Genome analysis

3.2.1. General genome properties

The genomes of the typical strains M. plutonius 21.1, 49.3, 60, 82, 90.0, 119, B5, H6, L9, S1, 764-5B, and 765-6B range from 2.021 to 2.101 Mbp and comprise between 1,589 and 1,686 predicted protein-coding genes (Supplementary Table 2). For comparison, the genome sequences of the typical strain M. plutonius ATCC 35311 and atypical strain DAT561 were derived and treated in the same way as that from the isolates. Gene content comparisons were performed by using the genome M. plutonius 49.3 as reference (Figure 2). In general, the genomes are very similar in their gene content, except for a plasmid pMP19 that is

missing in nine typical strains (82, 90.0, 119, B5, L9, S1, 764-5B, 765-6B, ATCC 35311) and the atypical strain DAT561. Based on this results, a phylogenetic tree was obtained via the Harvest software suite (Treangen et al., 2014) through a core-genome alignment with SNP detection (Figure 3). This phylogeny can resolve the relationships of the strains in more detail than MLST analysis. It confirms the close relationship between ST3 and ST7 strains, but shows that M. plutonius 82 is actually a sister taxon of M. plutonius ATCC35311 within the monophyletic lineage of CC13 strains. Interestingly, a high amount of putative pseudogenes could be detected (between 75 to 156) (Supplementary Table 2). Pseudogenes emerged mostly due to small mutational events in coding regions like SNPs and Indels, which lead to frameshifts and premature stop codons (Tutar, 2012).

All strains harbor a plasmid with high DNA sequence similarity to the recently published plasmid pMP1 (NC_015517) of M. plutonius ATCC 35311. Moreover, several strains such as 21.1, 49.3, 60, B5, and H6 might contain additional plasmids. Strain B5 harbors a 42.7 kbp plasmid (pMP43) encoding phage proteins, which are also present in the chromosome of B5, H6, L9, 49.3, S1, 60, 21.1, 764-5B, and 765-6B (see prophage region 1, Figure 2). In addition, we identified a 19.4-kbp plasmid (pMP19) present in strain 49.3. Contigs of strains 21.1, 60 and H6 show high sequence similarity to this plasmid, indicating that it is also present in these strains.

Through the identification of orthologous proteins we calculated the core genome of this set of M. plutonius strains to 1304 proteins, which represents on average approx. 71% of the proteins encoded by a M. plutonius genome. The pan genome has a total of 1846 proteins.

Figure 2: M. plutonius 49.3 artificial circular genome map. Comparison of the M. plutonius 49.3 genome to the genomes of strains H6, 21.1, 60, S1, L9, B5, 765-6B, 764-5B, 119, 90.0, 82, ATCC 35311, and DAT561 using the BRIG software (Alikhan et al., 2011). The inner circle shows the positions of pseudogenes in the M. plutonius 49.3 genome, while virulence factors and prophage regions are depicted on the outer circle and marked with red and blue blocks, respectively.

Furthermore, pathogenicity (PI) and genomic islands (GI) are encircled and numbered in red and black. The plasmid pMP19 of M. plutonius 49.3 is indicated as well.

Figure 3: Phylogenetic tree based on core-genome SNP-typing of typical M. plutonius strains used in this study. The phylogenetic tree on the left was obtained via the Harvest software suite (Treangen et al., 2014) and is based on a core-genome SNP typing. M. plutonius 49.3 was set as the reference strain. The strains are marked in the same color code used in Figure B. The SNP and Indel positions in relation to the reference are shown on the right hand side as violet lines. Sequence types are shown as well for comparison purposes.

3.2.2. Detection of putative virulence factors

In their assay, Arai et al. (2012) showed that atypical strains are more virulent than typical strains. The atypical strain DAT561 was shown to kill 94% of EFB-infected honey bee larvae five days after infection while in contrast, typical strains killed approx. 6% of the EFB-infected larvae within this timeframe. In order to correlate the functional capabilities of the strains to their phylogeny, we calculated a gene content tree based on the presence and absence of proteins in each strain (Figure 4). The gene content tree showed high similarity to the SNP-based phylogeny. The atypical strain DAT561 formed an outgroup while ST3/ST7 strains cluster together. Interestingly, the typical strains originate from different phylogenetic cluster (Figure 3). Therefore, we focused our study on the identification of M.

plutonius putative virulence factors.

Atypical and typical strains have phenotypic differences (Arai et al., 2012) and Takamatsu et al. (2014) suggested different regulation mechanisms for virulence. To verify these differences on a genomic level, orthologous proteins integrated in all strains were analyzed.

We obtained 132 proteins, which are present in the atypical strain but are absent in all typical strains (Supplementary Data 2, Sheet 1a, Figure 4). Some of these orthologous proteins potentially represent virulence factors or variations in metabolic properties necessary for a pathogenic lifestyle. Typical M. plutonius strains have 275 orthologs in common, which are absent in the atypical strain DAT561 (Supplementary Data 2, Sheet 1b). The majority of

these orthologs are hypothetical or phage-related proteins but several putative virulence factors could be identified. In addition, putative virulence factors were determined by identifying genomic and pathogenicity islands and their associated virulence determinants (Supplementary Data 2, Sheet 2, Sheet 3a, and Sheet 3b). A summary of all identified putative virulence factors is depicted in Supplementary Data 2, Sheet 4. Putative virulence factors include a tyrosine decarboxylase, bacteriocins, PlCBP49-like protein, enhancin, collagenase, cell surface and adhesion-associated proteins, capsule and antigen-forming proteins, and a toxin.

Figure 4: Gene content tree based on presence or absence of orthologous proteins. For constructing the phylogenetic tree a presence/absence binary matrix was created from orthologous groups to calculate a phylogeny with RAxML v8.1.3 (Stamatakis, 2014). M. plutonius DAT561 was used as an outgroup. Numbers at nodes are bootstrap values calculated from 1,000 resamplings to generate a majority consensus tree. The scale bar indicates divergence in presence or absence of proteins. STs are shown on the right. Color-filled boxes to the right of the organisms show the presence of the indicated proteins. Genes encoding virulence factors in brown are located on pMP19. An “N”

symbolizes that the respective ORF is not complete due to a gap in the DNA sequence. Epa is the abbreviation for “enterococcal polysaccharide antigen”.

3.3. Identification and analysis of putative virulence factors