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

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

<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

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

TEM micrographs of negatively stained self-assembly products of recombinant purified S-layer protein SplA.

<p>(A) Cylindrical self-assembly products of the recombinant purified S-layer protein SplA of <i>P. larvae</i> 04-309 (ERIC II); (B) Enlarged region of a cylinder showing the regular lattice; (C) Power spectrum of the S-layer patch indicated in (B); (D) S-layer lattice reconstruction.</p

Analysis of the virulence potential of SplA using exposure bioassays.

<p>(A) Total mortality of <i>P. larvae</i> 04-309 Δ<i>spl</i>A and the parent wild-type strain <i>P. larvae</i> 04-309 was assessed in exposure bioassays. Larvae infected with the SplA-knockout mutant showed a significantly decreased mortality (paired T Test, p-value = 0.0098) although the same amount of spores was fed. Data represent mean values ± SEM of three independent infection assays. (B) To obtain the time course of infection and the LT (lethal time), cumulative mortality of <i>P. larvae</i> 04-309 Δ<i>spl</i>A (open circles) and the parent wild-type strain <i>P. larvae</i> 04-309 (filled circles) was assessed in exposure bioassays and plotted against time post infection (days). 100% represented all larvae that died from <i>P. larvae</i> infection during the course of the experiment. No significant difference between both strains in the time course of infection could be observed (p-value = 0.6767; data represent mean values ± SD of three independent exposure bioassays and were analyzed by an unpaired Student's T Test). (C) To evaluate the stability of the knockout mutation and to verify that bacteria killing the larvae in the mutant groups still carried the mutation <i>P. larvae</i> isolated from dead larvae of the mutant groups were analyzed for the presence of the intron cassette. The amplicon of the knockout mutant <i>P. larvae</i> 04-309 Δ<i>spl</i>A is about 900 bp longer than the amplicon from the wild-type <i>P. larvae</i> strain (04-309 wt). Representative results for ten isolates are shown.</p

Adhesion of <i>P. larvae</i> 04-309 Δ<i>spl</i>A and <i>P. larvae</i> 04-309 wild-type to primary pupal gut cells.

<p>(A) Vitality of primary pupal gut cells after 6 days of cultivation was analyzed using Mitotracker Red FM (Invitrogen) to specifically label active mitochondria indicative for live cells. Cells were microscopically analyzed using Differential Interference Contrast (DIC) (left) as well as TexRed (mitochondria) and DAPI (nuclei) filters to visualize fluorescence signals (right). (B) Percentage of <i>P. larvae</i> 03-189 wild-type (03-189 wt; ERIC I, naturally SplA-deficient) and <i>P. larvae</i> 04-309 Δ<i>spl</i>A (SplA knockout mutant) associated to pupal gut cells after 60 min of incubation, extensive washing, and cell lysis; results are presented as mean ± SEM of three independent-experiments and are related to cell-associated bacteria of the wild-type strain <i>P. larvae</i> 04-309 (04-309 wt; ERIC II), i.e. the amount of <i>P. larvae</i> 04-309 cell-associated bacteria in each experiment equalled 100%. Data were analyzed by one-way analysis of variance (ANOVA) followed by the Bonferroni post-hoc test (***p<0.001).</p

Analysis of the genomic and protein sequence of <i>P. larvae</i> SplA.

<p>(A) In contrast to <i>P. larvae</i> ERIC II representatives, all <i>P. larvae</i> ERIC I strains investigated showed an additional adenin at position 894 (bold and highlighted by a star), leading to a frameshift mutation causing a premature translational stop at TAA (bold and underlined). This obviously results in the observed lack of a functional SplA in representatives of <i>P. larvae</i> ERIC I. (B) For SLH-domain prediction of SplA the CDD (Conserved domains and protein classification, NCBI) was applied. Two predicted domains with homology to SLH domains of other <i>Bacillaceae</i> (<i>B. anthracis</i>, <i>B. weihenstephanensis</i>) but also to SLH domains of other genera were identified. <i>P. larvae</i> SLH 1 domain of SplA (upper panel) includes residues 117–164 of SplA and exhibits homologies to several SLH domains of <i>Bacillaceae</i>. <i>P. larvae</i> SLH 2 domain of SplA (lower panel) includes residues 188–220 and exhibits homologies not only to other <i>Bacillaceae</i> but also to SLH domains of other genera. Identical amino acids are marked in black, conserved amino acids are highlighted in dark grey and blocks of similar residues are presented with a light grey background. Strains showing homologous domains are listed alphabetically.</p

Verification of the knockout of <i>spl</i>A in 04-309 (ERIC II) on genome and proteome level.

<p>(A) Verification of the successful knockout of <i>spl</i>A in <i>P. larvae</i> 04-309 (ERIC II) by the targetron knockout system is demonstrated on genome level. Primers flanking insertion site 101 in <i>spl</i>A amplify a 1530 bp product in the wild-type (04-309 wt), whereas the amplicon of the mutant <i>P. larvae</i> strain (04-309 Δ<i>spl</i>A) migrated at 2445 bp as expected. (B) Proteome analyses of <i>P. larvae</i> knockout mutant 04-309 Δ<i>spl</i>A and the parent wild-type <i>P. larvae</i> strain (04-309 wt) is shown. Cytosolic proteins extracted from bacteria in the stationary growth phase were separated by 2D-SDS-PAGE and stained with PageBlue (Fermentas). The protein patterns differed significantly in the occurrence of the highlighted protein spot (red circle) recently identified as a putative S-layer protein by mass spectrometry. The conspicuous spot of the <i>P. larvae</i> 04-309 wild-type was lacking in the knockout mutant <i>P. larvae</i> 04-309 Δ<i>spl</i>A.</p