A two-component T7 system for the overexpression of genes in Pseudomonas aeruginosa

Abstract

A two-component T7 expression system was developed for efficient expression of genes in the nonenteric bacterium, Pseudomonas aeruginosa. The first component of the expression system is a bacteriophage-based transposable element that contains a lacUV5/ lacI q-regulated T7 RNA polymerase gene and a selectable antibiotic-resistance determinant. This element, designated miniD-180, was stably integrated into the P. aeruginosa PAO1 chromosome. The second component of this system includes several improved broad-host-range expression vectors containing the T7 gene 10 promoter and multiple cloning site (MCS). These vectors (pEB8, pEB11, and pEB12) contain transcriptional terminators ( Tl 4) upstream from the T7 promoter, and T7 terminators downstream from the MCS. Because the T7 promoter is somewhat leaky in these vectors, pEB14 was constructed to decrease transcription of target genes bybasal levels of T7 RNA polymerase. This vector contains a core sequence of the lac operator located by 19 bp downstream from the transcriptional start point of the T7 promoter, thereby providing a dually regulated system. The utility of this system was demonstrated by placing a promoterless chloramphenicol acetyltransferase (CAT) cassette under control of the T7 promoter and monitoring the isopropyl-β- d-thiogalactopyranoside-dependent accumulation of CAT in cell-free extracts of P. aeruginosa. We observed up to nearly a 60-fold increase in CAT levels 4 h post-induction, at which time this polypeptide represented up to 20% of the total soluble protein.