A novel quinone biosynthetic pathway illuminates the evolution of aerobic metabolism

Abstract

The dominant organisms in modern oxic ecosystems rely on respiratory quinones with high redox potential (HPQs) for electron transport in aerobic respiration and photosynthesis. The diversification of quinones, from low redox potential (LPQ) in anaerobes to HPQs in aerobes, is assumed to have followed Earth's surface oxygenation ~2.3 billion years ago. However, the evolutionary origins of HPQs remain unresolved. Here, we characterize the structure and biosynthetic pathway of an ancestral HPQ, methyl-plastoquinone (mPQ), that is unique to bacteria of the phylum Nitrospirota. mPQ is structurally related to the two previously known HPQs, plastoquinone from Cyanobacteriota/chloroplasts and ubiquinone from Pseudomonadota/mitochondria, respectively. We demonstrate a common origin of the three HPQ biosynthetic pathways that predates the emergence of Nitrospirota, Cyanobacteriota, and Pseudomonadota. An ancestral HPQ biosynthetic pathway evolved ≥ 3.4 billion years ago in an extinct lineage and was laterally transferred to these three phyla ~2.5 to 3.2 billion years ago. We show that Cyanobacteriota and Pseudomonadota were ancestrally aerobic and thus propose that aerobic metabolism using HPQs significantly predates Earth's surface oxygenation. Two of the three HPQ pathways were later obtained by eukaryotes through endosymbiosis forming chloroplasts and mitochondria, enabling their rise to dominance in modern oxic ecosystems.

Keywords: biosynthesis; electron transport chain; great oxygenation event; nitrospirota; quinone.

Fig. 1.

Quinones detected in aerobic Nitrospirota. (A–D) Chromatograms showing presence of a distinct quinone type (methyl-plastoquinone, mPQ) in aerobic Nitrospirota (Nitrospira marina, Leptospirillum ferrooxidans, Ca. Manganitrophus noduliformans) and canonical menaquinones (MK) in the anaerobic Nitrospirota species Thermodesulfovibrio islandicus. Ubiquinone (UQ_8:8) in the Ca. Manganitrophus-Ramlibacter coculture derives from Ramlibacter (SI Appendix, Fig. S1). (E–G) High-resolution mass spectrometric characterization of mPQ_9:9 and PQ_9:9 showing similar fragmentation patterns but suggesting the presence of a trimethyl-benzoquinone moiety in mPQ_9:9 (SI Appendix, Fig. S1); structure and fragmentation pattern of UQ_8:8 from Ramlibacter shown in G for reference.

Fig. 2.

Characterization of the mPQ biosynthetic pathway. (A) Biosynthetic pathways of quinones showing homology of pathways for mPQ₉ in Nitrospirota (purple), PQ₉ in the cyanobacterium Synechocystis sp. PCC6803 (green), and UQ₈ in the gammaproteobacterium Escherichia coli (blue). Biosynthetic steps are numbered, and homologous steps are connected by colored lines. (B–D) Heterologous complementation experiments using mPQ biosynthesis gene candidates to restore UQ₈ production in E. coli mutants lacking key genes for ubiquinone biosynthesis (∆ubiC + mpqC, ∆ubiA + mpqA, ∆ubiX + mpqX, ∆ubiD + mpqD). (E) PQ production in E. coli ∆ubiIFE mutants complemented with mpqQ from N. inopinata as well as PQ and mPQ in E. coli ∆ubiIFE mutants complemented with mpqQ from N. inopinata and mpqE from other Nitrospirota. WT = wild type; vec = empty vector; thick bars represent means and error bars represent SD of the means, n = 3 to 5; AU = arbitrary units. Abbreviations: Ca. N. nitrificans (Nnit), N. moscoviensis (Nmos), N. inopinata (Nino), L. ferrooxidans (Lfer), Ca. M. noduliformans (Mnod). The numbering of the carbon atoms on the 4-HBA precursor (panel a, light gray) defines the nomenclature for all intermediates described in the text. The octaprenyl and nonaprenyl chains are abbreviated with R₈ and R₉, respectively. SI Appendix, Fig. S7–S9 for details on compound identification and quantification. Stars indicate **P < 0.01, ***P < 0.001, and ****P < 0.0001 for unpaired Student’s t tests relative to the empty vector.

Fig. 3.

Phylogenetic tree of bacteria showing the occurrence of respiratory quinones. Quinones with high redox potential (UQ, PQ, mPQ) occur only in aerobic Nitrospirota, Pseudomonadota, and Cyanobacteriota. Low potential quinones occur in anaerobic Nitrospirota (MK), some Pseudomonadota (MK), and all Cyanobacteriota (PhQ). Asterisks indicate strains in which presence of mPQ has been verified experimentally. SI Appendix, Fig. S11–S13 for detailed trees. The maximum-likelihood phylogenetic tree was constructed from 120 concatenated single copy marker proteins (59) of 547 isolate genomes and metagenome-assembled genomes, covering all bacterial phyla, and rooted using the DST group to approximate the bacterial root (60, 61). Quinone occurrences were derived from instrumental analysis of isolates or inferred from the presence of key biosynthesis genes (SI Appendix, Results and Discussion and Dataset S3; including literature data). Phenotype oxytolerance was curated from strain descriptions. Selected classes/orders denoted inside of rings. Selected phyla denoted outside of rings: ACD, Aquificota-Campylobacterota-Deferribacterota; Desulfob., Desulfobacterota; DST, Deinococcota-Synergistota-Thermotogota; BA, Bacillota-Actinomycetota; FCB, Fibrobacterota-Chloroflexota-Bacteroidota; Marg., Candidatus Margulisbacteria; Myxoc., Myxococcota; Nitrospin., Nitrospinota; PVC, Planctomycetota-Verrucomicrobiota-Chlamydiota; Seri., Candidatus Sericytochromatia; Vamp., Vampirovibrionophyceae. Circles indicate ultrafast bootstrap support ≥95%.

Fig. 4.

High-potential quinones (HPQs) share a single origin predating the great oxygenation event. (A) Phylogenetic trees of HPQ biosynthesis proteins demonstrating that prenyltransferases and decarboxylases of the ubiquinone (UbiA, UbiD), plastoquinone (PlqA, PlqD), and methylplastoquinone (MpqA, MpqD) pathways form sister clades of the archaeal and bacterial futalosine pathway for biosynthesis of menaquinone (MK, MqnPL). (B) Phylogenetic trees of quinone C5/C6 (PlqQ, MpqQ) and C2 methyltransferases (UbiE, MpqE), showing a nested topology of C5/C6 methyltransferases and that C2 methyltransferases form a sister lineage of menaquinone-associated methyltransferases (MqnK). Outgroups used for rooting the trees are not shown but discussed in SI Appendix. Scale bar indicate 0.5 substitutions per site. Open circles indicate ultrafast bootstrap support ≥95%. (C) Conceptual sketch of HPQ evolution and resulting redox potentials, based on the trees in panels (A and B). (D) Timescale of LPQ and HPQ evolution (colors as in panel C; based on panels A and B and the observation that the last common ancestors of Pseudomonadota, Cyanobacteriota, and aerobic Nitrospirota contained UQ, PQ, and mPQ, respectively) in relation to geochemical changes [evidence for localized O₂ oases (–64), the great oxygenation event, GOE (7)] and biological innovations [Archean rapid genetic expansion (10), evolution of enzymes protecting against reactive oxygen species (ROS) (12), expansion of O₂ reductase diversity (11)]. Shaded hexagons indicate minimum and maximum estimates of HPQ evolution timescale. Open symbols indicate median ages (colored bars: uncertainty range; quinone symbols: Upper/Lower estimate) of relevant clades estimated by previous molecular clock analyses (, –70). The earliest date of UQ/PQ/mPQ emergence is set as the earliest estimate of the radiation of crown Cyanobacteria, Pseudomonadota, and (aerobic) Nitrospirota assuming that UQ, PQ, or mPQ were present in the last common ancestor of each clade.