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Characterization and Regulation of the Resistance-Nodulation-Cell Pletzer and Weingart BMC Microbiology 2014, 14:185 http://www.biomedcentral.com/1471-2180/14/185 RESEARCH ARTICLE Open Access Characterization and regulation of the Resistance-Nodulation-Cell Division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora Daniel Pletzer and Helge Weingart* Abstract Background: 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. 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. Results: To 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. Conclusions: The 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. Keywords: Plant pathogen, Fire blight, Erwinia amylovora, Multidrug efflux, RND transporter, MdtABC, MdtUVW, BaeR, CpxR * Correspondence: [email protected] School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany © 2014 Pletzer and Weingart; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pletzer and Weingart BMC Microbiology 2014, 14:185 Page 2 of 15 http://www.biomedcentral.com/1471-2180/14/185 Background of heavy metals, in particular, in resistance to zinc, copper Fire blight, caused by the Gram-negative enterobacterium and tungstate [15-17]. Erwinia amylovora, is a devastating disease of rosaceous In E. coli, the expression of the multidrug efflux pump plants in the subfamily Maloideae that has global eco- MdtABC is regulated by two stress response systems, Bae nomic importance for apple and pear production [1]. and Cpx. The BaeSR and CpxARP two-component signal Typical symptoms include flower necrosis, immature fruit transduction systems respond to damage of the cell enve- rot, shoot curvature (shepherd’s crook), blackened leaves lope; however, they differ with regard to specific inducers. (these generally remain attached to the plant), bacterial The BaeSR regulon responds to a wide range of environ- ooze secretion, and cankers on woody tissues. mental stresses, including spheroplast formation, overex- Production of phenolic compounds with antimicrobial pression of the PapG pilin under conditions leading to activity (e.g. flavonoids, tannins) is an important part of misfolding, and exposure to indole, tannins, flavonoids, the plant defense repertoire to limit the spread of patho- tungstate, and zinc [16,18-20]. The small core regulon of gens [2]. Successful phytopathogens must be able to BaeSR includes the BaeSR two-component system itself, circumvent the toxic effects of antimicrobial plant com- the RND-type transporters AcrD and MdtABC, and the pounds. An important resistance mechanism of bacteria periplasmic chaperone Spy [19]. The CpxRA regulon of against plant-borne antimicrobials involves efflux of these E. coli contains hundreds of genes, including periplas- compounds by so-called multidrug transporters. Among mic protein folding and degrading factors, peptidogly- the transporter families containing multidrug efflux trans- can metabolic enzymes, inner membrane proteins and porters, the resistance-nodulation-cell division (RND) regulators, and envelope-localized protein complexes, family has been identified as the most relevant in terms of such as pili and flagella [21-23]. The Cpx system com- resistance to clinically important agents in Gram-negative prises the sensor histidine kinase CpxA, the cytoplasmic bacteria [3,4]. Members of the RND family are able to response regulator CpxR, and CpxP, a periplasmic in- recognize and expel a broad range of antimicrobials from hibitor of CpxA [24]. The Cpx pathway is activated by a the cell [5,6]. The RND-type efflux pump AcrAB has been variety of stresses within the bacterial cell envelope, e.g., shown to be involved in virulence of E. amylovora confer- alkaline pH, alterations to the composition of the inner ring resistance to plant-borne antimicrobial compounds membrane, overexpression of misfolded envelope proteins, like apple phytoalexins [7]. and adhesion to hydrophobic surfaces sensed by the lipo- The functional RND-type efflux pump is a tripartite protein NlpE [23,25]. Hirakawa et al. [26] reported that the complex, consisting of the RND-type transporter protein overexpression of BaeR and CpxR causes up-regulation of located in the inner membrane, a periplasmic membrane RND pumps AcrD and MdtABC in E. coli.Analysisofthe protein, and an outer membrane channel [5]. The inner induction of multidrug transporter genes by indole re- membrane RND transporter is a homotrimer that uses vealed that the CpxR-mediated induction of acrD and the proton gradient as an energy source [8,9]. Associated mdtABC genes depends on the BaeSR two-component sys- with this homotrimeric structure is the so-called ‘rotat- tem [27]. These results indicate that BaeR is a primary ing mechanism’, a conformational change in the periplas- regulator, and CpxR enhances the effect of BaeR. mic core domain to export drug molecules [10]. The aim of this study was to characterize the hetero- However, not all members of the RND family follow a multimeric RND-type multidrug efflux pumps MdtABC homotrimeric organization. For example, the RND- and MdtUVW from E. amylovora Ea1189 and to deter- family efflux system MdtABC from E. coli possesses two mine the role of the two-component system regulators different RND transporters, MdtB and MdtC, which are BaeR and CpxR in regulation of these efflux systems. co-transcribed in an operon with the membrane fusion protein MdtA. It has previously been shown that the Results functional pump consists of a heteromultimeric unit Computational analysis of RND-type transporters from formed by two subunits of MdtB and one subunit of E. amylovora Ea1189 MdtC [11]. Furthermore, it has been suggested that MdtC Analysis of the genome sequence of E. amylovora revealed is involved in binding and transport of drugs and that the presence of two, hitherto not characterized, operons MdtB presumably induces the conformational change encoding heterotrimer-type RND-type efflux pumps. Both needed for transport using the proton translocation as an show homology to the mdtABC operon of E. coli.The energy source [12]. mdtABC genes of E. coli encode an RND system that is Previous genetic studies have demonstrated that the unusual in that it contains two different RND pump deletion of the heteromultimeric RND pump MdtABC genes, mdtB and mdtC, in addition to the membrane abolishes the resistance of E. coli and Salmonella enterica fusion protein gene, mdtA [11]. One of the heterotrimer- to β-lactams, novobiocin, SDS, and bile salts [13-15]. type RND efflux systems of E. amylovora shows a high de- Moreover, MdtABC has been implicated in detoxification gree of homology to the E. coli MdtABC transporter as Pletzer and Weingart BMC Microbiology 2014, 14:185 Page 3 of 15 http://www.biomedcentral.com/1471-2180/14/185 depicted in the phylogenetic tree in Figure 1. We accord- BaeSR is involved in a unique envelope stress response in ingly propose to name the operon encoding this trans- E. coli [18]. porter as mdtABC. The second, more distantly related Topological analysis of the RND
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