Generation of Xylose-inducible promoter tools forPseudomonasspecies and their use in implicating a role for the Type II secretion system protein XcpQ in inhibition of corneal epithelial wound closure

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>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 <jats:italic>Pseudomonas aeruginosa</jats:italic> and <jats:italic>P. fluorescens</jats:italic>. The <jats:italic>P</jats:italic><jats:sub><jats:italic>xut</jats:italic></jats:sub> promoter derived from the <jats:italic>P. flurorescens xut</jats:italic> operon was incorporated into a broad host-range pBBR1-based plasmid and compared to the <jats:italic>Escherichia coli</jats:italic>-derived <jats:italic>P</jats:italic><jats:sub><jats:italic>BAD</jats:italic></jats:sub> promoter using <jats:italic>gfp</jats:italic> as a reporter. GFP-fluorescence from the <jats:italic>P</jats:italic><jats:sub><jats:italic>xut</jats:italic></jats:sub> promoter was inducible in both <jats:italic>Pseudomonas</jats:italic> species, but not in <jats:italic>E. coli,</jats:italic> which may facilitate cloning of toxic genes using <jats:italic>E. coli</jats:italic> to generate plasmids. The <jats:italic>P</jats:italic><jats:sub><jats:italic>xut</jats:italic></jats:sub> promoter was expressed at a lower inducer concentration than <jats:italic>P</jats:italic><jats:sub><jats:italic>BAD</jats:italic></jats:sub> in <jats:italic>P. fluorescens</jats:italic> and higher <jats:italic>gfp</jats:italic> levels were achieved using <jats:italic>P</jats:italic><jats:sub><jats:italic>xut</jats:italic></jats:sub>. Flow cytometry analysis indicated that <jats:italic>P</jats:italic><jats:sub><jats:italic>xut</jats:italic></jats:sub> was more leaky than <jats:italic>P</jats:italic><jats:sub><jats:italic>BAD</jats:italic></jats:sub> in the tested <jats:italic>Pseudomonas</jats:italic> species, but was expressed in a higher proportion of cells when induced. D-xylose did not support growth of <jats:italic>P. aeruginosa</jats:italic> or <jats:italic>P. fluorescens</jats:italic> as a sole carbon source and is less expensive than many other commonly used inducers which could facilitate large scale applications. The efficacy of this system aided in demonstrating a role for the <jats:italic>P. aeruginosa</jats:italic> type II secretion system gene from <jats:italic>xcpQ</jats:italic> in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in <jats:italic>Pseudomonas</jats:italic> species.</jats:p><jats:sec><jats:title>Importance</jats:title><jats:p><jats:italic>Pseudomonas</jats:italic> species are enormously important in human infections, biotechnology, and as a model system for interrogating basic science questions. In this study we have developed a xylose-inducible promoter system and evaluated it in <jats:italic>P. aeruginosa</jats:italic> and <jats:italic>P. fluorescens</jats:italic> 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 2 secretion system protein from <jats:italic>P. aeruginosa</jats:italic>, XcpQ, is important for host-pathogen interactions in a corneal wound closure model.</jats:p></jats:sec>