American foulbrood is one of the most devastating diseases of the honey bee. It is caused by the spore-forming, Gram-positive rod-shaped bacterium Paenibacillus larvae.

Paenibacillus larvae, a Gram positive bacterial pathogen, causes American Foulbrood (AFB), which is the most serious infectious disease of honey bees. In order to investigate the genomic potential of P. larvae, two strains belonging to two different genotypes were sequenced and used for comparative genome analysis. The complete genome sequence of P. larvae strain DSM 25430 (genotype ERIC II) consisted of 4,056,006 bp and harbored 3,928 predicted protein-encoding genes. The draft genome sequence of P. larvae strain DSM 25719 (genotype ERIC I) comprised 4,579,589 bp and contained 4,868 protein-encoding genes. Both strains harbored a 9.7 kb plasmid and encoded a large number of virulence-associated proteins such as toxins and collagenases. In addition, genes encoding large multimodular enzymes producing nonribosomally peptides or polyketides were identified. In the genome of strain DSM 25719 seven toxin associated loci were identified and analyzed. Five of them encoded putatively functional toxins. The genome of strain DSM 25430 harbored several toxin loci that showed similarity to corresponding loci in the genome of strain DSM 25719, but were non-functional due to point mutations or disruption by transposases. Although both strains cause AFB, significant differences between the genomes were observed including genome size, number and composition of transposases, insertion elements, predicted phage regions, and strain-specific island-like regions. Transposases, integrases and recombinases are important drivers for genome plasticity. A total of 390 and 273 mobile elements were found in strain DSM 25430 and strain DSM 25719, respectively. Comparative genomics of both strains revealed acquisition of virulence factors by horizontal gene transfer and provided insights into evolution and pathogenicity.

Summary: A merican foulbrood is the most destructive brood disease of honeybees ( A pis mellifera ) globally. The absence of a repeatable, universal typing scheme for the causative bacterium P aenibacillus larvae has restricted our understanding of disease epidemiology. We have created the first multilocus sequence typing scheme ( MLST ) for P . larvae , which largely confirms the previous enterobacterial repetitive intergenic consensus ( ERIC )–polymerase chain reaction ( PCR )‐based typing scheme's divisions while providing added resolution and improved repeatability. We have used the new scheme to determine the distribution and biogeography of 294 samples of P . larvae from across six continents. We found that of the two most epidemiologically important ERIC types, ERIC I was more diverse than ERIC II . Analysis of the fixation index ( F _ST ) by distance suggested a significant relationship between genetic and geographic distance, suggesting that population structure exists in populations of P . larvae . Interestingly, this effect was only observed within the native range of the host and was absent in areas where international trade has moved honeybees and their disease. Correspondence analysis demonstrated similar sequence type ( ST ) distributions between native and non‐native countries and that ERIC I and II ST s mainly have differing distributions. The new typing scheme facilitates epidemiological study of this costly disease of a key pollinator.

The Gram-positive, spore-forming bacterium Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a globally occurring, deathly epizootic of honey bee brood. AFB outbreaks are predominantly caused by two genotypes of P. larvae, ERIC I and ERIC II, with P. larvae ERIC II being the more virulent genotype on larval level. Recently, comparative proteome analyses have revealed that P. larvae ERIC II but not ERIC I might harbour a functional S-layer protein, named SplA. We here determine the genomic sequence of splA in both genotypes and demonstrate by in vitro self-assembly studies of recombinant and purified SplA protein in combination with electron-microscopy that SplA is a true S-layer protein self-assembling into a square 2D lattice. The existence of a functional S-layer protein is novel for this bacterial species. For elucidating the biological function of P. larvae SplA, a genetic system for disruption of gene expression in this important honey bee pathogen was developed. Subsequent analyses of in vivo biological functions of SplA were based on comparing a wild-type strain of P. larvae ERIC II with the newly constructed splA-knockout mutant of this strain. Differences in cell and colony morphology suggest that SplA is a shape-determining factor. Marked differences between P. larvae ERIC II wild-type and mutant cells with regard to (i) adhesion to primary pupal midgut cells and (ii) larval mortality as measured in exposure bioassays corroborate the assumption that the S-layer of P. larvae ERIC II is an important virulence factor. Since SplA is the first functionally proven virulence factor for this species, our data extend the knowledge of the molecular differences between these two genotypes of P. larvae and contribute to explaining the observed differences in virulence. These results present an immense advancement in our understanding of P. larvae pathogenesis. Paenibacillus larvae is the most devastating bacterial pathogen of honey bees. However, the molecular interactions between infected larvae and P. larvae are poorly understood and little more than speculation exist concerning virulence factors. Recently, a putative S-layer protein has been identified in P. larvae. We here demonstrate that only representatives of P. larvae genotype ERIC II harbor a functional splA-gene and that SplA is a true S-layer protein with self-assembly capability. The presence of a functional S-layer protein is novel for P. larvae. When elucidating the biological function of SplA we broke new ground by establishing primary cell culture for pupal gut cells and by developing a genetic system for disruption of gene expression in this important honey bee pathogen. By using these novel methods we were able to prove that SplA serves as a shape-determining factor, mediates adhesion to host cells, and is a key virulence factor of P. larvae ERIC II. These results present an immense advancement in our understanding of P. larvae pathogenesis. Furthermore, we propose P. larvae as a model system for the analysis of the in vivo functions of S-layer proteins because P. larvae SlpA knockout-mutants retain viability and are thus suitable for functional studies.