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Journal of Bacteriology, September 2005, p. 5937-5945, Vol. 187, No. 17
0021-9193/05/$08.00+0     doi:10.1128/JB.187.17.5937-5945.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Proteomic Analysis Reveals the Participation of Energy- and Stress-Related Proteins in the Response of Pseudomonas putida DOT-T1E to Toluene

Ana Segura,1* Patricia Godoy,1 Pieter van Dillewijn,1 Ana Hurtado,1 Nuria Arroyo,2 Simon Santacruz,2 and Juan-Luis Ramos1

Estación Experimental del Zaidin, Department of Biochemistry, Profesor Albareda 1, 18008 Granada,1 Proteomika S.L., Parque Tecnológico de Bizkaia, Edificio 801, 48160 Derio, Bizkaia, Spain2

Received 22 April 2005/ Accepted 13 June 2005

Pseudomonas putida DOT-T1E is tolerant to toluene and other toxic hydrocarbons through extrusion of the toxic compounds from the cell by means of three efflux pumps, TtgABC, TtgDEF, and TtgGHI. To identify other cellular factors that allow the growth of P. putida DOT-T1E in the presence of high concentrations of toluene, we performed two-dimensional gel analyses of proteins extracted from cultures grown on glucose in the presence and in the absence of the organic solvent. From a total of 531 spots, 134 proteins were observed to be toluene specific. In the absence of toluene, 525 spots were clearly separated and 117 proteins were only present in this condition. Moreover, 35 proteins were induced by at least twofold in the presence of toluene whereas 26 were repressed by at least twofold under these conditions. We reasoned that proteins that were highly induced could play a role in toluene tolerance. These proteins, identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry, were classified into four categories: 1, proteins involved in the catabolism of toluene; 2, proteins involved in the channeling of metabolic intermediates to the Krebs cycle and activation of purine biosynthesis; 3, proteins involved in sugar transport; 4, stress-related proteins. The set of proteins in groups 2 and 3 suggests that the high energy demand required for solvent tolerance is achieved via activation of cell metabolism. The role of chaperones that facilitate the proper folding of newly synthesized proteins under toluene stress conditions was analyzed in further detail. Knockout mutants revealed that CspA, XenA, and Tuf-1 play a role in solvent tolerance in Pseudomonas, although this role is probably not specific to toluene, as indicated by the fact that all mutants grew more slowly than the wild type without toluene.

* Corresponding author. Mailing address: EEZ-CSIC, C/Prof. Albareda, 1, E-18008 Granada, Spain. Phone: 34 958 181600, ext. 266. Fax: 34 958 129600. E-mail: ana.segura{at}eez.csic.es.

Journal of Bacteriology, September 2005, p. 5937-5945, Vol. 187, No. 17
0021-9193/05/$08.00+0     doi:10.1128/JB.187.17.5937-5945.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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