The predicted domain structure and function of B and C-subunits.

<p>(<b>A</b>) Comparisons of domain predictions of B+C sub-complex proteins and the <i>Salmonella</i> SpvB protein. Note, neither the B nor C -subunits contain a predicted type II signal leader present in SpvB. Note the “core” domain represents a highly conserved sequence common to all Rhs/TccC family proteins located adjacent to the variable C-terminal regions. The predicted OspA-like structural domains of the B and C proteins are shown in addition to YD-repeat sequences common to these protein families. The labels “inner membrane” and “outer membrane” on the N-terminal domains of the C and B -subunits reflect their secretion roles. (<b>B</b>) A diagrammatic summary of the fates of B and C-subunit proteins when produced either independently or together. Cellular compartments shown include supernatant (SUP), outer membrane (OM), periplasm (PERI), inner membrane (IM) and cytoplasm (CYT). The C and N-termini of the TcdB and TccC proteins are indicated. The straight and curved arrows show the ability (or not) of N-terminal domains to facilitate aspects of the export process.</p

The cosmid model for heterologous TC synthesis and secretion in <i>E. coli</i>.

<p>(<b>A</b>) Map of cosmid (c1AH10) containing a portion of the <i>P. luminescens</i> W14 <i>tcd</i> pathogenicity island (bounded by dotted lines). Both strands are shown and the ORFs encoded on these are show as boxes. TC genes are shaded in grey and the <i>pdl1</i> release lipase gene is hatched <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003644#ppat.1003644-Yang1" target="_blank">[27]</a>. Inverted triangles show the location of transposon insertion mutants. The AKO, BKO and CKO insertion points are at nucleotides 271, 730 and 562 respectively, with respect to the first nucleotides of the ORFs. Note “H3” represents a cosmid clone in which the transposon has inserted into the vector backbone outside of the insert and therefore contains the full intact functional locus. (<b>B</b>) Western blot analysis of whole cells (200 µl culture pellet) and concentrated supernatants (1.2 ml) from the cosmid model of various transposon insertion mutant clones. Supernatants are from 2 day old cultures grown at 28°C in LB medium. Antibodies used were raised against the C-terminus of the B-subunit, TcdB1. Two independent B1 KO clones where tested (bar). An antibody directed against β-lactamase (Anti-Bla) is used as a loading control for the quality of the soluble and membrane fractions in this and subsequent blots. Note that knock out (KO) of the C-subunit <i>tccC5</i> gene (CKO) prevents secretion of the B-subunit (*), while KO of the A-subunit <i>tcdA1</i> gene (AKO) does not prevent B-subunit secretion (arrow). Bioassays of these same samples can be seen in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003644#ppat.1003644.s001" target="_blank">Figure S1</a>.</p

The N-terminus of the C-subunit is essential for secretion of the B+C sub-complex.

<p>(<b>A</b>) Arabinose inducible (P<i>ara</i>) <i>E. coli</i> heterologous B+C sub-complex expression constructs (in pBAD30) containing an intact copy of <i>tcdB1</i> and different modified <i>tccC</i> genes. Native translation initiation regions were used. The <i>tccC5</i> genes each contain a C-terminal FLAG tag at different locations and are designated; BC5, BC<i>sm</i> and BC<i>osp</i>. The hatched region on the <i>tccC</i> genes represents a region highly conserved in “Rhs/C-subunit” family proteins. Construct “BC5” encodes the full length of <i>tccC5</i>; “BC<i>sm</i>” encodes the first 668 amino acids of TccC5 and “BC<i>osp</i>” the first 600 amino acids of TccC5. “B” represents a <i>tcdB1</i> only construct and “BC1” represents a construct for co-expression of the <i>tcdB1</i> and <i>tccC1</i> genes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003644#ppat.1003644-Waterfield4" target="_blank">[22]</a>. The “V” negative control samples are from <i>E. coli</i> harbouring the empty expression plasmid pBAD30 only. (<b>B</b>) Western blot analysis of cells and concentrated supernatants of these constructs using an anti-peptide antibody raised against the C-terminus of B-subunit, TcdB1. The arrow indicator shows the secreted TcdB1. An anti β-lactamase (Anti-Bla) Western blot is included to provide a control for sample quality and loading amount. (<b>C</b>) Western blot analysis of these same cell and concentrated supernatant samples using an anti-FLAG tag antibody to determine the location of the tagged truncated C-subunit proteins, as indicated by the asterisk.</p

Cosmid complementation bioassay showing the B+C sub-complex secretion is independent of the A-subunit.

<p>Mean larval weight (MLW) gain of cohorts of <i>M. sexta</i> neonates (n = 10) fed mixtures of sonicated cell extracts (L) and culture supernatants (S), at dilutions of 1∶5, 1∶10 or 1∶20. Samples were mixed at a ratio of 50 µl of lysate (L) to 950 µl of supernatants (S). Samples were taken from the cosmid mutant AKO (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003644#ppat-1003644-g001" target="_blank">figure 1</a>) and pBAD30 expression constructs containing either nothing, “V”, or the <i>tcdA1</i> gene only, “pA” <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003644#ppat.1003644-Waterfield4" target="_blank">[22]</a>. Error bars represent the standard error. The more potent the toxic effect, the smaller the mean larval weight. A key using the data column fill pattern is given above the graph to assist in the interpretation of the predicted TC subunit contents of the test samples. The indicators * and ** are discussed in text. Two sample <i>t</i>-test comparisons were used to confirm statistical significance in mean weight differences at 99% confidence between the following samples discussed in the text; [AKO(S)+V(L) 1∶5] vs. [AKO(S)+pA(L) 1∶5]: t = 47.75, P<0.001, df = 18 and [AKO(S)+pA(L) 1∶20] vs. [V(S)+pA(L) 1∶20]: t = 11.84, P<0.001, df = 18.</p

The N-terminus of the B-subunit is required for normal B+C sub-complex synthesis and secretion.

<p>(<b>A</b>) Arabinose inducible <i>E. coli</i> heterologous production constructions encoding full length or N-terminal truncated <i>tcdB1</i> with <i>tccC5</i> containing C-terminal FLAG-tag fusions. Construct “BC5” encodes full-length <i>tcdB1</i> and tagged <i>tccC5</i>, “BΔC” encodes an N-terminal truncated copy of <i>tcdB1</i> and a tagged <i>tccC5</i>, “B” encodes full-length <i>tcdB1</i> only and “C5” encodes the tagged <i>tccC5</i> only. “V” represents pBAD30 vector only control. All constructs use the native translation initiation regions. (<b>B</b>) Western blot analysis of concentrated supernatants, membrane (inner and outer) and soluble fractions (cytoplasm and periplasmic contents) of these induced constructs using an anti-peptide antibody that was raised against the C-terminus of B-subunit. (<b>C</b>) Western blot analysis of these same samples using an anti-FLAG tag antibody. The asterisk and arrow indicators are discussed in the text. An anti β-lactamase (Anti-Bla) Western blot is included to provide a control for fractionation sample quality and loading amount.</p

The N-terminal 361 amino acids of TcdB1 are not essential for toxicity.

<p>Mean larval weight (MLW) gain of cohorts of <i>M. sexta</i> neonates (n = 10) fed 100 µl of mixtures of sonicated cell extracts (L) and culture supernatants (S). Arabinose inducible expression constructs tested include the pBAD30 vector only, “V”, the <i>tcdA1</i> only construct, “pA” (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003644#ppat-1003644-g004" target="_blank">figure 4</a>) and the BC5 and BΔC constructs (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003644#ppat-1003644-g006" target="_blank">figure 6</a>). Sample mixes were as either (i) 50 µl lysed pA sample complemented with 950 µl induced supernatant (left panel) or (ii) 50 µl lysed pA sample complemented with 50 µl lysed test sample and diluted with 900 µl of PBS. Error bars represent the standard error. The more potent the toxic effect, the smaller the mean larval weight. A key using the data column fill pattern is given above the graph to assist in the interpretation of the predicted TC subunit contents of the test samples. The indicators * and ** are discussed in text. Two sample <i>t</i>-test comparisons were used to confirm statistical significance in mean weight differences at 99% confidence between the following samples discussed in the text; [pA(L)+BΔC(S)] vs. [pA(L)+BC5(S)]: t = 26.68, P<0.001, df = 18. [pA(L)+V(L)] vs. [pA(L)+BΔC(L)]: t = 8.95, P<0.001, df = 18.</p

C-subunit secretion requires the B-subunit.

<p>(<b>A</b>) Arabinose inducible <i>E. coli</i> heterologous <i>tccC5</i> expression constructions in pBAD30. The <i>tccC5</i> genes each contain a C-terminal FLAG tag at different locations and are designated; C5, C<i>sm</i> and C<i>osp</i>. Native translation initiation regions were used. Construct “C5” encodes the full length of <i>tccC5</i>; “C<i>sm</i>” encodes the first 668 amino acids of TccC5 and “C<i>osp</i>” the first 600 amino acids of TccC5. (<b>B</b>) Western blot analysis of the cells and concentrated supernatants of these induced constructs using both an anti-peptide antibody raised against the N-terminus of TccC and an anti-FLAG tag antibody. An anti β-lactamase (Anti-Bla) Western blot is included to provide a control for sample quality and loading amount. Note despite good intracellular synthesis levels, no C-subunit protein could be detected in supernatant in the absence of the B-subunit.</p

Amorphous Nickel Hydroxide Nanosheets with Ultrahigh Activity and Super-Long-Term Cycle Stability as Advanced Water Oxidation Catalysts

Good conductivity is conventionally considered as a typical reference standard in terms of selecting water electrolysis catalysts. Electrocatalyst research so far has focused on crystal rather than amorphous due to poor conductivity. Here, we demonstrate that the amorphous electrocatalyst made of 3D honeycomb-like amorphous nickel hydroxide (Ni­(OH)₂) nanosheets synthesized by a simple, facile, green, and low-cost electrochemistry technique possesses ultrahigh activity and super-long-term cycle stability in the oxygen evolution reaction (OER). The amorphous Ni­(OH)₂ affords a current density of 10 mA cm^–2 at an overpotential of a mere 0.344 V and a small Tafel slope of 46 mV/dec, while no deactivation is detected in the CV cycles even up to 5000 times. We also establish that the short-range order, i.e., nanophase, of amorphous creates a lot of active sites for OER, which can greatly promote the electrochemical performance of amorphous catalysts. These findings show that the conventional understanding of selecting electrocatalysts with conductivity as a typical reference standard seems out of date for developing new catalysts at the nanometer, which opens a door ever closed to applications of amorphous nanomaterials as advanced catalysts for water oxidation

Promoting the Performance of Layered-Material Photodetectors by Alloy Engineering

The successful peeling of graphene heralded the era of van der Waals material (vdWM) electronics. However, photodetectors based on semiconducting transition metal dichalcogenides (TMDs), formulated as MX₂ (M = Mo, W; X = S, Se), often suffer either poor responsivity or long response time because of their high density of deep-level defect states (DLDSs). Alloy engineering, which can shift the DLDSs to shallow-level defect states, is proposed to be an efficient strategy to solve this problem. However, proof-of-concept is still lacking, which is probably because of the absence of a facile technology to grow high-quality alloyed TMDs. Here, we report the growth of large-scale and high-quality Mo_0.5W_0.5S₂ alloy films via pulsed laser deposition (PLD). We demonstrate that the resulting Mo_0.5W_0.5S₂ photodetector possesses a stable photoresponse from 370 to 1064 nm. The photocurrent exhibits positive dependence on both the source–drain voltage and incident power density, providing good tunability for multifunctional photoelectrical applications. We also establish that, because of the suppression of DLDSs with alloy engineering, the Mo_0.5W_0.5S₂ photodetector achieves a good responsivity of 5.8 A/W and a response time shorter than 150 ms. The working mechanism for the suppression of DLDSs in Mo_0.5W_0.5S₂ is unveiled by qualitatively analyzing the alloying-dressed band structure. In conclusion, the excellent performance of the PLD-grown Mo_0.5W_0.5S₂ photodetector may pave the way for next-generation photodetection. The approach shown here represents a fundamental and universal scenario for the development of alloyed TMDs

In Situ Growth of the Ni₃V₂O₈@PANI Composite Electrode for Flexible and Transparent Symmetric Supercapacitors

Because of the poor specific capacitance of transparent and flexible supercapacitors reported recently, exploring electrode materials with high electrochemical properties for such devices is still a big challenge. We reported that the Ni₃V₂O₈@PANI composite has been synthesized using an in situ chemical bath method. It was found that the synthesized Ni₃V₂O₈@PANI composite has outstanding electrochemical behaviors (specific capacitance value of 2565.7 F/g at 5 mV/s, wide potential window, good rate capability), which are much superior to those of Ni₃V₂O₈ and PANI electrodes. The improved electrochemical behaviors result from the synergistic effect between Ni₃V₂O₈ and PANI. A symmetric flexible and transparent supercapacitor was fabricated using the Ni₃V₂O₈@PANI composite as the working electrode. The device demonstrated a maximum areal capacitance of 58.5 mF/cm² at 5 mV/s and an energy density of 20.8 μW h/cm² with 1.6 V potential window. Furthermore, the capacitance retained nearly 88% of its original value after 20 000 galvanostatic charging and discharging cycles. These results favor the promising potential of the Ni₃V₂O₈@PANI composite as the electrode material for the application in flexible and transparent supercapacitors