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. 2014 Sep 19;9(9):e108272.
doi: 10.1371/journal.pone.0108272. eCollection 2014.

Production of the catechol type siderophore bacillibactin by the honey bee pathogen Paenibacillus larvae

Affiliations

Production of the catechol type siderophore bacillibactin by the honey bee pathogen Paenibacillus larvae

Gillian Hertlein et al. PLoS One. .

Abstract

The Gram-positive bacterium Paenibacillus larvae is the etiological agent of American Foulbrood. This bacterial infection of honey bee brood is a notifiable epizootic posing a serious threat to global honey bee health because not only individual larvae but also entire colonies succumb to the disease. In the recent past considerable progress has been made in elucidating molecular aspects of host pathogen interactions during pathogenesis of P. larvae infections. Especially the sequencing and annotation of the complete genome of P. larvae was a major step forward and revealed the existence of several giant gene clusters coding for non-ribosomal peptide synthetases which might act as putative virulence factors. We here present the detailed analysis of one of these clusters which we demonstrated to be responsible for the biosynthesis of bacillibactin, a P. larvae siderophore. We first established culture conditions allowing the growth of P. larvae under iron-limited conditions and triggering siderophore production by P. larvae. Using a gene disruption strategy we linked siderophore production to the expression of an uninterrupted bacillibactin gene cluster. In silico analysis predicted the structure of a trimeric trithreonyl lactone (DHB-Gly-Thr)3 similar to the structure of bacillibactin produced by several Bacillus species. Mass spectrometric analysis unambiguously confirmed that the siderophore produced by P. larvae is identical to bacillibactin. Exposure bioassays demonstrated that P. larvae bacillibactin is not required for full virulence of P. larvae in laboratory exposure bioassays. This observation is consistent with results obtained for bacillibactin in other pathogenic bacteria.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The dhb gene cluster of P. larvae.
(A) Gene arrangement of the dhb gene cluster of P. larvae in comparison to the dhb gene clusters of B. subtilis and B. cereus involved in the synthesis of bacillibactin and of the pae gene cluster of P. elgii involved in the synthesis of paenibactin. (B) Domain arrangement within the dimodular genes paeF (P. elgii) and dhbF (B. subtilis, B. cereus, P. larvae) 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 .
Figure 2
Figure 2. Siderophore production in P. larvae.
(A) Overlaid CAS agar plate assays for detection of siderophore production by differentially cultured P. larvae 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 P. larvae 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.
Figure 3
Figure 3. Linking siderophore production and dhb gene cluster expression in P. larvae.
(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 ΔdhbF and DSM25430 ΔdhbF. 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 P. larvae dhb 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 ΔdhbF and DSM25430 ΔdhbF. 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.
Figure 4
Figure 4. Liquid chromatography (LC) ESI-negative mass spectrometry (MS) analytics of ethyl acetate extracts of P. larvae secretomes.
(A) Extracted ion chromatogram (m/z 881) of ethyl acetate extract P. larvae ATCC9545 wildtype (ERIC I; black line) and P. larvae ATCC9545 ΔdhbF (red line). (B) Extracted ion chromatogram (m/z 881) of ethyl acetate extract of P. larvae DMS25430 wildtype (ERIC II; black line) and P. larvae DMS25430 ΔdhbF (red line).
Figure 5
Figure 5. Liquid chromatography (LC) ESI-negative tandem mass spectrometry (MS/MS) analytics of bacillibactin.
MS/MS analytics of ethyl acetate extract of P. larvae ATCC9545 (A; ERIC I), P. larvae 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 m/z 249.1 is attributed to the decarboxylation of DHB-Gly-Thr.
Figure 6
Figure 6. Exposure bioassays for assessing the role of bacillibactin during pathogenesis.
Honey bee larvae were infected with wild type P. larvae (ATCC9545, DSM25430) or the corresponding mutant strains (ATCC9545 ΔdhbF, DSM25430 ΔdhbF) 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 P. larvae infection was not significantly different (Mann–Whitney U test) between ATCC9545 wild type and ATCC9545 ΔdhbF (A; p-value = 0.800) and between DSM25430 wild type and DSM25430 ΔdhbF (B; p-value = 0.800). Cumulative mortality due to P. larvae infection was not significantly different (Kolmogorov–Smirnow test) between ATCC9545 wildtype (C, closed triangles) and ATCC9545 ΔdhbF (C, open triangles) (p-value = 0.660) and between DSM25430 wild type (D, closed circles) and DSM25430 ΔdhbF (D, open circles) (p-value = 0.999). Natural mortality in the control groups did not exceed 20% (E, closed squares).

References

    1. Aizen M, Garibaldi L, Cunningham S, Klein A (2008) Long-term global trends in crop yield and production reveal no current pollination shortage but increasing pollinator dependency. Curr Biol 18: 1572–1575. - PubMed
    1. Aizen MA, Harder LD (2009) The global stock of domesticated honey bees is growing slower than agricultural demand for pollination. Curr Biol 19: 915–918. - PubMed
    1. Garibaldi LA, Steffan-Dewenter I, Winfree R, Aizen MA, Bommarco R, et al. (2013) Wild pollinators enhance fruit set of crops regardless of honey bee abundance. Science 339: 1608–1611. - PubMed
    1. Klein A-M, Vaissiere BE, Cane JH, Steffan-Dewenter I, Cunningham SA, et al. (2007) Importance of pollinators in changing landscapes for world crops. Proc R Soc B 274: 303–313. - PMC - PubMed
    1. Genersch E (2010) Honey bee pathology: Current threats to honey bees and beekeeping. Appl Microbiol Biotechnol 87: 87–97. - PubMed

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