Characterization of each aefR and mexT mutant in Pseudomonas syringae pv. tabaci 6605
Yuichiro Kawakita1, Fumiko Taguchi, Yoshishige Inagaki
1Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka 1-1-1, Okayama 700-8530, Japan.
Abstract:
To investigate the mechanism of activation of the genes for resistance-nodulation-division (RND) family members MexE, MexF, and OprN for multidrug resistance (MDR), we mutagenized aefR and mexT, the potential regulators of mexEF/oprN transcription in Pseudomonas syringae pv. tabaci 6605 (Pta 6605). AefR is a member of the TetR transcription factors, and is known to be required for production of the quorum-sensing molecules, acyl homoserine lactones (AHL), in P. syringae. Furthermore, we found that AHL-synthesis-defective mutant strains in Pta 6605 showed enhanced expression of mexEF/oprN, and were highly tolerant to antimicrobial compounds such as chloramphenicol. MexT is a LysR-type transcription factor and is known to positively regulate transcription of mexEF/oprN in Pseudomonas aeruginosa. The ∆aefR mutant reduced the amount of growth in in vitro culture, caused the loss of AHL production, reduced the swarming motility, virulence and expression of psyI (AHL synthase) and psyR (AHL transcriptional regulator), and enhanced mexEF/oprN expression and tolerance to chloramphenicol, whereas the ∆mexT mutant retained the ability to produce AHL and did not show remarkable changes in in vitro growth, tolerance to antimicrobial compounds or virulence. Furthermore, unlike P. aeruginosa, the expression of mexEF/oprN is independent of MexT. These results indicate that (1) AefR is a regulator for the quorum-sensing system and MDR, and is required for swarming motility and virulence toward the host tobacco plant, and (2) MexT is not involved in the expression of mexEF/oprN in this bacterium.
Insights
AefR regulates quorum sensing and multidrug resistance (MDR) in Pseudomonas syringae, impacting virulence and antimicrobial tolerance. MexT does not influence mexEF/oprN gene expression in this bacterium.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The resistance-nodulation-division (RND) family contributes to multidrug resistance (MDR) in bacteria.
- Quorum sensing (QS) regulates bacterial behaviors, including virulence and biofilm formation.
- MexEF/OprN is an RND efflux pump system implicated in MDR.
Purpose of the Study:
- To elucidate the regulatory mechanisms of mexEF/oprN gene expression in Pseudomonas syringae pv. tabaci 6605 (Pta 6605).
- To investigate the roles of AefR and MexT transcription factors in MDR and QS.
- To determine the involvement of QS in the regulation of MDR.
Main Methods:
- Mutagenesis of aefR and mexT genes in Pta 6605.
- Analysis of mexEF/oprN gene expression using quantitative methods.
- Assessment of antimicrobial susceptibility, swarming motility, and virulence in mutant strains.
- Quantification of acyl homoserine lactone (AHL) production.
Main Results:
- Mutations in aefR led to enhanced mexEF/oprN expression and increased tolerance to chloramphenicol.
- AefR mutants exhibited reduced AHL production, swarming motility, and virulence.
- Mutations in mexT did not significantly alter mexEF/oprN expression, AHL production, growth, or virulence.
- mexEF/oprN expression in Pta 6605 was independent of MexT, contrasting with findings in Pseudomonas aeruginosa.
Conclusions:
- AefR acts as a key regulator of both the QS system and MDR in Pta 6605.
- AefR is essential for swarming motility and virulence in Pta 6605.
- MexT is not involved in the regulation of mexEF/oprN expression in this specific bacterial strain.
