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The RpoT Regulon of Pseudomonas putida DOT-T1E and Its Role in Stress Endurance against Solvents

Estrella Duque, José-Juan Rodríguez-Herva, Jesús de la Torre, Patricia Domínguez-Cuevas, Jesús Muñoz-Rojas, and Juan-Luis Ramos^*

CSIC-Estación Experimental del Zaidín, Department of Environmental Protection, Profesor Albareda, 1, E-18008 Granada, Spain

Received 30 June 2006/ Accepted 5 October 2006

Pseudomonas putida encodes 20 extracytoplasmic sigma factors (ECFs). In this study, we show that one of these ECFs, known as ECF-Pp12 (PP3006), plays a role in tolerance of toluene and other organic solvents. Based on this finding, we have called the gene that encodes this new ECF rpoT. The rpoT gene forms an operon with the preceding gene and with the gene located downstream. The translated gene product of the open reading frame PP3005 is an inner membrane protein, whereas the PP3007 protein is periplasmic. A nonpolar rpoT mutant was generated by homologous recombination, and survival of the mutant was tested under various stress conditions. The mutant strain was hypersensitive to toluene and other solvents but just as tolerant as the wild type of stress imposed by heat, antibiotics, NaCl, paraquat, sodium dodecyl sulfate, H₂O₂, and benzoate. In the rpoT mutant background, expression of around 50 transcriptional units was affected: 31 cistrons were upregulated, and 23 cistrons were downregulated. This indicates that about 1% of all P. putida genes are under the direct or indirect influence of RpoT. The rpoT gene controls the expression of a number of membrane proteins, including components of the respiratory chains, porins, transporters, and multidrug efflux pumps. Hypersensitivity of the P. putida RpoT-deficient mutant to organic solvents can be attributed to the fact that in the rpoT strain, expression of the toluene efflux pump ttgGHI genes is severalfold lower than in the parental strain.

Published ahead of print on 27 October 2006.

Present address: Laboratorio de Microbiología de Suelos, Departamento de Microbiología, Instituto de Ciencias, Edificio 76, tercer piso, complejo de Ciencias, Ciudad Universitaria, Colonia San Manuel, C. P. 72570, Puebla, México.

E.D. and J.-J.R.-H. contributed equally to this work.

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