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
| Autor: | Jake D. Callaghan, Nicholas A. Stella, Kara M. Lehner, Benjamin R. Treat, Kimberly M. Brothers, Anthony J. St. Leger, Robert M. Q. Shanks |
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| Rok vydání: | 2020 |
| Předmět: |
0303 health sciences
Ecology Type II secretion system biology 030306 microbiology Pseudomonas aeruginosa Chemistry Operon Pseudomonas PBAD promoter medicine.disease_cause biology.organism_classification Applied Microbiology and Biotechnology Microbiology 03 medical and health sciences Plasmid Gene expression medicine Gene 030304 developmental biology Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 86 |
| ISSN: | 1098-5336 0099-2240 |
| Popis: | 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 P. fluorescens. The Pxut promoter derived from the P. fluorescens xut operon was incorporated into a broad host-range pBBR1-based plasmid and compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. GFP-fluorescence from the Pxut promoter was inducible in both Pseudomonas species, but not in E. coli, which may facilitate cloning of toxic genes using E. coli to generate plasmids. The Pxut promoter was activated at a lower inducer concentration than PBAD in P.fluorescens and higher gfp levels were achieved using Pxut. Flow cytometry analysis indicated that Pxut was more leaky than PBAD in the tested Pseudomonas species, but was expressed in a higher proportion of cells when induced. D-xylose did not support growth of P. aeruginosa or P. fluorescens 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 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. ImportancePseudomonas 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 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. |
| Databáze: | OpenAIRE |
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