<i>P. alvei</i> CCM 2051^T Δ<i>slh</i>A cells lose the ability to swarm on LB-agar plates.

<p>The upper panel shows swarming cells of wild-type (first column), <i>P. alvei</i> Δ<i>slh</i>A (second column), <i>P. alvei</i> Δhag (third column), wild-type (pEXALV) (fourth column), <i>P. alvei</i> Δ<i>slh</i>A (pEXALV) (fifth column) and the complemented strain <i>P. alvei</i> Δ<i>slh</i>A_comp (sixth column) on 0.4% (upper panel), 1% (middle panel) and 1.5% (lower panel) LB-agar plates. The pictures represent one of three independent experiments.</p

One SLH domain is sufficient for binding of SlhA to native cell wall sacculi.

<p>Binding of (A) native SlhA and SlhA truncations to PG(+) and (B) PG(-) cell wall sacculi of <i>P. alvei</i> was tested. SlhA was truncated for either one (SlhA-SLH₁₂, lacking SLH domain 3), two (SlhA-SLH₁, lacking SLH domains 2 and 3) or all three (SlhA-w/o SLH) SLH domains. Cell extracts containing the SlhA protein versions were incubated (a) with and (b) without cell wall sacculi. After incubation the reactions were centrifuged to separate cell walls (with bound protein) from unbound protein. Analysis was done by SDS-PAGE (8-10% gels) followed by Western-immunoblotting using anit-His₆-antibody. The integrated intensity of the detected bands was determined using the Li‑Cor Odyssey Application Software 3.0.21 applying automatic background subtraction. 10 µl of each sample were loaded onto the gel. L, PageRuler^TM Plus Prestained Protein Ladder (Fermentas); S, supernatant; P, pellet; w/o, without. Results of the Western blots used for quantification are summarized in Table 3. The figure represents one of at least two independent repeats of the experiment.</p

Knockout of <i>slhA</i> and <i>hag</i> decreases biofilm formation of <i>P. alvei</i> CCM 2051^T cells.

<p>(A) Evaluation of the ability of cells of <i>P. alvei</i> CCM 2051^T wild-type, Δ<i>slh</i>A, Δ<i>hag</i>, wild-type (pEXALV), Δ<i>slh</i>A (pEXALV) carrying the pEXALV vector, and the complemented strain <i>P. alvei</i> Δ<i>slh</i>A_comp for biofilm formation using Crystal violet (CV) staining. Data represent mean values <u>+</u> SD of at least four independent experiments with each four replicates and were analyzed by the unpaired Student’s T Test. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) SEM analysis of <i>P. alvei</i> CCM 2051^T wild-type, Δ<i>slh</i>A, Δ<i>hag</i> and the complemented strain Δ<i>slh</i>A_comp showing an overview and enlarged view of the biofilm. Size bars are 20 µm for the upper panel and 5 µm for the lower panel. (C) CSLM analysis of <i>P. alvei</i> CCM 2051^T wild-type, Δ<i>slh</i>A, Δ<i>hag</i> and the complemented strain Δ<i>slh</i>A_comp stained with Hoechst 33258 showing a diagonally above view (upper panel) and a side view (lower panel) of a three day biofilm. Size bars are 20 µm.</p

Knockout of <i>slhA</i> does not alter flagella production of <i>P. alvei</i> CCM 2051^T cells.

<p>Electron microscopic view of <i>P. alvei</i> CCM 2051^T wild-type (first column), Δ<i>slh</i>A (second column) and Δ<i>hag</i> cells (third column). Flagella are clearly visible for wild-type and Δ<i>slh</i>A cells but no flagella are present for Δ<i>hag</i> cells.</p

Immunofluorescence microscopy of <i>P. alvei</i> CCM 2051^T Δ<i>slh</i>A cells co-displaying SlhA_EGFP and SpaA_His₆.

<p>For immunofluorescence staining of surface-located SpaA_His₆, a penta-His Alexa Fluor 532 conjugate for direct detection of the His₆-tagged SpaA was used. The TRITC and the GFP Long pass filter blocks were used for detection of Alexa Fluor 532 and EGFP, respectively. The upper three rows show the immunofluorescence microscopy pictures of cells harboring pSURF and co-displaying SlhA_EGFP (upper three rows, second pictures) and SpaA_His₆ (upper three panels, third pictures). <i>P. alvei</i> CCM 2051^T Δ<i>slh</i>A cells harboring pEXALV are shown as a control in the fourth panel. Corresponding brightfield images of the same cells are shown on the very left and overlays are shown on the very right of each panel.</p

Comprehensive Size-Determination of Whole Virus Vaccine Particles Using Gas-Phase Electrophoretic Mobility Macromolecular Analyzer, Atomic Force Microscopy, and Transmission Electron Microscopy

Biophysical properties including particle size distribution, integrity, and shape of whole virus vaccine particles at different stages in tick-borne encephalitis (TBE) vaccines formulation were analyzed by a new set of methods. Size-exclusion chromatography (SEC) was used as a conservative sample preparation for vaccine particle fractionation and gas-phase electrophoretic mobility macromolecular analyzer (GEMMA) for analyzing electrophoretic mobility diameters of isolated TBE virions. The derived particle diameter was then correlated with molecular weight. The diameter of the TBE virions determined after SEC by GEMMA instrumentation was 46.8 ± 1.1 nm. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) were implemented for comparison purposes and to gain morphological information on the virion particle. Western blotting (Dot Blot) as an immunological method confirmed biological activity of the particles at various stages of the developed analytical strategy. AFM and TEM measurements revealed higher diameters with much higher SD for a limited number of virions, 60.4 ± 8.5 and 53.5 ± 5.3 nm, respectively. GEMMA instrumentation was also used for fractionation of virions with specifically selected diameters in the gas-phase, which were finally collected by means of an electrostatic sampler. At that point (i.e., after particle collection), AFM and TEM showed that the sampled virions were still intact, exhibiting a narrow size distribution (i.e., 59.8 ± 7.8 nm for AFM and 47.5 ± 5.2 nm for TEM images), and most importantly, dot blotting confirmed immunological activity of the collected samples. Furthermore dimers and virion artifacts were detected, too