Oxygen context and relative origins of quinones biosynthetic pathways – QUINEVOL

A combination of bioinformatics and experimental methodologies was employed:

1. Large-scale genomic annotation of quinone biosynthetic pathways

- Use of high-quality public bacterial genome databases from the Genome Taxonomy Database (GTDB) or NCBI, representing known bacterial diversity.

- Similarity search: Identification of genes involved in quinone biosynthetic pathways (ubiquinone/UQ, menaquinone/MK, plastoquinone/PQ, rhodoquinone/RQ) using Hidden Markov Model (HMM) profiles developed by the team.

- Pathway presence criteria: A pathway is considered present if a minimum number of genes required for its biosynthesis is identified in a genome (e.g., 3 proteins for the PQ pathway, 5 for the oxygen-dependent UQ pathway, etc.).

2. Phylogenetic and comparative analysis

- Mapping of biosynthetic pathways: Overlaying annotation data of pathways onto a bacterial phylogenetic tree to visualize the distribution of biosynthetic pathways and identify evolutionary mechanisms underlying pathway diversity (losses, gains, horizontal transfers).

- Phylogenetic analyses to determine the origin of pathway genes.

- Correlation with energy metabolisms: Study of the links between quinone diversity and metabolic adaptations (aerobic/anaerobic).

3. Biochemical characterization of produced quinones

- Extraction of quinones from bacterial cultures, using liquid chromatography coupled with mass spectrometry (LC-MS) to identify and quantify quinones produced by different bacterial species.

4. Functional validation of enzymes

- Heterologous expression: Cloning and expression of candidate genes in model strains (such as mutants of Escherichia coli) to confirm their role in quinone biosynthesis.

Among the major results, we can highlight the following, the details of which are discussed in four scientific articles and one pre-print article:

- The development of a genome annotation tool for quinone biosynthetic pathways (ubiquinone UQ, menaquinone MK/Men, and futalosine, plastoquinone PQ), which has been continuously expanded and improved through a combination of comparative genomics, phylogenetic, and experimental approaches

- The elucidation of the evolutionary scenario for the diversification of hydroxylation steps in O₂-dependent and O₂-independent ubiquinone biosynthetic pathways

- The demonstration of strong conservation in Pseudomonadota of the regulation by FNR (a major regulator of anaerobiosis) of the O₂-independent UQ biosynthetic pathway

- The identification of the link between the quinone repertoire of Pseudomonadota and their respiratory metabolism, providing an answer to one of the major questions of the project and opening rich perspectives for studying the diversification of prokaryotic metabolism in light of quinones

- The global scenario of the evolution of the quinone repertoire in Pseudomonadota, with the now-demonstrated ancestrality of the UQ pathway and secondary lateral acquisitions of the Men pathway of MK

- The involvement of an FMO enzyme (flavin monooxygenase) requiring O₂ to produce plastoquinone in photosynthetic Cyanobacteria, and the demonstration of its acquisition from FMO of the UQ pathway of Pseudomonadota

- The discovery of a new bacterial lineage containing UQ and possessing the O₂-independent pathway, which, after phylogenetic analyses, allows us to push back the origin of the O₂-independent ubiquinone production pathway and propose that it predates the O₂-dependent biosynthetic pathway

The last two points provide answers to major questions of the project regarding the respective order of appearance of quinone biosynthetic pathways: UQ versus PQ (at least in its contemporary version), and O₂-dependent versus O₂-independent UQ.

- A global overview of the distribution of quinone biosynthetic pathways in bacteria

- The extraction of literature data on the nature and chain lengths of quinones for thousands of bacterial species using text mining approaches

- The discovery, in collaboration with Dr. Felix Elling (U. Kiel, Germany), of a new high-potential quinone in Nitrospirota, methylplastoquinone, as well as the genomic and experimental characterization of the enzymes involved in the corresponding new biosynthetic pathway

The discoveries made during the QUINEVOL project have opened up numerous perspectives that will be explored in a newly ANR-funded project, the PRCI ToLQuin project.

Notably, it has emerged that 1) high-potential quinones (methylplastoquinone and ubiquinone) are more widespread in the tree of life than previously expected, 2) certain lineages lack quinones, and 3) the quinone repertoire appears to be associated with the diversification of bacterial metabolism.

In particular, this new project will focus on thoroughly exploring the still uncharacterized steps in quinone biosynthetic pathways, cataloging quinones in laboratory-cultivated archaeal and bacterial lineages, and investigating the diversity of quinones in environmental samples. Ultimately, this new perspective on quinone diversity across the tree of life will help elucidate the role of quinone diversification in the evolution of bacterial and archaeal metabolism within a biogeochemical context.

By provoking a shift from globally reducing to oxidizing conditions on Earth’s surface, the great oxidation event (GOE) had a profound impact on the bioenergetics of ancestral microorganisms. Curiously, in spite of their crucial role in bioenergetics processes, the nearly-universal quinone molecules have been overlooked in the context of O2 rise on Earth. This is the topic of the QUINEVOL project.
Quinones are central to cellular energy production, and have been subject to intense evolutionary pressures provoked by the GOE. Low-potential quinones, which are widely spread and thought to be ancestral, are sensitive to O2 as they quickly get oxidized when O2 levels rise. High-potential quinones are thought to have appeared in response to increased O2 levels. They are found in two lineages that remarkably succeeded in their colonization of oxic environments: the oxygenic Cyanobacteria and Proteobacteria.
Cyanobacteria are considered to be responsible for the GOE as they use oxygenic photosynthesis to generate cellular energy, a process that releases O2 and involves the high-potential quinone plastoquinone (PQ). Until now, Proteobacteria were thought to have emerged after oxygenic Cyanobacteria, as they required O2 to synthetize their essential high-potential quinone ubiquinone (UQ).
Recent discoveries by our team however leads to reconsider the relative timing of appearance of high-potential quinones, and of the organisms that adopted them. We found 1) a very widespread pathway in Proteobacteria that does not require O2 for UQ production and can be involved in anaerobic processes; 2) that the pathway for PQ production in Cyanobacteria requires O2 in at least one of its steps. Moreover, uncertainties on the tempo of Earth’s oxygenation, and the recent discovery of new lineages – including non-oxygenic Cyanobacteria – further question the O2-context of Proteobacteria and Cyanobacteria emergence, and their relative timing of origins.
In the QUINEVOL project, we propose to tackle these questions by performing an unprecedented integrative study of the evolution of quinone biosynthetic pathways. This will allow to shed lights on ancestral microorganisms’ adaptation to rising O2 levels, from a bioenergetics perspective. We propose to 1) design annotation tools to identify quinone biosynthetic pathways in genomes, 2) revise the classical divide between low-potential/high-potential quinones and the environmental conditions they are used in, 3) decipher how UQ and PQ appeared in Proteobacteria and Cyanobacteria respectively, and 4) build a global scenario for quinone evolutionary origins in the context of Earth’s oxygenation.
We propose to use an original combination of phylogenomic and experimental approaches. a) The developed annotation tools will be validated based on text mining approaches on the existing literature and based on experimental characterization of quinones from unexplored Bacterial lineages. This will enable to re-assess the quinones’ repertoire for the “new Tree of Life” while proposing candidate enzymes for missing steps in quinones’ pathways. b) Quinones’ ecophysiology will be re-evaluated using a meta-analysis of the environmental distribution of quinones, and the analysis of an original environmental dataset that will combine lipidomics and metagenomics data. c) A global timed-scenario for quinones’ origins will be built by combining classical phylogenetic analyses with the use of lateral gene transfers events as tools for “molecular dating”. Overall, the original approaches of the QUINEVOL project will enable to shed new lights on adaptations of bioenergetics processes in relation to Earth’s oxygenation.