Transcriptome profiling defines a novel regulon modulated by the LysR-type transcriptional regulator MexT in Pseudomonas aeruginosa

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.

MexT Functions as a Redox-Responsive Regulator Modulating Disulfide Stress Resistance in Pseudomonas aeruginosa

“…Due to its pleiotropic effect on P. aeruginosa physiology, MexT has been the subject of intense investigation (4,8,23,24,29). In addition to MexEF-OprN, several MexT-regulated target genes in P. aeruginosa have been identified, highlighting the role of MexT as a global transcriptional regulator (66). Interestingly, not all MexTassociated phenotypes are dependent on MexEF-OprN, and other members of the MexT regulon have come into focus as potential players in pathogen-host interactions (9,19,66,67).…”

“…In addition to MexEF-OprN, several MexT-regulated target genes in P. aeruginosa have been identified, highlighting the role of MexT as a global transcriptional regulator (66). Interestingly, not all MexTassociated phenotypes are dependent on MexEF-OprN, and other members of the MexT regulon have come into focus as potential players in pathogen-host interactions (9,19,66,67). The molecular mechanism by which MexT activates expression of its targets is thus an important physiological process in the context of understanding P. aeruginosa pathogenesis.…”

“…MexT is activated by the nitrosative stress elicitors S-nitrosoglutathione (GNSO) and diethylaminetriamine NONOate (DETA) as well as the antibiotic chloramphenicol, and transcriptome analysis has revealed that the MexT regulon is induced in response to airway epithelial cells (9,11). This has led to the hypothesis that MexT is activated in vivo and serves a physiological function related to pathogen-host interactions (9,60,66). In addition, mutations in both mexT and mexS are common in clinical isolates and occur during colonization of the cystic fibrosis lung (38,59).…”

“…His-tagged MexT protein was purified to homogeneity by nickel affinity chromatography as previously described (66). Purified protein was analyzed by SDS-PAGE under oxidizing and reducing conditions to assess whether oligomerization of MexT could be observed.…”

“…EMSAs. DY-682 infrared-labeled primers (PA4881EF, 5=-CGGCTTT TCTTTTCGCCTTA-3=; PA4881ER, 5=-TGCGTTGGAGTCATTTTCA G-3=; Eurofins MWG Operon, United Kingdom) were used to amplify a 207-bp fragment from the PA4881 promoter region containing the previously characterized MexT binding site (66). Amplified fragments were purified using the Qiagen PCR purification kit (Qiagen, United Kingdom), and 10 fmol of each was used as a DNA probe in electrophoretic mobility shift binding assays (EMSAs).…”