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

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

Knockout of <i>slhA</i> causes changes in colony morphology and adhesion of <i>P. alvei</i> CCM 2051^T cells to agar plates.

<p>(A) Colony morphology of the wild-type strain (A, first picture), <i>P. alvei</i> CCM 2051^T Δ<i>slh</i>A (A, second picture), <i>P. alvei</i> CCM 2051^T Δ<i>slh</i>A_comp (A, third picture) and <i>P. alvei</i> CCM 2051^T Δ<i>hag</i> (A, fourth picture) on LB agar plates. (B) Adhesion test of wild-type, <i>P. alvei</i> Δ<i>slh</i>A, the complemented strain and <i>P. alvei</i> Δhag. The upper panel shows the wild-type (B, upper panel first picture), <i>P. alvei</i> Δ<i>slh</i>A (B, upper panel second picture), the complemented strain ((B, upper panel third picture) and <i>P. alvei</i> Δhag (B, upper panel third picture) before washing, the lower panel shows the same strains after the washing step. The pictures represent one of four independent experiments.</p

Schematic drawing of the SlhA protein (A) and genetic localization of the <i>slhA</i> gene in the SCWP biosynthesis locus of <i>P. alvei</i> CCM 2051^T (B).

<p>bp, base pairs; aa, amino acids; SP, signal peptide; CBD, carbohydrate binding domain; SLH, S-layer homology domain.</p

Knockout of <i>slhA</i> decreases Congo red staining of <i>P. alvei</i> CCM 2051^T cells.

<p>(A) Congo red plates of <i>P. alvei</i> CCM 2051^T wild-type, Δ<i>slhA</i>, Δ<i>slh</i>A_comp and Δ<i>hag</i> cultures. (B) Quantification of Congo red-binding in planktonic cultures at 37°C. Data represent mean values <u>+</u> SD of at least four independent experiments and were analyzed by the unpaired Student’s T Test. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001).</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

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

<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

Primers used for sequence analysis of <i>spl</i>A, screening of <i>P. larvae</i> isolates for <i>spl</i>A, gene knockout in <i>P. larvae</i> 04-309, and recombinant production of SplA.

<p>Primers used for sequence analysis of <i>spl</i>A, screening of <i>P. larvae</i> isolates for <i>spl</i>A, gene knockout in <i>P. larvae</i> 04-309, and recombinant production of SplA.</p

Analysis of <i>P. larvae</i> 04-309 versus <i>P. larvae</i> 04-309 Δ<i>spl</i>A.

<p>(A) Differences in colony morphology of the <i>spl</i>A knockout mutant <i>P. larvae</i> 04-309 Δ<i>spl</i>A on CSA and MYPGP agar in comparison to the parent wild-type strain <i>P. larvae</i> 04-309 were evident. (B) Growth of wild-type strain <i>P. larvae</i> 04-309 (black squares) and the <i>spl</i>A knockout mutant <i>P. larvae</i> 04-309 Δ<i>spl</i>A (black triangles) in liquid broth did not differ significantly (p-value = 0.878; unpaired Student's t-test). (C) Electron microscopy analysis revealed differences in cell morphology between the S-layer knockout mutant <i>P. larvae</i> 04-309 Δ<i>spl</i>A and the parent wild-type strain <i>P. larvae</i> 04-309. The mutant cells were rather elongated and assembled to longer chains than the wild-type cells. Bars represent 50 µm (2,000 fold) and 5 µm (16,000 fold).</p