Liquid chromatography (LC) ESI-negative mass spectrometry (MS) analytics of ethyl acetate extracts of <i>P. larvae</i> secretomes.

<p>(A) Extracted ion chromatogram (<i>m/z</i> 881) of ethyl acetate extract <i>P. larvae</i> ATCC9545 wildtype (ERIC I; black line) and <i>P. larvae</i> ATCC9545 Δ<i>dhb</i>F (red line). (B) Extracted ion chromatogram (<i>m/z</i> 881) of ethyl acetate extract of <i>P. larvae</i> DMS25430 wildtype (ERIC II; black line) and <i>P. larvae</i> DMS25430 Δ<i>dhb</i>F (red line).</p

Exposure bioassays for assessing the role of bacillibactin during pathogenesis.

<p>Honey bee larvae were infected with wild type <i>P. larvae</i> (ATCC9545, DSM25430) or the corresponding mutant strains (ATCC9545 Δ<i>dhb</i>F, DSM25430 Δ<i>dhb</i>F) and total mortality (A, B) as well as cumulative mortality (C, D) were calculated for each group. Groups with non-infected larvae served as controls (E). All data represent mean values ± SD of three independent infection assays with 30 larvae each. Total mortality due to <i>P. larvae</i> infection was not significantly different (Mann–Whitney U test) between ATCC9545 wild type and ATCC9545 Δ<i>dhb</i>F (A; p-value = 0.800) and between DSM25430 wild type and DSM25430 Δ<i>dhb</i>F (B; p-value = 0.800). Cumulative mortality due to <i>P. larvae</i> infection was not significantly different (Kolmogorov–Smirnow test) between ATCC9545 wildtype (C, closed triangles) and ATCC9545 Δ<i>dhb</i>F (C, open triangles) (p-value = 0.660) and between DSM25430 wild type (D, closed circles) and DSM25430 Δ<i>dhb</i>F (D, open circles) (p-value = 0.999). Natural mortality in the control groups did not exceed 20% (E, closed squares).</p

Siderophore production in <i>P. larvae</i>.

<p>(A) Overlaid CAS agar plate assays for detection of siderophore production by differentially cultured <i>P. larvae</i> strains. One colony, each of ATCC9545 (ERIC I) or DSM25430 (ERIC II), was streaked out on different agar plates prepared from MYPGP, Sf900 and Insect-XPRESS media without (-) and with (+) Chelex 100 pre-treatment. Plates were incubated for 72 h at 37°C. Subsequently, the plates were overlaid with CAS agar and incubated for another 2 h. An orange halo around the bacteria grown on Chelex 100 pre-treated Insect-XPRESS agar plates (lower row) indicated the production of a siderophore. (B) Growth kinetics of <i>P. larvae</i> in Insect-XPRESS medium without and with pre-treatment with Chelex 100. Bacterial growth was monitored over 24 hours by measuring the optical density at 600 nm (OD₆₀₀) every hour. Three independent experiments (biological replicates) with four technical replicates each were performed; a representative curve is shown. Error bars represent SD from mean values.</p

Primers used for construction of gene knock-outs in <i>P. larvae</i> ATCC9545 (ERIC I) and DSM25430 (ERIC II).

<p>Primers used for construction of gene knock-outs in <i>P. larvae</i> ATCC9545 (ERIC I) and DSM25430 (ERIC II).</p

Liquid chromatography (LC) ESI-negative tandem mass spectrometry (MS/MS) analytics of bacillibactin.

<p>MS/MS analytics of ethyl acetate extract of <i>P. larvae</i> ATCC9545 (A; ERIC I), <i>P. larvae</i> DSM25430 (B; ERIC II), and of commercial bacillibactin (C) are shown. The single charged molecular ion of bacillibactin (m/z 881) was chosen for fragmentation with collision-induced dissociation (CID; 30 eV). Fragments are indicated in the spectrum; fragment <i>m/z</i> 249.1 is attributed to the decarboxylation of DHB-Gly-Thr.</p

The <i>dhb</i> gene cluster of <i>P. larvae</i>.

<p>(A) Gene arrangement of the <i>dhb</i> gene cluster of <i>P. larvae</i> in comparison to the <i>dhb</i> gene clusters of <i>B. subtilis</i> and <i>B. cereus</i> involved in the synthesis of bacillibactin and of the <i>pae</i> gene cluster of <i>P. elgii</i> involved in the synthesis of paenibactin. (B) Domain arrangement within the dimodular genes <i>pae</i>F (<i>P. elgii</i>) and <i>dhb</i>F (<i>B. subtilis</i>, <i>B. cereus</i>, <i>P. larvae</i>) and the predicted amino acids activated by the A-domains. A, adenylation domain; C condensation domain; T, thiolation domain; TE, thioesterase domain. Domain prediction was performed using SBSPKS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108272#pone.0108272-Anand1" target="_blank">[44]</a>.</p

Linking siderophore production and <i>dhb</i> gene cluster expression in <i>P. larvae</i>.

<p>(A) Overlaid CAS agar plate assays for detection of siderophore production in the wild type strains ATCC9545 and DSM25430 and in the corresponding mutant strains ATCC9545 Δ<i>dhb</i>F and DSM25430 Δ<i>dhb</i>F. Bacteria (one colony of each strain) were streaked out on agar plates prepared from Insect-XPRESS medium pre-treated with Chelex 100. Plates were incubated for 72 h at 37°C. Subsequently, the plates were overlaid with CAS agar and incubated for another 2 h. An orange halo around ATCC9545 and DSM25430 wild type bacteria indicated the production of a siderophore. This halo is missing in the corresponding mutant strains lacking bacillibactin expression thus linking siderophore synthesis to expression of the <i>P. larvae dhb</i> cluster. (B) Growth on MYPGP agar plates supplemented with increasing concentrations of the iron chelator 2,2′-dipyridyl (0 – 800 µM) of the wild type strains ATCC9545 and DSM25430 and the corresponding mutant strains ATCC9545 Δ<i>dhb</i>F and DSM25430 Δ<i>dhb</i>F. 10 µl of the cell suspension were spotted onto the plates, dried for 15 minutes, and incubated at 37°C for 72 h. For each culture, three biological replicates with three technical replicates each were performed. Representative data is shown.</p

Analysis of paenilarvin as putative virulence factor during pathogenesis.

<p>(A) At the age of 12 hours after egg hatching, honey bee larvae were infected with <i>P</i>. <i>larvae</i> DSM25430 wt and DSM25430 Δ<i>itu</i>. Daily mortality due to <i>P</i>. <i>larvae</i> infection was recorded and total mortality after 15 days was determined. Bars represent the percentage of exposed larvae that died from American Foulbrood after 15 days. Bars represent mean values ± SD of three independent exposure bioassays for each group and 30 larvae per group. No significant difference in total mortality (Student´s t-test; p-value = 0.89) was observed. (B) Daily mortality due to <i>P</i>. <i>larvae</i> infection was recorded and cumulative mortality per day was calculated. Curves represent mean cumulative mortality ± SD for each day of 3 replicates with 30 larvae each. Analysis of the data by two-way ANOVA did not reveal any significant difference between the two curves (p-value = 0.15).</p

Identification of the paenilarvins in infected larvae and in <i>P</i>. <i>larvae</i> secretomes.

<p>(A) Extracted ion chromatogram (EIC) for paenilarvins detected in experimentally infected bee larvae. Paenilarvins appear at retention times R_t = 9.06 and R_t = 9.69 minutes; the peak at 8.00 min does not correspond to the paenilarvins. The figure is representative for 4 independent experiments. (B) EICs for paenilarvins A/B/C of culture supernatants of <i>P</i>. <i>larvae</i> DSM25430 wt (red dotted line) producing paenilarvins and the knockout mutant <i>P</i>. <i>larvae</i> DSM25430 Δ<i>itu</i> (solid black line) not able to produce paenilarvins. (C) Masses for paenilarvins A/B extracted from the corresponding peak (R_t = 5 min) in (B).</p

Verification of the paenilarvin gene cluster inactivation mutant <i>P</i>. <i>larvae</i> DSM25430 Δ<i>itu</i>.

<p>(A) PCR analysis of the genomic region of <i>P</i>. <i>larvae</i> DSM25430 Δ<i>itu</i> with primers flanking the insertion site in the malonyl CoA-ACP transacylase gene of the paenilarvin gene cluster revealed successful intron insertion. The amplicons of the wild-type strain and of the mutant strain carrying the insertion migrated at their expected sizes of 1005 bp and 1905 bp, respectively. (B) Growth curves of the wild-type strain <i>P</i>. <i>larvae</i> DSM25430 wt (closed circles) and the mutant strain DSM25430 <i>Δitu</i> (open circles) in liquid broth did not differ significantly (two-way-ANOVA, p-value = 0.15). Data points represent the mean ± SEM and consisted of three biological replicates with three technical replicates each.</p