The guanine-hypoxanthine permease GhxP of Erwinia amylovora facilitates the influx of the toxic guanine derivative 6-thioguanine.
Erwinia amylovora
6-thioguanine
guanine
nucleobase cation symporter 2
transport
Journal
Journal of applied microbiology
ISSN: 1365-2672
Titre abrégé: J Appl Microbiol
Pays: England
ID NLM: 9706280
Informations de publication
Date de publication:
Jun 2021
Jun 2021
Historique:
revised:
02
11
2020
received:
11
09
2020
accepted:
02
11
2020
pubmed:
6
11
2020
medline:
27
5
2021
entrez:
5
11
2020
Statut:
ppublish
Résumé
Erwinia amylovora is the causal agent of fire blight, a devastating disease of apples and pears. This study determines whether the E. amylovora guanine-hypoxanthine transporter (EaGhxP) is required for virulence and if it can import the E. amylovora produced toxic analogue 6-thioguanine (6TG) into cells. Characterization of EaGhxP in guanine transport deficient Escherichia coli reveals that it can transport guanine, hypoxanthine and the toxic analogues 8-azaguanine (8AG) and 6TG. Similarly, EaGhxP transports 8AG and 6TG into E. amylovora cells. EaGhxP has a high affinity for 6TG with a K EaGhxP is not required for virulence, but it does import 6TG into E. amylovora cells. As part of the disease establishment process, E. amylovora synthesizes and exports a toxic guanine derivative 6TG. Our results are counter intuitive and show that EaGhxP, an influx transporter, can move 6TG into cells raising questions regarding the role of 6TG in disease establishment.
Substances chimiques
Nucleobase Transport Proteins
0
Recombinant Proteins
0
Hypoxanthine
2TN51YD919
Guanine
5Z93L87A1R
Thioguanine
FTK8U1GZNX
Azaguanine
Q150359I72
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
2018-2028Subventions
Organisme : U.S. Department of Agriculture
ID : Hatch Fund CONH00652
Organisme : Indiana University-Purdue University Fort Wayne
Informations de copyright
© 2020 The Society for Applied Microbiology.
Références
Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M. et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2, 1-11. https://doi.org/10.1038/msb4100050.
Bittner, A.J., Huntley, R.B., Mourad, G.S. and Schultes, N.P. (2020) An Erwinia amylovora uracil transporter mutant retains virulence on immature apple and pear fruit. Microb Pathog 147, https://doi.org/10.1016/j.micpath.2020.104363.
Botou, M., Lazou, P., Papakostas, K., Lambrinidis, G., Evangelidis, T., Mikros, E. and Frillingos, S. (2018) Insite on the specificity of uracil permeases of the NAT/NCS2 family from analysis of the transporter encoded in the pyrimidine utilization operon of Escherichia coli. Mol Microbiol 108, 204-219. https://doi.org/10.1111/mmi.13931.
Burse, A., Weingart, H. and Ullrich, M.S. (2004) The phytoalexin-inducible multi-drug efflux pump, AcrAB, contributes to virulence in the fire blight pathogen, Erwinia amylovora. Mol Plant-Microbe Interact 17, 43-54.
Chaliotis, A., Vlastaridis, P., Ntountoumi, C., Botou, M., Yalelis, V., Lazou, P., Tatsaki, E., Mossialos, D. et al. (2018) NAT/NCS2-hound: a webserver for the detection and evolutionary classification of prokaryotic and eukaryotic nucleobase-cation symporters of the NAT/NCS2 family. GigaScience 7, https://doi.org/10.1093/gigascience/giy133.
Coyne, S., Chizzali, C., Khalil, M.N.A., Litomska, A., Richter, K., Beerhues, L. and Hertweck, C. (2013) Biosynthesis of the antimetabolite 6-thioguanine in Erwinia amylovora plays a key role in fire blight pathogenesis. Angew Chem Int Ed 52, 10564-10568.
Coyne, S., Litomska, A., Chizzali, C., Khalil, M.N.A., Richter, K., Beerhues, L. and Hertweck, C. (2014) Control of plant defense mechanisms and fire blight pathogenesis through the regulation of 6-thioguanine biosynthesis in Erwinia amylovora. ChemBioChem 15, 373-376. https://doi.org/10.1002/cbic.201300684.
Datsenko, K.A. and Wanner, B.L. (2000) One-step inactivation of chromosomal genes for Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97, 6640-6645.
de Koning, H. and Diallinas, G. (2000) Nucleobase transporters. Mol Membr Biol 17, 75-94. https://doi.org/10.1080/09687680050117101
Eastgate, J.A., Thompson, L., Milner, J., Cooper, R.M., Pollitt, C.E. and Roberts, I.S. (1997) Identification of a nonpathogenic Erwinia amylovora guaB mutant. Plant Pathol 46, 594-599.
Feistner, G. and Staub, C.M. (1986) 6-Thioguanine from Erwinia amylovora. Curr Microbiol 13, 95-101.
Funk, A., Huntley, R.B., Mourad, G.S. and Schultes, N.P. (2020) A nucleobase cation symporter 2, EaXanP, from Erwinia amylovora transports xanthine. J Plant Pathol https://doi.org/10.1007/s42161-020-00584-5.
Karatza, P. and Frillingos, S. (2005) Cloning and functional characterization of two bacterial members of the NAT/NCS2 family in Escherichia coli. Mol Membr Biol 22, 251-261.
Klee, S.M., Sinn, J.P., Finley, M., Allman, E.L., Smith, P.B., Aimufua, O., Sitther, V., Lehman, B.L. et al. (2019a) Erwinia amylovora auxotrophic mutant exometabolomics and virulence on apples. Appl Environ Microbiol https://doi.org/10.1128/AEM.00935-19.
Klee, S.M., Sinn, J.P. and McNellis, T.W. (2019b) The apple fruitlet model system for fire blight disease. In Plant Innate Immunity, Methods in Molecular Biology ed. Gassmann, W. V1991, pp. 187-198. New York, NY: Humana.
Kourkoulou, A., Scazzochio, C., Frillingos, S., Mikros, E., Byrne, B. and Diallinas, G. (2018) Nucleobase-ascorbate-transporter (NAT) family. In Encyclopedia of Biophysics ed. Roberts, G. and Watts, A. Berlin, Heidelberg: Springer. https://doi.org/10.1007/978-3-642-35943-9_10090-1.
Kozmin, S.G., Stepchenkova, E.I., Chow, S.C. and Schaaper, R.M. (2013) A critical role for the putative NCS2 nucleobase permease YjcD in the sensitivity of Escherichia coli to cytotoxic and mutagenic purine analogs. MBio 4, https://doi.org/10.1128/mBio.00661-13.
Lu, F., Li, S., Jiang, Y., Jiang, J., Fan, H., Lu, G., Deng, D., Dang, S. et al. (2011) Structure and mechanism of the uracil transporter UraA. Nature 472, 243-246.
Mansfield, T.A., Schultes, N.P. and Mourad, G.S. (2009) AtAzg1 and AtAzg2 comprise a novel family of purine transporters in Arabidopsis. FEBS Lett 583, 481-486.
Mourad, G.S., Tippmann-Crosby, J., Hunt, K.A., Gicheru, Y., Bade, K., Mansfield, T.A. and Schultes, N.P. (2012) Genetic and molecular characterization reveals a unique nucleobase cation symporter 1 in Arabidopsis. FEBS Lett 586, 1370-1378.
Nguyen, J.N., Schein, J.R., Hunt, K.A., Tippmann-Feightner, J.A., Rapp, M., Stoffer-Bittner, A.J., Nalam, V.J., Funk, A.M. et al. (2020) Functional characterization of the sole nucleobase cation symporter 1 of Nicotiana sylvestris reveals a broad solute specificity profile. Plant Gene 22, 100226. https://doi.org/10.1016/j.plgene.2020.100226.
Papakostas, K. and Frillingos, S. (2012) Substrate selectivity of YgfU, a uric acid transporter from Escherichia coli. J Biol Chem 287, 15684-15695.
Papakostas, K., Botou, M. and Frillingos, S. (2013) Functional identification of the hypoxanthine/guanine transporters YjcD and YgfQ and the adenine transporters PurP and YicO of Escherichia coli K-12. J Biol Chem 288, 36827-36840.
Patching, S.G. (2018) Recent developments in nucleobase cation symporter-1 (NCS1) family transport proteins from bacteria, archaea, fungi and plants. J Biosci 43, 797-815.
Piqué, N., Miñana-Galbis, D., Merino, S. and Tomás, J.M. (2015) Virulence factors of Erwinia amylovora: a review. Int J Mol Sci 16, 12836-12854.
Pulawska, J., Kalužna, M., Warabieda, W. and Mikicinski, A. (2017) Comparative transcriptomic analysis of a lowly virulent strain of Erwinia amylovora in shoots of two apple cultivars -susceptible and resistant to fire blight. BMC Genom 18, 868. https://doi.org/10.1186/s12864-017-4251-z.
Ramos, L.S., Lehman, B.L., Peter, K.A. and McNellis, T.W. (2014) Mutation of the Erwinia amylovora argD gene causes arginine auxotrophy, nonpathogenicitgy in apples and reduced virulence in pears. Appl Environ Microbiol 80, 6739-6749.
Ramos, L.S., Sinn, J.P., Lehman, B.L., Pfeufer, E.E., Peter, K.A. and McNellis, T.W. (2015) Erwinia amylovora pyrC mutant causes fire blight despite pyrimidine auxotrophy. Lett Appl Microbiol 60, 572-579.
Rapp, M., Schein, J., Hunt, K.A., Nalam, V., Mourad, G.S. and Schultes, N.P. (2016) The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility. Protoplasma 253, 611-623.
Ritchie, R.J. and Prvan, T. (1996) Current statistical methods for estimating the Km and Vmax of Michaelis-Menten kinetics. Biochem Ed 24, 16-206.
Schein, J., Hunt, K.A., Minton, J., Schultes, N.P. and Mourad, G.S. (2013) The nucleobase cation symporter 1 from Chlamydomonas reinhardtii and the evolutionary distant Arabidopsis thaliana share function and establish a plant-specific solute transport profile. Plant Physiol Biochem 70, 52-60.
Schröpfer, S., Böttcher, C., Wöhner, T., Richter, K., Norelli, J., Rikkerink, E.H.A. et al. (2018) A single effector protein, AvrRpt2EA, from Erwinia amylovora can cause fire blight disease symptoms and induces a salicylic acid-dependent defense response. Mol Plant-Microbe Interact 31, 1179-1191. https://doi.org/10.1094/MPMI-12-17-0300-R.
Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.
van der Zwet, T., Orolaza-Halbrendt, N. and Zeller, W. (2012) Fire Blight: History, Biology, and Management. St. Paul, MN: American Phytopathological Society Press.
Wang, L. and Beer, S.V. (2006) Application of signature-tagged mutagenesis to the study of virulence of Erwinia amylovora. FEMS Microbiol Lett 265, 164-171. https://doi.org/10.1111/j.1574-6968.2006.00476.x.
Wensing, A., Gernold, M., Jock, S. and Jansen, R. (2014) Identification and genetics of 6-thioguanine secreted by Erwinia species and its interference with the growth of other bacteria. Mol Genet Genomics 289, 215-223. https://doi.org/10.1007/s00438-013-0805-1.
Wöhner, T.W., Richter, K., Sundin, G.W., Zhao, Y., Stockwell, V.O., Sellmann, J., Flachowsky, H., Hanke, M.-V. et al. (2018) Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene-for-gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus ‘Evereste’. Plant Pathol 67, 938-947. https://doi.org/10.1111/ppa.12784.
Yu, X., Yang, G., Yan, C., Baylon, J.L., Jiang, J., Fan, H., Lu, G., Hasegawa, K. et al. (2017) Dimeric structure of the uracil:proton symporter UraA provides mechanistic insights into the SLC4/23/26 transporters. Cell Res 27, 1020-1033. https://doi.org/10.1038/cr.2017.83