Transcriptional Organization of the Pseudomonas putida tol-oprL Genes

Abstract

ABSTRACT 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 σ 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 σ 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 β-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).