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http://dx.doi.org/10.14348/molcells.2019.0168

Crystal Structure of the Regulatory Domain of MexT, a Transcriptional Activator of the MexEF-OprN Efflux Pump in Pseudomonas aeruginosa  

Kim, Suhyeon (Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, Seoul National University)
Kim, Songhee H. (Institute of Molecular Biology and Genetics, Seoul National University)
Ahn, Jinsook (Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, Seoul National University)
Jo, Inseong (Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, Seoul National University)
Lee, Zee-Won (Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, Seoul National University)
Choi, Sang Ho (Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, Seoul National University)
Ha, Nam-Chul (Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, Seoul National University)
Publication Information
Molecules and Cells / v.42, no.12, 2019 , pp. 850-857 More about this Journal
Abstract
The Gram-negative opportunistic pathogen, Pseudomonas aeruginosa, has multiple multidrug efflux pumps. MexT, a LysR-type transcriptional regulator, functions as a transcriptional activator of the MexEF-OprN efflux system. MexT consists of an N-terminal DNA-binding domain and a C-terminal regulatory domain (RD). Little is known regarding MexT ligands and its mechanism of activation. We elucidated the crystal structure of the MexT RD at 2.0 Å resolution. The structure comprised two protomer chains in a dimeric arrangement. MexT possessed an arginine-rich region and a hydrophobic patch lined by a variable loop, both of which are putative ligand-binding sites. The three-dimensional structure of MexT provided clues to the interacting ligand structure. A DNase I footprinting assay of full-length MexT identified two MexT-binding sequence in the mexEF-oprN promoter. Our findings enhance the understanding of the regulation of MexT-dependent activation of efflux pumps.
Keywords
antibiotic resistance; crystal structure; lysR-type transcriptional regulator; MexT; Pseudomonas aeruginosa;
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1 Poole, K. and Srikumar, R. (2001). Multidrug efflux in Pseudomonas aeruginosa : components, mechanisms and clinical significance. Curr. Top. Med. Chem. 1, 59-71.   DOI
2 Sobel, M.L., Neshat, S., and Poole, K. (2005). Mutations in PA2491 (mexS) promote MexT-dependent mexEF-oprN expression and multidrug resistance in a clinical strain of Pseudomonas aeruginosa. J. Bacteriol. 187, 1246-1253.   DOI
3 Terwilliger, T.C., Adams, P.D., Read, R.J., McCoy, A.J., Moriarty, N.W., Grosse-Kunstleve, R.W., Afonine, P.V., Zwart, P.H., and Hung, L.W. (2009). Decisionmaking in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr. D Biol. Crystallogr. 65, 582-601.   DOI
4 Tian, Z.X., Fargier, E., Mac Aogain, M., Adams, C., Wang, Y.P., and O'Gara, F. (2009). Transcriptome profiling defines a novel regulon modulated by the LysR-type transcriptional regulator MexT in Pseudomonas aeruginosa. Nucleic Acids Res. 37, 7546-7559.   DOI
5 Xu, Y., Moeller, A., Jun, S.Y., Le, M., Yoon, B.Y., Kim, J.S., Lee, K., and Ha, N.C. (2012). Assembly and channel opening of outer membrane protein in tripartite drug efflux pumps of Gram-negative bacteria. J. Biol. Chem. 287, 11740-11750.   DOI
6 Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66(Pt 2), 213-221.   DOI
7 Choi, H., Kim, S., Mukhopadhyay, P., Cho, S., Woo, J., Storz, G., and Ryu, S.E. (2001). Structural basis of the redox switch in the OxyR transcription factor. Cell 105, 103-113.   DOI
8 Dallakyan, S. and Olson, A.J. (2015). Small-molecule library screening by docking with PyRx. Methods Mol. Biol. 1263, 243-250.   DOI
9 Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010). Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66(Pt 4), 486-501.   DOI
10 Fargier, E., Mac Aogain, M., Mooij, M.J., Woods, D.F., Morrissey, J.P., Dobson, A.D., Adams, C., and O'Gara, F. (2012). MexT functions as a redox-responsive regulator modulating disulfide stress resistance in Pseudomonas aeruginosa. J. Bacteriol. 194, 3502-3511.   DOI
11 Horna, G., Lopez, M., Guerra, H., Saenz, Y., and Ruiz, J. (2018). Interplay between MexAB-OprM and MexEF-OprN in clinical isolates of Pseudomonas aeruginosa. Sci. Rep. 8, 16463.   DOI
12 Fetar, H., Gilmour, C., Klinoski, R., Daigle, D.M., Dean, C.R., and Poole, K. (2011). mexEF-oprN multidrug efflux operon of Pseudomonas aeruginosa : regulation by the MexT activator in response to nitrosative stress and chloramphenicol. Antimicrob. Agents Chemother. 55, 508-514.   DOI
13 Galie, S., Garcia-Gutierrez, C., Miguelez, E.M., Villar, C.J., and Lombo, F. (2018). Biofilms in the food industry: health aspects and control methods. Front. Microbiol. 9, 898.   DOI
14 Goethals, K., Van Montagu, M., and Holsters, M. (1992). Conserved motifs in a divergent nod box of Azorhizobium caulinodans ORS571 reveal a common structure in promoters regulated by LysR-type proteins. Proc. Natl. Acad. Sci. U. S. A. 89, 1646-1650.   DOI
15 Hinchliffe, P., Symmons, M.F., Hughes, C., and Koronakis, V. (2013). Structure and operation of bacterial tripartite pumps. Annu. Rev. Microbiol. 67, 221-242.   DOI
16 Hirai, K., Suzue, S., Irikura, T., Iyobe, S., and Mitsuhashi, S. (1987). Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 31, 582-586.   DOI
17 Jang, Y., Choi, G., Hong, S., Jo, I., Ahn, J., Choi, S.H., and Ha, N.C. (2018). A novel tetrameric assembly configuration in VV2_1132, a LysR-type transcriptional regulator in Vibrio vulnificus. Mol. Cells 41, 301-310.   DOI
18 Jeong, H., Kim, J.S., Song, S., Shigematsu, H., Yokoyama, T., Hyun, J., and Ha, N.C. (2016). Pseudoatomic structure of the tripartite multidrug efflux pump AcrAB-TolC reveals the intermeshing cogwheel-like interaction between AcrA and TolC. Structure 24, 272-276.   DOI
19 Jo, I., Chung, I.Y., Bae, H.W., Kim, J.S., Song, S., Cho, Y.H., and Ha, N.C. (2015). Structural details of the OxyR peroxide-sensing mechanism. Proc. Natl. Acad. Sci. U. S. A. 112, 6443-6448.   DOI
20 Jin, Y., Yang, H., Qiao, M., and Jin, S. (2011). MexT regulates the type III secretion system through MexS and PtrC in Pseudomonas aeruginosa. J. Bacteriol. 193, 399-410.   DOI
21 Kim, J.S., Jeong, H., Song, S., Kim, H.Y., Lee, K., Hyun, J., and Ha, N.C. (2015). Structure of the tripartite multidrug efflux pump AcrAB-TolC suggests an alternative assembly mode. Mol. Cells 38, 180-186.   DOI
22 Jo, I., Kim, D., No, T., Hong, S., Ahn, J., Ryu, S., and Ha, N.C. (2019). Structural basis for HOCl recognition and regulation mechanisms of HypT, a hypochlorite-specific transcriptional regulator. Proc. Natl. Acad. Sci. U. S. A. 116, 3740-3745.   DOI
23 Juarez, P., Broutin, I., Bordi, C., Plesiat, P., and Llanes, C. (2018). Constitutive activation of MexT by amino acid substitutions results in MexEFOprN overproduction in clinical isolates of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 62, e02445-17.
24 Juarez, P., Jeannot, K., Plesiat, P., and Llanes, C. (2017). Toxic electrophiles induce expression of the multidrug efflux pump MexEF-OprN in Pseudomonas aeruginosa through a novel transcriptional regulator, CmrA. Antimicrob. Agents Chemother. 61, e00585-17.
25 Kim, S., Ahn, J., and Ha, N. (2018). Purification and preliminary analysis of the regulatory domain of MexT from Pseudomonas aeruginosa, a LysRtype transcriptional activator of the MexEF-OprN multidrug efflux pump. Biodesign 6, 42-45.
26 Kohler, T., Epp, S.F., Curty, L.K., and Pechere, J.C. (1999). Characterization of MexT, the regulator of the MexE-MexF-OprN multidrug efflux system of Pseudomonas aeruginosa. J. Bacteriol. 181, 6300-6305.   DOI
27 Maseda, H., Uwate, M., and Nakae, T. (2010). Transcriptional regulation of the mexEF-oprN multidrug efflux pump operon by MexT and an unidentified repressor in nfxC-type mutant of Pseudomonas aeruginosa. FEMS Microbiol. Lett. 311, 36-43.   DOI
28 Kohler, T., Michea-Hamzehpour, M., Henze, U., Gotoh, N., Curty, L.K., and Pechere, J.C. (1997). Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Mol. Microbiol. 23, 345-354.   DOI
29 Lister, P.D., Wolter, D.J., and Hanson, N.D. (2009). Antibacterial-resistant Pseudomonas aeruginosa : clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin. Microbiol. Rev. 22, 582-610.   DOI
30 Maddocks, S.E. and Oyston, P.C. (2008). Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology 154(Pt 12), 3609-3623.   DOI
31 Muraoka, S., Okumura, R., Ogawa, N., Nonaka, T., Miyashita, K., and Senda, T. (2003a). Crystal structure of a full-length LysR-type transcriptional regulator, CbnR: unusual combination of two subunit forms and molecular bases for causing and changing DNA bend. J. Mol. Biol. 328, 555-566.   DOI
32 Muraoka, S., Okumura, R., Uragami, Y., Nonaka, T., Ogawa, N., Miyashita, K., and Senda, T. (2003b). Purification and crystallization of a LysR-type transcriptional regulator CBNR from Ralstonia eutropha NH9. Protein Pept. Lett. 10, 325-329.   DOI
33 Piddock, L.J. (2006). Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin. Microbiol. Rev. 19, 382-402.   DOI
34 Otwinowski, Z. and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307-326.   DOI