Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut

Abstract

Background: In the honeybee Apis mellifera, the bacterial gut community is consistently colonized by eight distinct phylotypes of bacteria. Managed bee colonies are of considerable economic interest and it is therefore important to elucidate the diversity and role of this microbiota in the honeybee. In this study, we have sequenced the genomes of eleven strains of lactobacilli and bifidobacteria isolated from the honey crop of the honeybee A. mellifera.

Results: Single gene phylogenies confirmed that the isolated strains represent the diversity of lactobacilli and bifidobacteria in the gut, as previously identified by 16S rRNA gene sequencing. Core genome phylogenies of the lactobacilli and bifidobacteria further indicated extensive divergence between strains classified as the same phylotype. Phylotype-specific protein families included unique surface proteins. Within phylotypes, we found a remarkably high level of gene content diversity. Carbohydrate metabolism and transport functions contributed up to 45% of the accessory genes, with some genomes having a higher content of genes encoding phosphotransferase systems for the uptake of carbohydrates than any previously sequenced genome. These genes were often located in highly variable genomic segments that also contained genes for enzymes involved in the degradation and modification of sugar residues. Strain-specific gene clusters for the biosynthesis of exopolysaccharides were identified in two phylotypes. The dynamics of these segments contrasted with low recombination frequencies and conserved gene order structures for the core genes. Hits for CRISPR spacers were almost exclusively found within phylotypes, suggesting that the phylotypes are associated with distinct phage populations.

Conclusions: The honeybee gut microbiota has been described as consisting of a modest number of phylotypes; however, the genomes sequenced in the current study demonstrated a very high level of gene content diversity within all three described phylotypes of lactobacilli and bifidobacteria, particularly in terms of metabolic functions and surface structures, where many features were strain-specific. Together, these results indicate niche differentiation within phylotypes, suggesting that the honeybee gut microbiota is more complex than previously thought.

Figure 1

Core genome phylogeny of lactobacilli. Phylogenetic tree inferred from a maximum likelihood analysis of a concatenated alignment of 303 pan-orthologous genes. Strains sequenced in the current study are highlighted in red, with their group names indicated to the right (“Firm4” and “Firm5”). The main groupings of lactobacilli as identified by Kant et al. [34] are indicated with bold letters. Accession numbers of all genomes are listed in Additional file 3: Table S2 (L. = Lactobacillus, Le. = Leuconostoc, W. = Weissella, O. = Oenococcus, P. = Pediococcus).

Figure 2

Core genome phylogeny of bifidobacterial strains. Phylogenetic tree inferred from a maximum likelihood analysis of a concatenated alignment of 400 pan-orthologous genes. Strains sequenced in the current study are highlighted in red, with their group names indicated to the right (“Bifido-1” and “Bifido-2”). Other strains isolated from the honeybee gut are shown in light blue, and strains isolated from the bumblebee gut are shown in dark blue. Accession numbers of all genomes are listed in Additional file 3: Table S2.

Figure 3

Venn diagram of shared protein families within the “Bifido-1” group. Numbers correspond to protein families of orthologous sequences, inferred with Ortho-MCL, plus singletons (proteins unique to a single strain). Similar plots for the “Firm-5” and “Bifido-2” groups can be found in Additional file 6: Figure S4 and Additional file 7: Figure S5.

Figure 4

Genome synteny plot of the “Firm-4″ strains. Comparative analysis of the “Firm-4” genomes. The genome and plasmid sequences are represented by horizontal grey lines. The similarity between genomes was inferred with blastn and is shown with connecting grey lines, where darker lines indicate higher similarity. Blue bars show the positions of conserved group-specific core genes. Yellow bars indicate the positions of genes, which are not shared between the two strains. Red bars indicate the conserved group-specific operon encoding the putative cscAB genes [52], whereas green bars show the position of putative eps-clusters.

Figure 5

Genome synteny plot of the “Firm-5” strains. Comparative analyses of the “Firm-5” genomes. The genome and plasmid sequences are represented by horizontal grey lines. The similarity between genomes was inferred with blastn and is shown with connecting grey lines, where darker lines indicate higher similarity. Blue bars show the positions of conserved group-specific core genes. Yellow bars indicate the positions of genes, which are strain-specific. The positions of the putative surface-exposed proteins are indicated in red. CRISPR genes are shown in purple. The tree topology is as in Figure 1.

Figure 6

Genome synteny plot of the “Bifido” strains. Comparative analyses of the “Bifido” genomes. The genome and plasmid sequences are represented by horizontal grey lines. The similarity between genomes was inferred with blastn and is shown with connecting grey lines, where darker lines indicate higher similarity. Blue bars show the positions of genes conserved within the “Bifido-1” and “Bifido-2” groups respectively. Yellow bars indicate the positions of genes, which are strain-specific. Red bars indicate the positions of genes containing the RCC1-repeat domain, where the largest region is indicated with text. CRISPR genes are shown in purple. Green bars show the positions of putative eps-clusters. The tree topology is as in Figure 2.

Figure 7

Comparative analysis of regions containing group-specific putative outer-surface proteins. A) Two genomic regions containing duplicated genes for novel putative surface exposed proteins in “Firm-5” strains. Genes are shown as boxes, where blue and grey boxes represent the putative outer surface proteins, and white boxes represent other genes in the regions. Grey boxes correspond to genes identified at contig borders in the genome assemblies (partially assembled). The tree topology is as in Figure 1. B) Comparative analysis of the main genomic region containing tandemly duplicated genes for RCC1-repeat domain proteins in “Bifido” strains.” Genes are shown as arrows, where genes corresponding to each of the two protein clusters indicated by OrthoMCL and phylogeny (Additional file 8: Figure S6) are shown in light and dark blue respectively. Genes containing RCC1-repeats that are members of other protein familes, as predicted by OrthoMCL, are shown in purple. Genes not containing the RCC1-repeat domains are shown in white. The genomic position of the region is indicated in Figure 6. The tree topology is as in Figure 2. C) Genomic regions containing the putative cscAB genes in “Firm-4″ strains. Genes are shown as arrows, where light-blue arrows represent the putative cscA genes, and dark-blue arrows represent the putative cscB genes. The gene with homologues in other lactobacilli strains used for the OrthoMCL search is shown with a red border. In all comparisons, the similarity between genomes was inferred with tblastx and is shown with connecting grey lines, where darker lines indicate higher similarity.

Figure 8

Genomic locations of PTS transporters in the genomes of the “Firm-5” strains. Genome sequences, similarity and phylogeny are as for Figure 5. PTS transporters are shown as red and yellow bars along the genomes, where red bars represent the Man family PTS transporters and yellow bars represent all other PTS transporters. Additionally, the Man PTS transporters have been numbered according to the order of their occurrence in the genomes. The green boxes show the position of the variable region analyzed in Figure 9.

Figure 9

Hyper-variable regions containing multiple diverse PTS transporters. Genes are shown with bars along the sequences, where PTS transporters are shown in colour, and all other genes are shown in white. Red = Glc family, blue = Man family, purple = Gat family, green = Fru family, orange = Lac family, pink = Asc family (family designation according to [55]). Numbers above Man family transporters indicate their annotations as shown in Figure 8. The strain phylogeny is as in Figure 1, and similarity between the sequences is shown with connecting grey lines as estimated using tblastx with no filtering.

Figure 10

Comparative analysis of putative eps-clusters. A) Putative eps-clusters for the “Bifido” strains. Phylogenetic tree is as in Figure 2. B) Putative eps-clusters for the “Firm-4″ strains. Similarity was estimated with tblastx, using a length filter of 100 bp. Pink: dTDP-rhamnose biosynthesis genes, Green: ABC transporter genes, Yellow: glycosyl-transferases or genes with orthology to known eps genes, Orange: glycosyl-hydrolases, Brown: C-terminal domain of priming glycosyl-transferase, Grey: other genes with a putative function in polysaccharide biosynthesis. Light blue: putative catalase, dark blue: putative manganese transporter and repressor. For a complete list of protein domain predictions, see Additional file 12: Table S3.