Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Interaction Mediated by the Putative Tip Regions of MdsA and MdsC in the Formation of a <i>Salmonella</i>-Specific Tripartite Efflux Pump

<div><p>To survive in the presence of a wide range of toxic compounds, gram-negative bacteria expel such compounds via tripartite efflux pumps that span both the inner and outer membranes. The <i>Salmonella</i>-specific MdsAB pump consists of MdsB, a resistance-nodulation-division (RND)-type inner membrane transporter (IMT) that requires the membrane fusion protein (MFP) MdsA, and an outer membrane protein (OMP; MdsC or TolC) to form a tripartite efflux complex. In this study, we investigated the role of the putative tip regions of MdsA and its OMPs, MdsC and TolC, in the formation of a functional MdsAB-mediated efflux pump. Comparative analysis indicated that although sequence homologies of MdsA and MdsC with other MFPs and OMPs, respectively, are extremely low, key residues in the putative tip regions of these proteins are well conserved. Mutagenesis studies on these conserved sites demonstrated their importance for the physical and functional interactions required to form an MdsAB-mediated pump. Our studies suggest that, despite differences in the primary amino acid sequences and functions of various OMPs and MFPs, interactions mediated by the conserved tip regions of OMP and MFP are required for the formation of functional tripartite efflux pumps in gram-negative bacteria.</p></div

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Sequence comparisons of the putative tip regions of MdsA and MdsC.

<p>(A) Sequence alignment of the RLS motifs from three distinct adapter proteins. The corresponding heptad positions are marked in bold. The three conserved residues are highlighted in the black box. (B) Conserved amino acid residues in repeat 1 and repeat 2 in the aperture tip region of OMP. Repeat sequences in the tip regions are shown in bold, and conserved residues are shown in black boxes. Se, <i>Salmonella enterica</i>; Ec, <i>Escherichia coli</i>; Pa, <i>Pseudomonas aeruginosa</i>; Vc, <i>Vibrio cholerae</i>. (C) A modeled complex structure of the α-hairpin tip regions of MdsA and MdsC structure, based on the adaptor bridging model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100881#pone.0100881-Xu4" target="_blank">[23]</a>. The residues mentioned in the text are shown in the stick representations.</p

Interaction between MdsC or MdsC variants and MdsA <i>in vivo</i>.

<p>(A) Protein expression of hexahistidine-tagged MdsC and MdsC variants (MdsC-G220A, MdsC-G433A, and MdsC-L441R), MdsA-Flag and MdsB-Myc was detected by western blotting. (B) The <i>in vivo</i> interaction between MdsA and MdsC or MdsC variants was detected by using the chemical-crosslinker DSP. <i>S. enterica</i> ATCC14028S<i>ΔacrABΔmdsABC::Cm5ΔtolC::Tn10</i> cells coexpressing c-Flag-tagged MdsA, c-Myc-tagged MdsB, and hexahistidine-tagged wild-type (WT) or mutant MdsC (G220A, G433A, and L441R) are shown. All cultures were treated with (+) or without (−) DSP. Affinity-purified MdsC or MdsC variants and crosslinked MdsA were separated by SDS-PAGE and immunoblotted using monoclonal anti-His-tag and anti-Flag-tag antibodies.</p

The <i>in vivo</i> effect of mutations at the aperture tip region of MdsC in the absence of functional MdsA, MdsB, and TolC^a.

a<p>The <i>in vivo</i> effect of mutations in the aperture tip region of MdsC when wild-type MdsA and MdsB were disrupted was determined by measuring the resistance of the ATCC14028S<i>ΔacrABΔmdsABC::Cm5ΔtolC::Tn10</i> strain to several substrates. MdsC and its variants were expressed from pMdsC2, pMdsC2-G220A, pMdsC2-G433A, and pMdsC2-L441R.</p>b<p>All MIC measurements were done in triplicate. The concentrations of crystal violet and acriflavine used to determine the MICs were 0, 1, 2, 4, 8, 16, and 32 µg/mL. The concentrations of rhodamine 6G and methylene blue used to determine the MICs were 0, 4, 8, 16, 32, 64, 128, and 256 µg/mL. The vancomycin concentrations used to determine the MICs were 0, 300, 400, 500, 600, 700, and 800 µg/mL.</p

The <i>in vivo</i> effect of mutations at the aperture tip region of MdsC^a in the presence of wild type MdsA and MdsB^a.

a<p>The <i>in vivo</i> effect of mutations in the aperture tip region of MdsC was determined by measuring the resistance of the ATCC14028S<i>ΔacrABΔmdsABC::Cm5ΔtolC::Tn10</i> strain to several substrates. MdsC and its variants were expressed from pMdsABC2, pMdsABC2 MdsC-G220A, pMdsABC2 MdsC-G433A, pMdsABC2 MdsC-L441R.</p>b<p>All MIC measurements were done in triplicate. The concentrations of crystal violet and acriflavine used to determine the MICs were 0, 1, 2, 4, 8, 16, and 32 µg/mL. The concentrations of rhodamine 6G and methylene blue used to determine the MICs were 0, 4, 8, 16, 32, 64, 128, and 256 µg/mL.</p>c<p>The vancomycin concentrations used to determine the MICs were 0, 300, 400, 500, 600, 700, and 800 µg/mL.</p>d<p>None, strain carrying the empty vector pKAN6B instead of a pMdsABC2 variant.</p

Physical interaction between MdsA or MdsA variants and MdsC <i>in vivo</i>.

<p>(A) Protein expression of Flag-tagged MdsA and MdsA variants (MdsA-R135D, MdsA-L139D, and MdsA-S146D), MdsB-Myc, and hexahistidine-tagged MdsC was detected by western blotting. (B) The <i>in vivo</i> interaction between MdsA and MdsC was analyzed by using the chemical-crosslinker DSP. <i>S. enterica</i> ATCC14028S<i>ΔacrABΔmdsABC::Cm5ΔtolC::Tn10</i> cells coexpressing c-flag-tagged wild-type (WT) or mutant MdsA (R135D, L139D, and S146D), c-myc-tagged MdsB, hexahistidine-tagged MdsC were tested. All cultures were treated with (+) or without (−) DSP. Affinity-purified MdsC and crosslinked MdsA and MdsA variants were separated by SDS-PAGE and immunoblotted using monoclonal anti-His-tag and anti-Flag-tag antibodies.</p

Membrane Fusion Proteins of Type I Secretion System and Tripartite Efflux Pumps Share a Binding Motif for TolC in Gram-Negative Bacteria

<div><p>The Hly translocator complex of <em>Escherichia coli</em> catalyzes type I secretion of the toxin hemolysin A (HlyA). In this complex, HlyB is an inner membrane ABC (ATP Binding Cassette)-type transporter, TolC is an outer membrane channel protein, and HlyD is a periplasmic adaptor anchored in the inner membrane that bridges HlyB to TolC. This tripartite organization is reminiscent of that of drug efflux systems such as AcrA-AcrB-TolC and MacA-MacB-TolC of <em>E. coli</em>. We have previously shown the crucial role of conserved residues located at the hairpin tip region of AcrA and MacA adaptors during assembly of their cognate systems. In this study, we investigated the role of the putative tip region of HlyD using HlyD mutants with single amino acid substitutions at the conserved positions. <em>In vivo</em> and <em>in vitro</em> data show that all mutations abolished HlyD binding to TolC and resulted in the absence of HlyA secretion. Together, our results suggest that, similarly to AcrA and MacA, HlyD interacts with TolC in a tip-to-tip manner. A general model in which these conserved interactions induce opening of TolC during drug efflux and type I secretion is discussed.</p> </div

Interaction between the HlyD RLT motif and the TolC α-barrel tip region.

<p>A. The MacA-TolCα hybrid (T) was coupled to the CNBr-activated resin or the inactivated resin by Tris and incubated with a MacA-HlyD hybrid protein (D24, D18, or D12; H). After washing, the resin was applied to the SDS-PAGE gel. Only the MacA-HlyD12 hybrid protein was bound to the MacA-TolCα-coupled resin. The <i>E. coli</i> MacA (M), which was known to bind to the MacA-TolCα hybrid protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040460#pone.0040460-Xu2" target="_blank">[22]</a>, was used as a positive control. B. Interaction of the <i>E. coli</i> MacA-HlyD hybrid (wild-type and mutants) and MacA-TolCα hybrid on a size-exclusion chromatographic column. Elution profiles of the wild-type MacA-HlyD12 hybrid and its mutants (R186A, L190A, T197A, and T197Y) were co-injected with the MacA-TolCα hybrid protein. The arrows with the molecular mass indicate the fractions corresponding to the calculated molecular size from the elution volume. The box around 560 kDa as a molecular size indicates complex formation from the two proteins. The elution profiles of MacA-HlyD12 hybrid proteins alone are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040460#pone.0040460.s001" target="_blank">Figure S1</a>. C. <i>In vitro</i> binding assay to confirm the results of the size-exclusion chromatography. The same proteins were used as in (B), and the experimental methods were used as in (A). Results were similar to those produced in (B), except for the augmented affinity between the MacA-HlyD12 hybrid T142A mutant and the MacA-TolCα hybrid.</p