Extreme mitochondrial reduction in a novel group of free-living metamonads

Abstract

Metamonads are a diverse group of heterotrophic microbial eukaryotes adapted to living in hypoxic environments. All metamonads but one harbour metabolically altered 'mitochondrion-related organelles' (MROs) with reduced functions relative to aerobic mitochondria, however the degree of reduction varies markedly over the metamonad tree. To further investigate metamonad MRO diversity, we generated high quality draft genomes, transcriptomes, and predicted proteomes for five recently discovered free-living metamonads. Phylogenomic analyses place these organisms in a clade sister to the Fornicata - a group of metamonads that includes parasitic and free-living diplomonads and Carpediemonas-like organisms. Extensive bioinformatic analyses of the manually curated gene models showed that these organisms have extremely reduced MROs in comparison to other free-living metamonads. Loss of the mitochondrial iron-sulfur cluster (ISC) assembly system in some organisms in this group appears to be linked to the acquisition in their common ancestral lineage of a SUF-like minimal system (SMS) Fe/S cluster pathway through lateral gene transfer (LGT). One of the isolates, named 'RC', appears to have undergone even more drastic mitochondrial reduction losing almost all other detectable MRO-related functions. The extreme mitochondrial reduction observed within this free-living anaerobic protistan clade is unprecedented and demonstrates that mitochondrial functions, under some conditions, can be almost completely lost even in free-living organisms.

Characterisation ofSkoliomonasgen. nov., a haloalkaliphilic anaerobe related to barthelonids (Metamonada)

“…The concatenated SSU-LSU rRNA gene phylogeny infers a clade containing fornicates, barthelonids, and skoliomonads with 76% non-parametric bootstrap support, the latter two in turn forming a clade with 60% bootstrap support (Fig S2). The ribosomal rRNA gene trees are consistent with phylogenomic results in (Williams et al, 2023), which show full support for the barthelonid+skoliomonad clade.…”

“…Ultrastructural studies of Skoliomonas spp., coupled with those of barthelonids, would be needed to better characterise ‘excavate’ groove morphology and evolution, particularly of the last metamonad common ancestor. Given the position of the skoliomonad+barthelonid clade as sister to fornicates (Williams et al, 2023), ultrastructural studies would help characterise the nature of the fornicate last common ancestor as well. Feeding, from prey selection to the role of the flagellar vane in particle ingestion, would be an additional topic to study in relation to groove ultastructure.…”

“…Genomic DNA for each of the cultured isolates was extracted using the Qiagen DNeasy Blood & Tissue kit (CAT#69504), except for GEM-RC, which was obtained using a combination of phenol:chloroform and CTAB extraction protocols, the QIAGEN Genomic-tip kit, and the QIAGEN MagAttract HMW DNA kit, further detailed in (Williams et al, 2023). Partial SSU rDNA sequences for TZLM1-RC and Soap18-RC, and GEM-RC and Soap20A-RC were amplified using EukA (5l⍰-AACCTGGTTGATCCTGCCAGT-3l⍰) and EukB (5’-TGATCCTTCTGCAGGTTCACCTAC-3’) primers (Medlin et al, 1988) at 60°C and 58°C annealing temperatures, respectively.…”

“…The amplified product was Sanger sequenced at Génome Québec, with the reads trimmed and assembled in Geneious Prime 2022.1.1 (https://www.geneious.com). For phylogenetic analyses (see below), the partial and lower quality PCR-derived TZLM3-RCL SSU rDNA sequence was replaced by one extracted from the TZLM3-RCL genome assembly (Williams et al, 2023) using barrnap (Seeman, 2018).…”

“…Ribosomal gene sequences were aligned to a manually curated eukaryote-wide alignment in SeaView (Gouy et al, 2010) using profile alignment via MUSCLE (Edgar, 2004), trimmed with a gblocks (Castresana, 2000) masking further curated by hand, with 1321 sites retained and 219 taxa. SSU and LSU rRNA gene sequences corresponding to the same operon were extracted from genomic data of skoliomonad isolates TZLM1-RC, GEM-RC, and TZLM3-RCL as well as barthelonid PCE from (Williams et al, 2023), then added to a concatenated SSU-LSU rDNA alignment derived from (Eglit et al, 2024), which was then trimmed with gblocks and manually edited to a total of 3064 sites and 141 taxa. For each dataset, a maximum likelihood phylogeny was inferred in RAxML v8.2.6 (Stamatakis, 2014) under the GTR+Г model with 50 starting trees and 500 non-parametric bootstraps.…”