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Journal of Bacteriology, January 2003, p. 184-195, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.184-195.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Transcriptional Organization of the Pseudomonas putida tol-oprL Genes

María A. Llamas,1 Juan L. Ramos,1 and José J. Rodríguez-Herva2*

Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain,1 Department of Molecular Microbiology, Utrecht University, Utrecht, The Netherlands2

Received 15 July 2002/ Accepted 1 October 2002

Proteins of the Tol system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. In Pseudomonas putida, the seven genes, orf1, tolQ, tolR, tolA, tolB, oprL, and orf2, which encode the proteins of this complex, are clustered in a 5.8-kb region of chromosomal DNA. Analysis of polar mutations, reverse transcriptase PCR assays, and transcriptional fusion constructs with a promoterless lacZ gene revealed that the genes are arranged in two operons: orf1 tolQ tolR tolA tolB and oprL orf2. We were also able to find a transcript that was initiated at the orf1 promoter and covered the two operons in a single mRNA. On the basis of the OprL protein level, we surmised that this transcript contributed only about 10 to 15% of the total OprL protein. Primer extension analysis identified the oprL orf2 operon promoter within the tolB gene, and the -10 and -35 regions exhibited some similarity to those of {sigma}70-recognized promoters. The transcription start point of orf1 was located 91 bp upstream of the orf1 start codon, and the -10/-35 region also exhibited {sigma}70 -10/-35 recognition sequences. The expression from both promoters in rich and minimal media was constitutive and was very little influenced by the growth phase or iron-deficient conditions. In addition, analyses of the ß-galactosidase activities of different translational fusion constructs revealed that translation of tolA and orf2 genes was dependent on the translation of their corresponding upstream genes (tolR and oprL, respectively).

* Corresponding author. Mailing address: Department of Molecular Microbiology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Phone: 31 302 533632. Fax: 31 302 513655. E-mail: J.J.Rodriguez-Herva{at}bio.uu.nl.

Journal of Bacteriology, January 2003, p. 184-195, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.184-195.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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  • Llamas, M. A., Rodriguez-Herva, J. J., Hancock, R. E. W., Bitter, W., Tommassen, J., Ramos, J. L. (2003). Role of Pseudomonas putida tol-oprL Gene Products in Uptake of Solutes through the Cytoplasmic Membrane. J. Bacteriol. 185: 4707-4716 [Abstract] [Full Text]  


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