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. 2020 Jul 2;86(14):e00250-20.
doi: 10.1128/AEM.00250-20. Print 2020 Jul 2.

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

Jake D Callaghan  1 , Nicholas A Stella  1 , Kara M Lehner  1 , Benjamin R Treat  2 , Kimberly M Brothers  1 , Anthony J St Leger  2 , Robert M Q Shanks  3
Affiliations

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

Jake D Callaghan et al. Appl Environ Microbiol. .

Abstract

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 Pxut promoter, derived from the P. fluorescensxut operon, was incorporated into a broad-host-range pBBR1-based plasmid and was compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. Green fluorescent protein (GFP) fluorescence from the Pxut 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 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 leakier than PBAD 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.IMPORTANCEPseudomonas 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.

Keywords: Pseudomonas; inducible promoter; plasmids; xylose.

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Figures

FIG 1
FIG 1
Xylose repressor genomic region from P. fluorescens strain Pf0-1. (A) Genetic map of the xutR-xutA region. The shaded bar represents the 188-bp intergenic region. (B) DNA sequence of the Pxut promoter upstream of Pf101_2303. The conserved operator inverted repeats are shown in boldface, and the –35 and –10 regions for the xutA promoter (Pxut) are underlined. A direct repeat of 13 bp is shown in italics. A putative ribosome binding site 9 to 12 bp upstream of the start codon is shown in lowercase. (C) Schematic diagrams of select plasmids used in this study.
FIG 2
FIG 2
Comparison of the PBAD and Pxut promoters in E. coli and Pseudomonas species. Shown is the expression of gfpmut3 from the two different promoters in a pBBR1-based plasmid. Cultures were prepared in LB medium with a range of inducer concentrations and were grown at 30°C for 25 h. Means and standard deviations are shown (n, ≥5 independent cultures). Pxut was not inducible in E. coli and was inducible at lower inducer concentrations than PBAD in P. fluorescens. The pMQ578 plasmid has Pxut-gfp, and pMQ80 has PBAD-gfp. Asterisks indicate statistically significant differences (P < 0.05) between groups by Student’s t test.
FIG 3
FIG 3
Expression of the PBAD and Pxut promoters in Pseudomonas species and E. coli over time with 10 mM inducer. Shown is the expression of gfpmut3 over time from the two different promoters in a pBBR1-based plasmid. Cultures were prepared in LB medium with an inducer (10 mM) and were grown at 30°C. Means and standard deviations are shown (n, ≥4 independent cultures for E. coli; n, 6 for Pseudomonas species). The pMQ578 plasmid has Pxut-gfp, and pMQ80 has PBAD-gfp. Xylose was used as an inducer for pMQ578, and arabinose was used for pMQ80. Asterisks indicate statistically significant differences (P < 0.05) between groups by Student’s t test.
FIG 4
FIG 4
The Pseudomonas species tested do not use d-xylose as a sole carbon source. Cultures were incubated in M9 minimal medium with the indicated sugars at 10 mM for 25 h at 30°C. Means and standard deviations are shown (n, 6 independent cultures). Asterisks indicate significant differences from the glucose group (P < 0.05) by ANOVA with Tukey’s posttest.
FIG 5
FIG 5
Evaluation of ribose for induction of the Pxut promoter in two Pseudomonas species. Shown is the expression of gfpmut3 in a pBBR1-based plasmid. Cultures were prepared in LB medium with a range of inducer concentrations and were grown at 30°C for 25 h. Means and standard deviations are given (n, 6 independent cultures). (A) Pxut-based GFP expression was minimally induced by different ribose concentrations. Asterisks indicate significant differences compared to the no-ribose group as determined by Student's t test (P < 0.05). (B) Ribose (10 mM) did not alter the ability of xylose (10 mM) to activate Pxut-based GFP expression in P. aeruginosa strain PA14. Asterisks indicate significant differences compared to the water group as determined by ANOVA with Tukey's posttest (P < 0.05).
FIG 6
FIG 6
Stationary-phase induction of the PBAD and Pxut promoters in Pseudomonas species. Bacteria were grown overnight in LB broth, washed, and adjusted to an OD600 of 2.0 in PBS with an inducer at 10 mM. GFP fluorescence was measured over time. Means and standard deviations are given (n, 3 to 4 independent cultures). Pxut was more highly inducible in P. fluorescens than in P. aeruginosa during stationary phase. Pxut and PBAD were indistinguishable in P. aeruginosa. The pMQ578 plasmid has Pxut-gfp, and pMQ80 has PBAD-gfp. The asterisk indicates a statistically significant difference (P < 0.05) by Student’s t test between normalized fluorescence levels for Pf0-1 with different plasmids; there were no differences between plasmids in fluorescence levels for PA14. At 25 h, Pf0-1 with pMQ578 produced significantly more fluorescence than PA14.
FIG 7
FIG 7
Flow cytometry analysis of PBAD and Pxut promoter-driven GFP expression in Pseudomonas species indicates increased leakiness and increased proportions of bacteria expressing GFP with an inducer. The expression of gfpmut3 from the two different promoters was measured by flow cytometry. Cultures were prepared in LB medium with an inducer (10 mM) and were grown at 30°C. Gray areas represent PA14 or Pf0-1 with no plasmid, used to determine background levels. Yellow peaks represent bacteria with pMQ578 or pMQ80 without an inducer, used to indicate leakiness. Blue (and green) peaks represent bacteria with pMQ578 or pMQ80 and with an inducer. The pMQ578 plasmid has Pxut-gfp, and pMQ80 has PBAD-gfp. Xylose was used as an inducer for pMQ578, and arabinose was used for pMQ80. Levels of GFP fluorescence above those for bacteria without a plasmid were used to determine the positive cutoff (noted as GFP+); horizontal green bars indicate positive values. Results of a representative experiment are shown (n, ∼100,000 cells per group).
FIG 8
FIG 8
Analysis of promoter leakiness and strength using flow cytometry. GFP expression in Pseudomonas species was analyzed by flow cytometry (n = 7). (A) The percentage of GFP-positive cells was determined relative to the level for bacteria without a GFP plasmid. Means and standard deviations are shown. pMQ578 has Pxut, and pMQ80 has PBAD, driving gfp expression. Asterisks indicate significant differences (P < 0.05) from the no-inducer group by Student’s t test. (B) Fluorescence intensity of GFP-positive cells that have been induced (inducer concentration, 10 mM). Means and standard deviations are shown. Asterisks indicate significant differences (P < 0.05) between species by Student’s t test.
FIG 9
FIG 9
XcpQ is required for the inhibition of stratified corneal cell migration in vitro. Shown are representative images of cells stained with the vital stain Calcein AM and imaged by confocal microscopy. (A to C) A cell-free zone (black circle) in layers of a stratified human corneal cell line (A) closes rapidly when treated with LB medium and incubated for 24 h (B), but this migration is inhibited by filtrates from PA14 cultures grown in LB medium (C). (D) Filtrates from isogenic ΔxcpQ mutants were unable to impede cell migration. (E and F) d-Xylose (50 mM)-induced expression of gfp from pMQ578 did not alter the migration phenotypes of cells exposed to PA14 or ΔxcpQ mutant filtrates. (G and H) Culture filtrates from the ΔxcpQ mutant with the wild-type xcpQ gene on a plasmid (pMQ644) that had been grown in LB medium with 5 mM d-xylose were unable to inhibit cell migration (G), but those grown with 50 mM d-xylose were complemented for the cell migration inhibition-defective phenotype (H). All cultures were grown to stationary phase and were adjusted to an OD600 of 2 with fresh LB, and bacteria were removed by centrifugation and filtration.
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