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, 37 (22), 7546-59

Transcriptome Profiling Defines a Novel Regulon Modulated by the LysR-type Transcriptional Regulator MexT in Pseudomonas Aeruginosa

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Transcriptome Profiling Defines a Novel Regulon Modulated by the LysR-type Transcriptional Regulator MexT in Pseudomonas Aeruginosa

Zhe-Xian Tian et al. Nucleic Acids Res.

Abstract

The LysR-family regulator MexT modulates the expression of the MexEF-OprN efflux system in the human pathogen Pseudomonas aeruginosa. Recently, we demonstrated that MexT regulates certain virulence phenotypes, including the type-three secretion system and early attachment independent of its role in regulating MexEF-OprN. In this study, transcriptome profiling was utilized to investigate the global nature of MexT regulation in P. aeruginosa PAO1 and an isogenic mexEF mutant. Twelve genes of unknown function were highly induced by overexpressing MexT independent of MexEF-OprN. A well-conserved DNA motif was identified in the upstream regulatory region of nine of these genes and upstream of mexE. Reporter fusion analysis demonstrated that the expression of the genes was significantly induced by MexT in P. aeruginosa and a heterogenous Escherichia coli strain and that the conserved sequence was required for this induction. The conserved DNA motif was further characterized as the MexT binding site by site-directed mutagenesis and electrophoretic mobility shift assays. Genes containing this conserved regulatory sequence were identified across other Pseudomonas species, and their expression was activated by MexT. Thus, a novel regulon directly modulated by MexT, that includes but is independent of mexEF-oprN, has been identified.

Figures

Figure 1.
Figure 1.
(A and B) MA-plots showing the relationship between the logarithmic mean signal ratios, M value, and the logarithmic mean signal intensities, A value, are used to spot genes whose expression is significantly altered in (A) PAO1 mexT++ compared to PAO1 and (B) PAO1 mexT++ compared to PAO1ΔmexEF mexT++. The spots representing genes altered more than 10-fold are labelled in red. A positive M value indicates increased gene expression, while a negative value indicates reduced expression. (C) 3D scatter-plot projecting the logarithmic mean signal intensities obtained from PAO1, PAO1 mexT++ and PAO1ΔmexEF mexT++ is used to spot genes whose expression was altered by overexpressing MexT dependent or independent of the MexEF-OprN efflux pump. (Left) The orientation of the main cloud body is adjusted towards the reader, genes highly activated by overexpressing MexT independent of the MexEF-OprN efflux pump are grouped in the red oval and genes highly reduced by overexpressing MexT dependent of the MexEF-OprN efflux pump are grouped in the green oval. (Right) The orientation of the main cloud body is adjusted to show the full scale.
Figure 2.
Figure 2.
Semi-quantitative reverse transcription PCR validation of selected genes identified by microarray analysis as significantly altered by MexT. Total RNA was isolated from PAO1 containing pME6032 (WT vector), PAO1 containing pME6032-mexT (WT mexT) or PAO1ΔmexEF containing pME6032-mexT (ΔEF mexT). The identity of the genes targeted in each set of PCRs is indicated at the bottom of the figure.
Figure 3.
Figure 3.
Alignment of the predicted conserved DNA motif in the upstream regulatory regions of P. aeruginosa genes highly induced by MexT (A) and sequence logo for the conserved DNA motif reflecting position-specific probability matrixes (B) deduced from MEME software analysis. Nucleotides with high probability (≥70%) are highlighted by red. The number of nucleotides between the conserved DNA motif and the start codon ATG is shown in brackets.
Figure 4.
Figure 4.
Purified MexT protein binds to the conserved DNA motif in the promoter region of PA4881. EMSAs were carried out applying 0 ng, 37.5 ng, 75 ng, 150 ng and 300 ng (lanes Ø, 1, 2, 3 and 4) of purified His6-tagged functional (A and B) or non functional (C) MexT proteins to 10 fmol of DIG-labelled DNA target containing the intact conserved DNA motif (A and C), or the mutated DNA target with the conserved DNA motif disrupted (B). Arrow indicates the free DNA probes and the band shifts of the MexT–DNA complex.
Figure 5.
Figure 5.
Upstream regulatory DNA sequences and amino acid sequences of PA4623 (A) and PA4881 (B) and tandem repeat proteins in other Pseudomonas (C–F). Locus IDs are from the Pseudomonas Genome Project (http://www.pseudomonas.com). The conserved DNA motif is underlined and the start codon ATG is boxed. The amino acid sequence of each protein is organized showing alignment of tandem repeats with the position number of the first amino acid residue at the beginning of each line.

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