title: Transcriptional organization of the Pseudomonas putida tol-oprL genes.
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 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 beta-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).
authors.affiliation: Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain.
authors.forename: María A
authors.initials: MA
authors.lastname: Llamas
authors.forename: Juan L
authors.initials: JL
authors.lastname: Ramos
authors.forename: José J
authors.initials: JJ
authors.lastname: Rodríguez-Herva
mesh_terms: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D003470:Culture Media; D029968:Escherichia coli Proteins; D007501:Iron / Q000378:metabolism; D008074:Lipoproteins / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009876:Operon; D010457:Peptidoglycan / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011509:Proteoglycans; D016958:Pseudomonas putida / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; C031163:ExcC protein, E coli; D008074:Lipoproteins; D008565:Membrane Proteins; D010457:Peptidoglycan; D011509:Proteoglycans; C069988:PplA protein, Legionella pneumophila; D007501:Iron