Comparative genomics of Mollicutes-related endobacteria supports a late invasion into Mucoromycota fungi

Abstract

Diverse members of early-diverging Mucoromycota, including mycorrhizal taxa and soil-associated Mortierellaceae, are known to harbor Mollicutes-related endobacteria (MRE). It has been hypothesized that MRE were acquired by a common ancestor and transmitted vertically. Alternatively, MRE endosymbionts could have invaded after the divergence of Mucoromycota lineages and subsequently spread to new hosts horizontally. To better understand the evolutionary history of MRE symbionts, we generated and analyzed four complete MRE genomes from two Mortierellaceae genera: Linnemannia (MRE-L) and Benniella (MRE-B). These genomes include the smallest known of fungal endosymbionts and showed signals of a tight relationship with hosts including a reduced functional capacity and genes transferred from fungal hosts to MRE. Phylogenetic reconstruction including nine MRE from mycorrhizal fungi revealed that MRE-B genomes are more closely related to MRE from Glomeromycotina than MRE-L from the same host family. We posit that reductions in genome size, GC content, pseudogene content, and repeat content in MRE-L may reflect a longer-term relationship with their fungal hosts. These data indicate Linnemannia and Benniella MRE were likely acquired independently after their fungal hosts diverged from a common ancestor. This work expands upon foundational knowledge on minimal genomes and provides insights into the evolution of bacterial endosymbionts.

Fig. 1. FISH imaging demonstrates intracellular localization of MRE in Benniella erionia GBAus27B.

a FISH imaging in wildtype and cured Benniella erionia GBAus27B. MRE and fungal mycelia are visualized using the universal 16S rRNA (Cyan) and 18S rRNA (Magenta) showing specificity of the bacterial probe in only the uncured fungus. Non-specific, amorphous staining was observed in the cured sample. b A high magnification image of the signals in (a) displays distinct bacterial morphologies. c Relative fluorescence intensity across the Z-axis shows that the peak intensities of the bacteria correlate with the 18S rRNA of fungal mycelia indicating that these symbionts are internalized symbionts.

Fig. 2. Mortierellaceae MRE differ in phylogenetic placement and genomic content.

a Maximum likelihood phylogeny of MRE and selected Phytoplasma and Spiroplasma taxa indicates that MRE form four distinct phylogenetic clades. Benniella MRE and Linnemannia MRE cluster in separate phylogenetic clades. b Percentage of each genome composed of pseudogenes, repeat regions, and phage sequences. c Coding density of each genome, (d) Host phylogeny created from beta-tubulin genes. Branch labels indicate bootstrap values (100 replicates). Bootstrap values >50 are shown.

Fig. 3. Phylogenetic evidence demonstrates horizontal transfer of the MGMT gene between fungal hosts and endobacteria.

Maximum likelihood phylogeny of Methylated-DNA-protein-cysteine (MGMT gene) from Mortierellaceae MRE and diverse fungal and bacterial BLAST hits. Phylogeny indicates fungal origin of MGMT gene in MRE. Bootstrap values (100 replicates) >50 are shown on branches.

Fig. 4. MRE from Linnemannia and Benniella differ in degrees of genomic rearrangement.

Synteny analysis of Linnemannia and Benniella MRE genomes. Dotplot showing whole genome synteny between (a) Linnemannia MRE and (b) Benniella MRE. Synteny of single copy orthologs identified by BUSCO analysis between (c) Linnemannia MRE (87 orthologs) and (d) Benniella MRE (53 orthologs).

Fig. 5. MRE are reduced in gene content related to DNA replication and repair.

Functional repertoire of MRE vs selected Mollicutes. Presence (red) / absence (white) heatmaps of genes associated with (a) DNA replication, (b) Base excision repair, (c) Nucleotide excision repair, (d) Homologous recombination, (e) Mismatch repair.

Fig. 6. Linnemannia MRE lack site-specific recombination genes which are enriched in other MRE genome.

Counts of XerC and XerD genes in MRE and selected Mollicutes genomes.