Fig 1.
Method summary of the monoxenic culture establishment.
The cells with 40% Optiprep was infused at the bottom of tube. And 20, 10 and 0% of Optiprep solutions were stratified on the cell solution. After centrifugation at 800x g for 20 min, Kipferlia bialata enriched fraction between 10–20% solutions were collected. Above procedures were repeated three times. K. bialata enriched fraction were inoculated to the liquid culture contanining single bacterium species, Pseudoalteromonas sp. Cells were cultivated 3–5 days. The triple centrifugations followed by cultivation were repeated for 11 times.
Fig 2.
(A) the original and (B) the monoxenic culture analyzed in this study. “n” indicates the numbers of the 16S rDNA clones sequenced.
Fig 3.
Sequencing depths analysis of the Kipferlia bialata genome.
Coverage depths distribution of the original scaffolds before bioinformatic filtration. Coverages were calculated based on the SPAdes output. Scaffolds were generated using 200 million paired-end reads (half of available reads).
Table 1.
Overview of nuclear genome sequences for Metamonada species.
Fig 4.
Functional annotation based on the eukaryotic orthologous groups (KOG).
(A) Venn diagram showing the number of shared unique KOG identifiers (IDs) under three-ways comparison amongst fornicates including Kipferlia bialata, Giardia intestinalis and Spironucleus salmonicida and (B) the four-ways comparison considering with Trichomonas vaginalis. The number under species names indicates the total number of unique KOG IDs (left) and the total number of proteins assigned into KOG (right). (C) Histogram showing the number of proteins assigned into KOGs and (D) the unique number of KOG IDs in each genome. KOG categories are as follows: A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell cycle control, cell division and chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination and repair; M, cell wall, membrane or envelope biogenesis; N, cell motility; O, post-translational modification, protein turnover, chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general function prediction only; S, function unknown; T, signal transduction; U, intracellular trafficking, secretion and vesicular transport; V, defence mechanisms; W, extracellular structures; Y, nuclear structure; Z, cytoskeleton. Higher KOG categories are as follows: CP, cellular processing and signaling; Hyp, poorly characterized; Inf, information storage and processing; Met, metabolism.
Table 2.
Protein list surveyed in this study.
Fig 5.
Maximum-likelihood tree inferred from (A) malic enzyme and (B) pyruvate:ferredoxin oxidoreductase. Trees were inferred by using IQ-tree with LG4X model. Numbers above branches are bootstrap support values. Bootstrap support values higher than 75% are shown. Arrowheads indicate the mitochondrial-targeting protein predicted by at least one prediction program.
Fig 6.
Reconstructed metabolic pathway of mitochondrion-related organelles in Kipferlia bialata.
1. H2-synthesis, 2. Pyruvate metabolism, 3. Substrate-level phosphorylation, 4. Amino acid metabolism, and 5. Fe-S cluster assembly. NuoE and F: 24 and 51 kDa of mitochondria NADH:ubiquinone oxidoreductase, respectively, Fe-Hyd: Fe-hydrogenase, HydE/F/G: Fe-hydrogenase maturases, Fdx: ferredoxin, ME: malic enzyme, SHMT: serine hydroxymethyltransferase, H/L/P/T: glycine cleavage system proteins H/L/P/T, Fe/S ISC: iron-sulfur cluster assembly ISC system, SCS: succinyl-CoA synthase, ASCT: acetate:succinyl-CoA transferase, PFO: pyruvate:ferredoxin oxidoreductase. Suc: Succinate, Ac-CoA: Acetyl-Coenzyme A, suc-CoA: succinyl-Coenzyme A.