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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

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 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 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).

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