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Transcript
Multidrug efflux (MDE) transporters are major contributors to bacterial resistance towards antibiotics. In contrast to the well-understood role of MDE in clinically relevant microbes, only few data are available about MDE transporters in environmental bacteria. Comparisons of genome sequences revealed that MDE transporters are ubiquitous in nature. The highest numbers are found in soil- or plantassociated bacteria. This finding argues against the tenet that these systems have arisen recently in clinically relevant pathogens as a result of extensive exposure to antibiotics. Instead, they may play important physiological roles in the extrusion of naturally occurring toxic substances. The overall goal of our research program is to identify and characterize MDE pumps in the plant pathogenic bacteria Pseudomonas syringae and Erwinia amylovora and to gain in-depth knowledge about their regulation and natural functions. Our model strains infect different plants, each producing a unique spectrum of antimicrobial metabolites. MDE pumps may play an important role in the adaptation of plant pathogenic bacteria to its respective host plants by protecting them against plant antimicrobials. The identification and characterization of new MDE pumps in environmental bacteria is important to learn more about their physiological role. Moreover, the genes encoding such pumps can easily be interchanged between bacterial species leading to new multidrug resistant pathogens. One aim of our research is to identify all MDE pumps in Pseudomonas syringae pv. tomato DC3000. We use a microarray approach to identify transporters that are expressed after treatment with antibiotics, antimicrobial plant metabolites, and in planta. Putative MDE proteins are cloned to determine substrate specificity, to search for natural substrates, and to study their transcriptional regulation. The multiplicity of multidrug transporter in the bacterial genomes is probably due to the fact that they are fulfilling a broad diversity of roles in the export of different hydrophobic substrates and many of them may only transport drugs opportunistically because of the accommodating nature of their substrate binding pockets. To learn more about their natural functions and their role during pathogenesis, we analyze the expression of these transporters during infection of the host plant. In another project we develop and establish new biochemical and biophysical methods to characterize MDE transporter. We would like to understand the molecular basis of how do MDE pumps bind and transport multiple structurally unrelated substrates. We will use NorM from Erwinia amylovora as model transporter. In our previous studies, we could show that NorM is involved in the resistance of E. amylovora towards antibiotics produced by other bacteria found on plants. NorM belongs to the multidrug and toxin extrusion (MATE) family of transporters. We use a microfluidic device to characterise transport through MDE transporters with fluorescence. Purified NorM is reconstituted into giant unilamellar liposomes that are forced to fuse with a small hole within the microfluidic device to form a planar lipidbilayer membrane. Confocal laser scanning microscopy is used to measure the transport of fluorescent molecules by the MDE pump from one side of the membrane to the other. To elucidate the molecular mechanism underlying the Na+/multidrug antiport by transporters of the MATE family, we will introduce a series of point mutations by site-directed mutagenesis and analyze the transport activities for different substrates and Na+. Figure Legends Fig. 1. Minimal Inhibitory Concentration (MIC) - the lowest concentration of an antibiotic that completely stops visible cell growth. Fig. 2. Antibiotic-resistance mechanisms in bacteria.