3154CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • María-Victoria Grosso-Becerra
    • 1
  • Abigail González-Valdez
    • 1
  • María-Jessica Granados-Martínez
    • 1
  • Estefanía Morales
    • 1
  • Luis Servín-González
    • 1
  • José-Luis Méndez
    • 2
  • Gabriela Delgado
    • 2
  • Rosario Morales-Espinosa
    • 2
  • Gabriel-Yaxal Ponce-Soto
    • 3
  • Miguel Cocotl-Yañez
    • 1
  • Gloria Soberón-Chávez
    • 1
    Email author
  1. 1.Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones BiomédicasUniversidad Nacional Autónoma de México, Ciudad UniversitariaMéxico, DFMexico
  2. 2.Departamento de Microbiología y Parasitología, Facultad de MedicinaUniversidad Nacional Autónoma de México, Ciudad UniversitariaMéxico, DFMexico
  3. 3.Departamento de Ecología Evolutiva, Instituto de EcologíaUniversidad Nacional Autónoma de México, Ciudad UniversitariaMéxico, DFMexico

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