A Weekend of One Act Plays

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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

Tunable Physicomechanical and Drug Release Properties of In Situ Forming Thermoresponsive Elastin-like Polypeptide Hydrogels

With the continued advancement in the design and engineering of hydrogels for biomedical applications, there is a growing interest in imparting stimuli-responsiveness to the hydrogels in order to control their physicomechanical properties in a more programmable manner. In this study, an in situ forming hydrogel is developed by cross-linking alginate with an elastin-like polypeptide (ELP). Lysine-rich ELP synthesized by recombinant DNA technology is reacted with alginate presenting an aldehyde via Schiff base formation, resulting in facile hydrogel formation under physiological conditions. The physicomechanical properties of alginate–ELP hydrogels can be controlled in a wide range by the concentrations of alginate and ELP. Owing to the thermoresponsive properties of the ELP, the alginate–ELP hydrogels undergo swelling/deswelling near the physiological temperature. Taking advantage of these highly attractive properties of alginate–ELP, drug release kinetics were measured to evaluate their potential as a thermoresponsive drug delivery system. Furthermore, an ex vivo model was used to demonstrate the minimally invasive tissue injectability

Bacterial strains and plasmids used in this study.

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

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

Likelihood ratio tests of selective pressure on catarrhine <i>MOXD2</i> gene^a.

a<p>Models in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104085#pone-0104085-t003" target="_blank">Table 3</a> were compared using likelihood ratio test.</p>b<p>Twice the difference in log likelihood values between the two models compared.</p>c<p>Degree of freedom.</p>d<p>***, <i>P</i><0.001; **, <i>P</i><0.01; *, <i>P</i><0.05; ns, not significant.</p