Affiliation:
1. Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain
2. Department of Molecular Microbiology, Utrecht University, Utrecht, The Netherlands
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).
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
26 articles.
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