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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.