Intracellular distribution of Hfq-MT-associated electron-dense particles.

<p>Transformed MC4100 <i>hfq⁻ E. coli</i> cells were induced with different concentrations of arabinose (ARA) and grown in the presence of gold salts. Ultrathin-sections of vitrified and freeze-substituted cells are shown in all panels. (A) A bacterium induced with 0.001% ARA. This concentration reproduces the expression levels of Hfq during the stationary growth phase. Electron-dense particles are abundant both in the interior (asterisk) and the periphery (arrow) of the cell. The inset is an enlargement of the peripheral region marked with the dashed rectangle. (B) A transformed bacterium induced with 0.01% ARA showing a heavy accumulation of particles both in the interior (asterisk) and periphery (arrows) of the cell. The inset is a higher magnification view of the area marked with the dashed rectangle where it can be appreciated that some particles are slightly bigger than in bacteria induced with lower ARA concentrations. (C) Detail of the wall of a bacterium induced with 0.01% ARA and stained for 30 s with uranyl acetate (UA). Staining shows the real location of the outer membrane (OM) and confirms that the peripheral electron-dense particles delineate the inner membrane (IM). Extracellular granules are due to deposition of uranium on resin protrusions. (D) and (E) A transformed bacterium induced with 0.001% ARA showing intense signal in intracellular regions compatible with being the nucleoid (asterisk). (E) A high magnification view of the central area of the cell in (D) shows abundant small electron-dense particles, apparently arranged with a filamentous-like pattern (the arrows point to some of several such fibers). Bars: 100 nm (A, B, C and D), 50 nm (E and insets in A and B).</p

Hfq-MT expressed in <i>E. coli</i> wild-type <i>E. coli</i> cells and induced with 0.0025% ARA.

<p>Localization patterns of the small electron-dense particles associated with the fusion protein expressed in the presence of endogenous Hfq show some differences with respect to Hfq⁻ cells. (A) Arrows point to a double layer of particles separated by a gap clearly seen on the cell periphery. (B) In this gap particles arranged with a filamentous pattern are frequently seen at higher magnification (arrow). (C) Immunogold labeling with anti-LamB antibodies and a 15 nm colloidal gold conjugate confirms that the external layer of electron-dense particles corresponds to the outer bacterial membrane. (D–F) Labeling with anti-RNase E antibodies and a 10 nm colloidal gold conjugate shows labelling associated with the internal layer of electron-dense particles. Inset in (D) is a higher magnification view of the area marked with the dashed rectangle. (F) Higher magnification of the area marked with a dashed rectangle in (E). Black Arrows point to RNase E-conjugated colloidal gold particles associated with the inner layer of small particles (Hfq-MT). Bars: 50 nm (A, C, inset in D, and F), 25 nm (B), 100 nm (D and E).</p

Immunogold labeling on cryo-sections.

<p>(A) and (B) Double immunogold labeling of <i>hfq⁻ E. coli</i> cells expressing Hfq-MT treated with gold salts and induced with 0.01% ARA with a rabbit anti-Hfq polyclonal antibody followed by a goat anti-rabbit secondary antibody conjugated with 15 nm colloidal gold particles and a mouse anti-MT monoclonal antibody followed by a goat anti-mouse secondary antibody conjugated with 10 nm colloidal gold particles. Signals co-exist both in the interior (arrowhead in A) and the peripheral membranes (arrows in A and B) of these cells, confirming the presence of the fusion protein in both locations. (C) and (D) Double labelling with rabbit anti-S1 antibodies (and a 15 nm colloidal gold conjugate of a goat anti-rabbit secondary antibody) and mouse anti-MT antibodies (and a 10 nm colloidal gold conjugate of a goat anti-mouse secondary antibody) showing co-localization of Hfq-MT and S1 protein on the cell periphery (arrows). Bars: 100 nm.</p

Expression of Hfq-MT protein.

<p>Hfq-MT construct (A). The pBAD-Hfq-MT plasmid allowing the expression of Hfq-MT under the control of the arabinose pBAD promoter. Semi-quantitative Western Blot (B) Relative quantification of intracellular Hfq was achieved by using crude extracts of <i>(a) Hfq⁺</i> MC4100 in exponential phase (EP) and stationary phase (SP). <i>hfq⁻</i> cells are shown as a control to confirm that, in agreement with previous work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008301#pone.0008301-Sledjeski1" target="_blank">[39]</a>, in the absence of arabinose very little Hfq accumulated (The designations <i>hfq⁺</i> and <i>hfq⁻</i> indicate whether the chromosomal copy of the MC4100 strains are wild-type (+) or mutant (−)). <i>(b) Hfq⁺</i> and <i>hfq⁻</i> cells transformed with the plasmid pBAD-Hfq-MTtag. Percentages indicate the final concentrations of arabinose when added to the culture to induce expression of Hfq-MT from the pBAD promoter. The relative amounts of Hfq are shown as rectangles with error bars at the top. EP and SP refer to cultures in exponential phase and stationary phase, respectively.</p

Controls.

<p>(A) <i>hfq⁻</i> transformed bacteria expressing the Hfq-MT protein induced with 0.001% ARA) and grown without gold salts. A diffused, non-particulated electron-density is observed. (B) Bacterium expressing MT alone (without fused Hfq) treated with gold salts. Arrows point to small electron-dense particles while the arrowhead points to one of the biggest gold clusters. Bars 50 nm.</p