MexEF-OprN multidrug efflux pump transporter negatively controls N-acyl-homoserine lactone accumulation in pseudomonas syringae pv. Tabaci 6605

  • Takahiro Sawada
  • Miho Eguchi
  • Seiya Asaki
  • Ryota Kashiwagi
  • Kousuke Shimomura
  • Fumiko Taguchi
  • Hidenori Matsui
  • Mikihiro Yamamoto
  • Yoshiteru Noutoshi
  • Kazuhiro Toyoda
  • Yuki Ichinose
Original Article
  • 73 Downloads

Abstract

Our previous studies revealed that flagellar-motility-defective mutants such as ∆fliC of Pseudomonas syringae pv. tabaci 6605 (Pta6605) have remarkably reduced production of N-acyl-homoserine lactones (AHL), quorum-sensing molecules. To investigate the reason of loss of AHL production in ∆fliC mutant, we carried out transposon mutagenesis. Among approximately 14,000 transconjugants, we found 11 AHL production-recovered (APR) strains. In these APR strains, a transposon was inserted into either mexE or mexF, genes encoding for the multidrug efflux pump transporter MexEF-OprN, and mexT, a gene encoding a putative transcriptional activator for mexEF-oprN. These results suggest that MexEF-OprN is a negative regulator of AHL production. To confirm the negative effect of MexEF-OprN on AHL production, loss- and gain-of-function experiments for mexEF-oprN were carried out. The ∆fliCmexF and ∆fliCmexT double mutant strains recovered AHL production, whereas the mexT overexpressing strain abolished AHL production, although the psyI, a gene encoding AHL synthase, is transcribed as wild type. Introduction of a mexF or mexT mutation into another flagellar-motility- and AHL production-defective mutant strain, ∆motCD, also recovered the ability to produce AHL. Furthermore, introduction of the mexF mutation into other AHL production-defective mutant strains such as ∆gacA and ∆aefR also recovered AHL production but not to the ∆psyI mutant. These results indicate that MexEF-OprN is a decisive negative determinant of AHL production and accumulation.

Keywords

N-Acyl-homoserine lactone Flagella motility MexEF-OprN Multidrug efflux pump transporter Quorum sensing 

Notes

Acknowledgements

We thank the Leaf Tobacco Research Laboratory, Japan Tobacco Inc., and Dr. D. Studholme, University of Exeter, UK, for providing Pta6605 and genome sequence information of Pta6605, respectively. This work was supported in part by Grants-in-Aid for Scientific Researches, 15H04458 and 16K14861 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Compliance with ethical standards

Conflict of interest

All of the authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with animals performed by any of the authors.

Supplementary material

438_2018_1430_MOESM1_ESM.ppt (4 mb)
Supplementary material 1 (PPT 4098 KB)
438_2018_1430_MOESM2_ESM.ppt (4 mb)
Supplementary material 2 (PPT 4098 KB)

References

  1. Alexeyev MF, Shokolenko IN, Croughan TP (1995) New mini-Tn5 derivatives for insertion mutagenesis and genetic engineering in gram-negative bacteria. Can J Microbiol 41:1053–1055CrossRefPubMedGoogle Scholar
  2. Buell CR, Joardar V, Lindeberg M, Selengut J, Paulsen IT, Gwinn ML, Dodson RJ, Deboy RT, Durkin AS, Kolonay JF, Madupu R, Daugherty S, Brinkac L, Beanan MJ, Haft DH, Nelson WC, Davidsen T, Zafar N, Zhou L, Liu J, Yuan Q, Khouri H, Fedorova N, Tran B, Russell D, Berry K, Utterback T, Van Aken SE, Feldblyum TV, D’Ascenzo M, Deng WL, Ramos AR, Alfano JR, Cartinhour S, Chatterjee AK, Delaney TP, Lazarowitz SG, Martin GB, Schneider DJ, Tang X, Bender CL, White O, Fraser CM, Collmer A (2003) The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. Tomato DC3000. Proc Natl Acad Sci USA 100:10181–10186CrossRefPubMedPubMedCentralGoogle Scholar
  3. Cha JY, Lee DG, Lee JS, Oh JI, Baik HS (2012) GacA directly regulates expression of several virulence genes in Pseudomonas syringae pv tabaci 11528. Biochem Biophys Res Commun 417:665–672CrossRefPubMedGoogle Scholar
  4. Devine JH, Countryman C, Baldwin TO (1988) Nucleotide sequence of the luxR and luxI genes and structure of the primary regulatory region of the lux regulon of Vibrio fischeri ATCC 7744. Biochemistry 27:837–842CrossRefGoogle Scholar
  5. Elasri M, Delorme S, Lemanceau P, Stewart G, Laue B, Glickmann E, Oger PM, Dessaux Y (2001) Acyl-homoserine lactone production is more common among plant-associated Pseudomonas spp. than among soilborne Pseudomonas spp. Appl Environ Microbiol 67:1198–1209CrossRefPubMedPubMedCentralGoogle Scholar
  6. Feil H, Feil WS, Chain P, Larimer F, DiBartolo G, Copeland A, Lykidis A, Trong S, Nolan M, Goltsman E, Thiel J, Malfatti S, Loper JE, Lapidus A, Detter JC, Land M, Richardson PM, Kyrpides NC, Ivanova N, Lindow SE (2005) Comparison of the complete genome sequences of Pseudomonas syringae pv. syringae B728a and pv. tomato DC3000 Proc Natl Acad Sci USA 102:11064–11069CrossRefPubMedPubMedCentralGoogle Scholar
  7. Fetar H, Gilmour C, Klinoski R, Daigle DM, Dean CR, 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 55:508–514CrossRefPubMedGoogle Scholar
  8. Humair B, Wackwitz B, Haas D (2010) GacA-controlled activation of promoters for small RNA genes in Pseudomonas fluorescens. Appl Environ Microbiol 76:1497–1506CrossRefPubMedPubMedCentralGoogle Scholar
  9. Ichinose Y, Taguchi F, Mukaihara T (2013) Pathogenicity and virulence factors of Pseudomonas syringae. J Gen Plant Pathol 79:285–296CrossRefGoogle Scholar
  10. Joardar V, Lindeberg M, Jackson RW, Selengut J, Dodson R, Brinkac LM, Daugherty SC, Deboy R, Durkin AS, Giglio MG, Madupu R, Nelson WC, Rosovitz MJ, Sullivan S, Crabtree J, Creasy T, Davidsen T, Haft DH, Zafar N, Zhou L, Halpin R, Holley T, Khouri H, Feldblyum T, White O, Fraser CM, Chatterjee AK, Cartinhour S, Schneider DJ, Mansfield J, Collmer A, Buell CR (2005) Whole-genome sequence analysis of Pseudomonas syringae pv. phaseolicola 1448A reveals divergence among pathovars in genes involved in virulence and transposition. J Bacteriol 187:6488–6498CrossRefPubMedPubMedCentralGoogle Scholar
  11. Kanda E, Tatsuta T, Suzuki T, Taguchi F, Naito K, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2011) Two flagellar stators and their roles in motility and virulence in Pseudomonas syringae pv. tabaci 6605. Mol Genet Genomics 285:163–174CrossRefPubMedGoogle Scholar
  12. Kawakita Y, Taguchi F, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2012) Characterization of each aefR and mexT mutant in Pseudomonas syringae pv. tabaci 6605 Mol Genet Genomics 287:473–484CrossRefPubMedGoogle Scholar
  13. Kay E, Humair B, Dénervaud V, Riedel K, Spahr S, Eberl L, Valverde C, Haas D (2006) Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa. J Bacteriol 188:6026–6033CrossRefPubMedPubMedCentralGoogle Scholar
  14. Keen NT, Tamaki S, Kobayashi D, Trollinger D (1988) Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria. Gene 70:191–197CrossRefPubMedGoogle Scholar
  15. King EO, Ward NK, Raney DE (1954) Two simple media for the demonstration of pyrocyanin and fluorescein. J Lab Clin Med 44:301–307PubMedGoogle Scholar
  16. Köhler T, Epp SF, Curty LK, Pechère J-C (1999) Characterization of MexT, the regulator of the MexE-MexF-OprN multidrug efflux system of Pseudomonas aeruginosa. J Bacteriol 181:6300–6305PubMedPubMedCentralGoogle Scholar
  17. Köhler T, van Delden C, Curty LK, Hamzehpour MM, Pechere JC (2001) Overexpression of the MexEF-OprN multidrug efflux system affects cell-to-cell signaling in Pseudomonas aeruginosa. J Bacteriol 183:5213–5222CrossRefPubMedPubMedCentralGoogle Scholar
  18. Lamarche MG, Déziel E (2011) MexEF-OprN efflux pump exports the Pseudomonas quinolone signal (PQS) precursor HHQ (4-hydroxy-2-heptylquinoline). PLoS One 6:e24310CrossRefPubMedPubMedCentralGoogle Scholar
  19. Li XZ, Nikaido H (2009) Efflux-mediated drug resistance in bacteria: an update. Drugs 69:1555–1623CrossRefPubMedPubMedCentralGoogle Scholar
  20. Marutani M, Taguchi F, Ogawa Y, Hossain MM, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2008) Gac two-component system in Pseudomonas syringae pv. tabaci is required for virulence but not for hypersensitive reaction. Mol Genet Genomics 279:313–322CrossRefPubMedGoogle Scholar
  21. Maseda H, Sawada I, Saito K, Uchiyama H, Nakae T, Nomura N (2004) Enhancement of the mexAB-oprM efflux pump expression by a quorum-sensing autoinducer and its cancellation by a regulator, MexT, of the mexEF-oprN efflux pump operon in Pseudomonas aeruginosa. Antimicrob Agents Chemother 48:1320–1328CrossRefPubMedPubMedCentralGoogle Scholar
  22. McClean KH, Winson MK, Fish L, Taylor A, Chhabra SR, Camara M, Daykin M, Lamb JH, Swift S, Bycroft BW, Stewart GS, Williams P (1997) Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143:3703–3711CrossRefPubMedGoogle Scholar
  23. Minogue TD, Wehland-von Trebra M, Bernhard F, von Bodman SB (2002) The autoregulatory role of EsaR, a quorum-sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function. Mol Microbiol 44:1625–1635CrossRefPubMedGoogle Scholar
  24. Moll S, Schneider DJ, Stodghill P, Myers CR, Cartinhour SW, Filiatrault MJ (2010) Construction of an rsmX co-variance model and identification of five rsmX non-coding RNAs in Pseudomonas syringae pv. tomato DC3000. RNA Biol 7:508–516CrossRefPubMedPubMedCentralGoogle Scholar
  25. Quiñones B, Pujol CJ, Lindow SE (2004) Regulation of AHL production and its contribution to epiphytic fitness in Pseudomonas syringae. Mol Plant-Microbe Interact 17:521–531CrossRefPubMedGoogle Scholar
  26. Sambrook T, Fritshch EF, Maniatis J (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  27. Sawada H, Suzuki F, Matsuda I, Saitou N (1999) Phylogenetic analysis of Pseudomonas syringae pathovars suggests the horizontal gene transfer of argK and the evolutionary stability of hrp gene cluster. J Mol Evol 49:627–644CrossRefPubMedGoogle Scholar
  28. Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73CrossRefPubMedGoogle Scholar
  29. Shimizu R, Taguchi F, Marutani M, Mukaihara T, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2003) The ∆fliD mutant of Pseudomonas syringae pv. tabaci, which secretes flagellin monomers, induces a strong hypersensitive reaction (HR) in non-host tomato cells. Mol Genet Genomics 269:21–30PubMedGoogle Scholar
  30. Sitnikov DM, Schineller JB, Baldwin TO (1995) Transcriptional regulation of bioluminesence genes from Vibrio fischeri. Mol Microbiol 17:801–812CrossRefPubMedGoogle Scholar
  31. Taguchi F, Takeuchi K, Katoh E, Murata K, Suzuki T, Marutani M, Kawasaki T, Eguchi M, Katoh S, Kaku H, Yasuda C, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2006) Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci. Cell Microbiol 8:923–938CrossRefPubMedGoogle Scholar
  32. Taguchi F, Yamamoto M, Ohnishi-Kameyama M, Iwaki M, Yoshida M, Ishii T, Konishi T, Ichinose Y (2010) Defects in flagellin glycosylation affect the virulence of Pseudomonas syringae pv. tabaci 6605 Microbiology 156:72–80CrossRefPubMedGoogle Scholar
  33. Taguchi F, Inoue Y, Suzuki T, Inagaki Y, Yamamoto M, Toyoda K, Noutoshi Y, Shiraishi T, Ichinose Y (2015) Characterization of quorum sensing-controlled transcriptional regulator MarR and Rieske (2Fe-2S) cluster-containing protein (Orf5), which are involved in resistance to environmental stresses in Pseudomonas syringae pv. tabaci 6605 Mol Plant Pathol 16:376–387CrossRefPubMedGoogle Scholar
  34. Wilson K (1989) Preparation of genomic DNA from bacteria. In: Ausubel FM, Brent R, Kingston RE, Moor DD, Smith JA, Seidman JG, Struhl K (eds) Current protocols in molecular biology. Wiley, New York pp. 2.4.1–2.4.5Google Scholar
  35. Yun S, Lee JS, Do MS, Jeon YJ, Cha JY, Baik HS (2015) Functional analysis of the aefR mutation and identification of its binding site in Pseudomonas syringae pv. tabaci 11528 Acta Biochim Biophys Sin 47:938–945CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Takahiro Sawada
    • 1
  • Miho Eguchi
    • 1
  • Seiya Asaki
    • 2
  • Ryota Kashiwagi
    • 2
  • Kousuke Shimomura
    • 2
  • Fumiko Taguchi
    • 1
    • 3
  • Hidenori Matsui
    • 1
  • Mikihiro Yamamoto
    • 1
  • Yoshiteru Noutoshi
    • 1
  • Kazuhiro Toyoda
    • 1
  • Yuki Ichinose
    • 1
  1. 1.Graduate School of Environmental and Life ScienceOkayama UniversityOkayamaJapan
  2. 2.Faculty of AgricultureOkayama UniversityOkayamaJapan
  3. 3.Department of Biotechnology, Graduate School of EngineeringNagoya UniversityNagoyaJapan

Personalised recommendations