Charakterisierung der Multidrug-Efflux-Transporter NorM und AcrAB in Erwinia amylovora

Frequently, plants are challenged by pathogenic microbes. Therefore, they developed an effective arsenal of defense mechanisms to combat these invaders. Secondary metabolites have been considered to be important players in the protection of plants against potential pathogens. They may be preformed compounds which are present in healthy plant tissue or synthesized de novo in response to pathogen attack. Successful pathogens must be able to circumvent the toxic effect of these phytoalexins. Multidrug efflux transporters which mediate resistance towards structurally unrelated compounds might confer tolerance to these phytoalexins. Particularly, studies on phytopathogenic fungi indicated that multidrug resistance transporters contribute to virulence by reducing the toxic effect of phytoalexins and only few data are available about transporters conferring multidrug resistance in phytopathogenic bacteria. In this work, the multidrug efflux pumps NorM and AcrAB of the plant pathogenic bacterium Erwinia amylovora were identified and their role in the pathogenesis of fire blight evaluated. AcrAB belongs to the RND-superfamily and NorM to the MATE-family which both utilize the transmembrane electrochemical gradient to drive transport. AcrAB conferred resistance to hydrophobic and amphiphilic toxins, and NorM particularly to hydrophobic cations. In contrast to NorM, the AcrAB transport system is characterized by broad substrate specificity. An acrB-deficient mutant was impaired in virulence on apple rootstock MM 106. Furthermore, it was susceptible towards extracts of leaves of MM 106 as well as against the apple phytoalexins phloretin, naringenin, quercetin, and (+)-catechin. The expression of acrAB was determined with the reporter gene egfp. The acrAB operon was up-regulated in vitro by phloretin and naringenin. The promoter activity of acrR was increased by naringenin. These results strongly suggest that the AcrAB transport system plays an important role as virulence factor of E. amylovora in resistance to apple phytoalexins and that this tolerance is required for successful colonization of a host plant. NorM does not contribute to virulence of E. amylovora on apple rootstock MM106. In planta assays revealed, that the norM mutant caused symptoms and established population densities in the plant tissue comparable to them of the wild type. In contrast, NorM of E. amylovora furnished protection against antibiotics produced by epiphytic P. agglomerans strains. Thus, the multidrug efflux transporter may be important for epiphytic fitness of the plant pathogen. It is of common knowledge that E. amylovora survives poorly on plant surfaces with exception of the stigmatic areas on pistils. The presence of high population densities of the fire blight pathogen provides for efficient dissemination from flower to flower by rain or flower visiting insects and more importantly, it favors the successful infection of a host plant. Mutation of norM in E. amylovora reduced tolerance to toxins produced by P. agglomerans strains which were isolated from apple and quince blossoms. Because temperature affects epiphytic multiplication of E. amylovora and the infection of the host plant, promoter activity of norM was analyzed by transcriptional fusion with the reporter gene uidA in vitro at 18°C and 28°C. The contribution of the multidrug efflux transporter NorM to resistance to epiphytic antibiotics is especially obvious at 18°C. This temperature is important for infection of a host because it warrants availability of moisture necessary for movement of the bacteria from the stigmas to natural openings into the floral cup. Thus, the plant pathogen needs to compete successfully with other microbes for site and nutrition resources to reach high population density at this temperature. Since P. agglomerans strains colonize similar habitats on the stigmatic surface as E. amylovora, the pathogen might has recruited multidrug efflux as a mechanism for the specific purpose of resistance to antibiotics produces by P. agglomerans strains.