Microbial eukaryotes have adapted to hypoxia by horizontal acquisitions of a gene involved in rhodoquinone biosynthesis

  1. Courtney W Stairs  Is a corresponding author
  2. Laura Eme
  3. Sergio A Muñoz-Gómez
  4. Alejandro Cohen  Is a corresponding author
  5. Graham Dellaire  Is a corresponding author
  6. Jennifer N Shepherd  Is a corresponding author
  7. James P Fawcett  Is a corresponding author
  8. Andrew J Roger  Is a corresponding author
  1. Dalhousie University, Canada
  2. Gonzaga University, United States
6 figures, 2 tables and 5 additional files

Figures

Structure and function of ubiquinone and rhodoquinone in mitochondria.

(A) Under aerobic conditions, electrons from NADH or succinate (Suc) are shuttled to ubiquinone (UQ) via Complex I (I) or Complex II (II) respectively to generate NAD (NAD+), fumarate (fum) and …

https://doi.org/10.7554/eLife.34292.002
Figure 2 with 3 supplements
Maximum-likelihood phylogeny of RquA proteins constructed from an alignment of homologs from 166 organisms and 197 aligned sites.

Eukaryotic proteins are coloured based on their phylogenetic affiliations: Obazoa (purple), Stramenopiles-Alveolata (orange), Excavata (green), Amoebozoa (magenta) and Rhizaria (blue). Hexagons …

https://doi.org/10.7554/eLife.34292.003
Figure 2—figure supplement 1
Intron positions in eukaryotic rquA gene sequences.

Nucleotide from both transcriptome and genomic sequencing projects (when available) were aligned using Sequencher (Gene Codes v 5.4.6) and introns were manually inspected for position, length and …

https://doi.org/10.7554/eLife.34292.004
Figure 2—figure supplement 2
Phyletic distribution of RQUA among alphaproteobacteria superimposed on the alphaproteobacterial species tree.

Maximum likelihood analysis of a 200 phylum-specific phylogenetic marker genes representing 54,400 sites from 210 representative alphaproteobacteria under the LG + C60 + F (PMSF)+ Γ4 model of …

https://doi.org/10.7554/eLife.34292.005
Figure 2—figure supplement 3
RquA homologues lack critical S-adenosyl methionine (SAM) binding site.

Conserved residues known to interact with the carbonyl group of SAM are shown with black triangles. A substitution from aspartate to glutamine was obversed in all bacterial (represented here by Rhodo…

https://doi.org/10.7554/eLife.34292.006
RquA co-occurs with quinone-utilizing enzymes.

Eukaryotic genomes and transcriptomes were surveyed for homologs of respiratory chain components (Complexes I-V, CI-CV), alternative oxidase (AOX), dihydroorotate dehydrogenase (DHOH), …

https://doi.org/10.7554/eLife.34292.011
Figure 4 with 3 supplements
Subcellular localization of RquA and rhodoquinone production in Pygsuia biforma.

(A) Antibodies raised against RquA (green) colocalized with MitoTracker (red). Confocal slices (0.3 μm) were deconvoluted (using a constrained interative algorithm) and combined to render a 3D …

https://doi.org/10.7554/eLife.34292.012
Figure 4—figure supplement 1
Antibodies directed against Pygsuia RquA (PbRquA) recognize purified recombinant PbRquA by western blot analysis.

Proteins isolated from whole cell extracts of E. coli induced to express pGEX4T-1 (1) and pGEX- Pb-rquA (2) and glutathione-S-transferase(GST)-Pb-rquA purified with glutathione magnetic beads (3) …

https://doi.org/10.7554/eLife.34292.013
Figure 4—figure supplement 2
Spectra represent the elution times of detection of the rhodoquinone head group (182.1 m/z; left) and ubiquinone head group (197.1 m/z) following fragmentation of parent ions of different isoprenyl chain lengths as indicated.
https://doi.org/10.7554/eLife.34292.014
Figure 4—figure supplement 3
Lipid extracts were separated by high-performance liquid chromatography and analyzed with mixed-reaction monitoring mass spectrometry from Pygsuia mixed culture, Pygsuia ‘food’ bacteria culture, glassware, and Rhodospirillum rubrum as indicated. 

Spectra represent the elution times of detection of the rhodoquinone head group (182.1 m/z; left) and ubiquinone head group (197.1 m/z) following fragmentation of parent ions of different isoprenyl …

https://doi.org/10.7554/eLife.34292.015
The interactions of rhodoquinone with other mitochondrial redox reactions in different eukaryotes with mitochondrion-related organelles.

Standard reduction potentials for each major reaction involved in rhodoquinone (R)) and ubiquinone (U) metabolism are shown in increasing order of potential. Half reaction equations are detailed in s…

https://doi.org/10.7554/eLife.34292.016
Author response image 1
Cumulative frequency plot of transcriptome completeness for the marine microbial eukaryotes sequencing projects.
https://doi.org/10.7554/eLife.34292.023

Tables

Table 1
Approximate unbiased topology tests for RquA analyses.
https://doi.org/10.7554/eLife.34292.007
Monophyly TestedTree fileCONSEL p-AUa
Group A and Group B
Maximum likelihood treeFigure 2; Supplementary file 2 - tree 20.743
Group A eukaryotes + Group B
eukaryotes
Supplementary file 2 - tree 33.00E-37***
Group A1 eukaryotes: Blastocystis, Proteromonas,
Neoparamoebids, Euglenids, Pygsuia
Supplementary file 2 - tree 40.622
Group A1 eukaryotes + BrevimastigamonasSupplementary file 2 - tree 50.46
Group A1 eukaryotes + Brevimastigamonas
+ Mastigamoeba
Supplementary file 2 - tree 60.294
Group A eukaryotesSupplementary file 2 - tree 70.253
Group B eukaryotesSupplementary file 2 - tree 80.179
Obazoa (Pygsuia + Monosiga)Supplementary file 2 - tree 90.002**
Amoerphea (Obazoa + Amoebozoa)Supplementary file 2 - tree 101.00E-32***
AmoebozoaSupplementary file 2 - tree 110.206
Stramenopiles + AlveolatesSupplementary file 2 - tree 120.004**
Stramenopiles + Alveolates +
Rhizaria (SAR)
Supplementary file 2 - tree 130.034*
Diaphoretickes
(SAR + Euglenids)
Supplementary file 2 - tree 140.018*
RhizariaSupplementary file 2 - tree 151.00E-60***
Eukaryotes + MAG
alphaproteobacteria
Supplementary file 2 - tree 162.00E-41***
Group A eukaryotes + MAG
alphaproteobacteria
Supplementary file 2 - tree 170.227
AlphaproteobacteriaSupplementary file 2 - tree 185.00E-34***
Eukaryotes + alphaproteobacteriaSupplementary file 2 - tree 198.00E-43***
Group A eukaryotes +
Group A alphaproteobacteria
Supplementary file 2 - tree 203.00E-31***
Group B eukaryotes +
Group B alphaproteobacteria
Supplementary file 2 - tree 214.00E-05***
Group A
Maximum likelihood treeSupplementary file 3 - tree 10.892
EukaryotesSupplementary file 3 - tree 20.225
AmoebozoaSupplementary file 3 - tree 30.315
Pygsuia + Amoebozoa (Amorphea)Supplementary file 3 - tree 40.22
Eukaryotes + alphaproteobacteriaSupplementary file 3 - tree 53.00E-59***
Eukaryotes + MAG
alphaproteobacteria
Supplementary file 3 - tree 60.226
Group B
Maximum likelihood treeSupplementary file 4 - tree 10.827
EukaryotesSupplementary file 4 - tree 20.081
Stramenopiles + AlveolatesSupplementary file 4 - tree 30.287
SARSupplementary file 4 - tree 40.281
Eukaryotes + alphaproteobacteriaSupplementary file 4 - tree 52.00E-75***
  1. aitalicized values indicate topologies that could not be rejected (p<0.05).

    * 0.05 > p > 0.01

  2. ** 0.01 > p > 0.001

    *** p < 0.001

Table 1—source data 1

Topology test output from CONSEL.

Trees 1-20 represent trees 2-21 from Supplementary file 2; trees 21-120 represent 100 bootstrap trees from the maximum likelihood analysis. Relevant column headers: Obs, observed log-likelihood …

https://doi.org/10.7554/eLife.34292.008
Table 1—source data 2

Topology test output from CONSEL.

Trees 1-6 represent trees1-6 from Supplementary file 3; trees 7-106 represent 100 bootstrap trees from the maximum likelihood analysis. Relevant column headers: Obs, observed log-likelihood value; au, topology test p-value; np, bootstrap probability. Details on the column headers can be found at http://stat.sys.i.kyoto-u.ac.jp/prog/consel/quick.html

https://doi.org/10.7554/eLife.34292.009
Table 1—source data 3

Topology test output from CONSEL.

Trees 1-5 represent trees 1-5 from Supplementary file 4; trees 6-105 represent 100 bootstrap trees from the maximum likelihood analysis. Relevant column headers: Obs, observed log-likelihood value; au, topology test p-value; np, bootstrap probability. Details on the column headers can be found at http://stat.sys.i.kyoto-u.ac.jp/prog/consel/quick.html

https://doi.org/10.7554/eLife.34292.010
Author response table 1
Numbers of complete genomes and transcriptome projects surveyed from each of the major lineage of eukaryotes.
https://doi.org/10.7554/eLife.34292.024

RquA detecteda

NCBI Genomesb

NCIB TSA nrc

MMETSPd

Alveolata710532134
Amoebozoa441108
Apusozoa0100
Breviatea1110
Centroheliozoa0010
Cryptophyta08125
Euglenozoa

2e

5535
Fornicata0220
Glaucocystophyceae0403
Haptophyceae05261
Heterolobosea0512
Jakobida0600
Malawimonadidae0200
Opisthokonta19,240139510
Oxymonadida0110
Parabasalia0210
Rhizaria412421
Rhodophyta0124158
Stramenopiles515049279
Viridiplantae02,46149972
unclassified00319
Multispecies with undefined taxa ID008219
Total24122252102647
a Source: this study
b Source: NCBI Taxonomy Browser, selecting “Genomes” – January 28, 2018

c Source NCBI Trace Archive for the transcriptome shotgun assembly database availble at https://www.ncbi.nlm.nih.gov/Traces/wgs/?term=tsa Taxonomy ID were extracted and taxonomy parsed with ete-toolkit. Only non-redundant taxonomy IDs are shown.

d Source: iMicrobe; Taxonomy ID were extracted and taxonomy assigned with ete-toolkit. Only non-redundant taxonomy IDs are shown.
e Three copies were identified in Eutriptiella and 1 in Euglena

Additional files

Supplementary file 1

Excel file with workbooks containing information about the number of genomes surveyed, PFAM domains of putative RquA homologues, gene accession numbers for RquA and Q-utilizing proteins, mitochondrial targeting sequence information, redox half-potentials used for Figure 5, bacterial genes with and without linkage to respiratory complexes.

https://doi.org/10.7554/eLife.34292.017
Supplementary file 2

PDF with all the trees for the full RquA phylogenetic analysis and associated topology tests

https://doi.org/10.7554/eLife.34292.018
Supplementary file 3

PDF with all the tree for the Group A phylogenetic analysis and associated topology tests

https://doi.org/10.7554/eLife.34292.019
Supplementary file 4

PDF with all the tree for the Group B phylogenetic analysis and associated topology tests

https://doi.org/10.7554/eLife.34292.020
Transparent reporting form
https://doi.org/10.7554/eLife.34292.021

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