A putative MexT regulon was identified by carrying out a whole-genome transcriptome profiling on PAO1 wild-type and an isogenic mexEF mutant overexpressing MexT. The validity of the microarray data was confirmed by a semi-quantitative reverse transcription PCR analysis of genes whose expression was significantly altered (PA4881, pqsA and lldP). A well-conserved DNA motif ATCA-N5-GTCGAT-N4-ACYAT was identified in the upstream regions of 10 genes by using MEME Suite online software. The transcriptional activation of the putative target genes by MexT was confirmed by constructing promoter-lacZ fusions of selected genes. To further investigate if these genes are direct targets of MexT, the ability of MexT to activate gene expression in a heterogeneous E. coli background was assessed. The results indicate that the conserved DNA motif is required for the induced expression of these genes by MexT. EMSAs and the site-directed mutagenesis showed that MexT protein bound to the conserved DNA motif and that a mutation in two of the most conserved nucleotides in the motif abolished the binding of the MexT protein. MexT binding to the conserved motif was abolished when a mutation in the helix–turn–helix motifwas introduced.
Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.
|Site sequence||Regulated genes||Gene diagram||Experimental techniques||TF function||TF type|