Abstract
BackgroundThe Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. In order to survive and multiply in a host, microbes must be able to circumvent the toxic effects of antimicrobial plant compounds, such as flavonoids and tannins. E. amylovora uses multidrug efflux transporters that recognize and actively export toxic compounds out of the cells. Here, two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump.ResultsTo find the substrate specificities of the two efflux systems, we overexpressed the transporters in a hypersensitive mutant lacking the major RND pump AcrB. Both transporters mediated resistance to several flavonoids, fusidic acid and novobiocin. Additionally, MdtABC mediated resistance towards josamycin, bile salts and silver nitrate, and MdtUVW towards clotrimazole. The ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock was reduced. Quantitative RT-PCR analyses revealed that the expression of the transporter genes was induced during infection of apple rootstock. The polyphenolic plant compound tannin, as well as the heavy metal salt tungstate was found to induce the expression of mdtABC. Finally, the expression of the mdtABC genes was shown to be regulated by BaeR, the response regulator of the two-component system BaeSR, a cell envelope stress response system that controls the adaptive responses to changes in the environment.ConclusionsThe expression of MdtABC and MdtUVW is induced during growth of E. amylovora in planta. We identified the plant polyphenol tannin as inducer of mdtABC expression. The reduced ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock suggests that the efflux pumps are involved in resistance to plant antimicrobials, maybe including flavonoids, which were identified as substrates of both pumps. Furthermore, we found that the mdtABC operon belongs to the regulon of the two-component regulator BaeR suggesting a role of this RND transporter in the cell envelope stress response of E. amylovora.
Highlights
The Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster
Induction of mdtABC and mdtUVW expression by the response regulators BaeR and CpxR Owing to the interaction between the mdtABC promoter region and BaeR in the electrophoretic mobility shift assay (EMSA), we investigated whether overexpression of BaeR and CpxR may induce the expression of efflux pump genes in vivo
The aim of the present study was the characterization of the resistance-nodulation-cell division (RND)-type multidrug efflux pumps MdtABC and MdtUVW from the plant pathogen E. amylovora, causing fire blight disease of apple, pear, and other members of the Rosaceae family
Summary
The Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. Two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump. Fire blight, caused by the Gram-negative enterobacterium Erwinia amylovora, is a devastating disease of rosaceous plants in the subfamily Maloideae that has global economic importance for apple and pear production [1]. Among the transporter families containing multidrug efflux transporters, the resistance-nodulation-cell division (RND) family has been identified as the most relevant in terms of resistance to clinically important agents in Gram-negative bacteria [3,4]. The RND-type efflux pump AcrAB has been shown to be involved in virulence of E. amylovora conferring resistance to plant-borne antimicrobial compounds like apple phytoalexins [7]
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