Xylose-Inducible Promoter Tools for Pseudomonas Species and Their Use in Implicating a Role for the Type II Secretion System Protein XcpQ in the Inhibition of Corneal Epithelial Wound Closure

摘要

Tunable control of gene expression is an invaluable tool for biological experiments. In this study, we describe a new xylose-inducible promoter system and evaluate it in both Pseudomonas aeruginosa and Pseudomonas fluorescens . The P_(xut) promoter, derived from the P. fluorescens xut operon, was incorporated into a broad-host-range pBBR1-based plasmid and was compared to the Escherichia coli -derived P_(BAD) promoter using gfp as a reporter. Green fluorescent protein (GFP) fluorescence from the P_(xut) promoter was inducible in both Pseudomonas species, but not in E. coli , which may facilitate the cloning of genes toxic to E. coli to generate plasmids. The P_(xut) promoter was activated at a lower inducer concentration than P_(BAD) in P. fluorescens , and higher gfp levels were achieved using P_(xut) . Flow cytometry analysis indicated that P_(xut) was leakier than P_(BAD) in the Pseudomonas species tested but was expressed in a higher proportion of cells when induced. d-Xylose as a sole carbon source did not support the growth of P. aeruginosa or P. fluorescens and is less expensive than many other commonly used inducers, which could facilitate large-scale applications. The efficacy of this system was demonstrated by its use to reveal a role for the P. aeruginosa type II secretion system gene xcpQ in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in Pseudomonas species.IMPORTANCE Pseudomonas species are enormously important in human infections, in biotechnology, and as model systems for investigating basic science questions. In this study, we have developed a xylose-inducible promoter system, evaluated it in P. aeruginosa and P. fluorescens , and found it to be suitable for the strong induction of gene expression. Furthermore, we have demonstrated its efficacy in controlled gene expression to show that a type II secretion system protein from P. aeruginosa , XcpQ, is important for host-pathogen interactions in a corneal wound closure model.