Characterization of the orf1-tolQRA operon in Pseudomonas aeruginosa

The tol-pal genes play important roles in maintaining outer membrane integrity, transmembrane transportation, and cell division in Gram-negative bacteria. In Pseudomonas aeruginosa, an important human opportunistic pathogen, the tol-oprL genes are organized uniquely in three operons, orf1-tolQRA, tolB and oprL-orf2, and are regulated by iron availability. Similarity between TolQRA and the iron transport system ExbBD-TonB also exists in P. aeruginosa and they can replace each other imperfectly. It is of importance to investigate the regulation and functions of this membrane complex. In the present study, we characterized the promoters and expression profiles of the orf1-tolQRA operon and investigated the function of Orf1. Primer extension was carried out by using both isotope-labeled and florescence labeled primers and the expression profiles were determined using both lacZ and luxCDABE-based transcriptional fusions. Our results revealed two distinct promoters at the upstream region of tolQRA; the one located in front of orf1 was constitutive whereas the other within the orf1 coding region was iron regulated. Expression profiles indicate the tol genes were also downregulated by the quorum-sensing systems during the late stage of growth. Unlike tolQ and tolA, we were able to construct a viable orf1 knockout strain, and the mutant exhibited altered cell and colony morphology, providing first evidence that Orf1 plays a non-essential role in the Tol-OprL complex in P. aeruginosa.