Testing the Ménage à Trois Hypothesis – MATHTEST

We carried out classical phylogeny and biochemistry approaches which allowed us on the one hand to highlight the transfer, within the genome of the ancestral plastid, of a cluster of genes involved in the synthesis of menaquinone and on the other, to analyse glycogen metabolism in chlamydia. In order to reassess the bacterial contribution to the emergence of lineages originating from plastid endosymbiosis, we set up an automatic unbiased pipeline to detect bacterial LGTs in Archaeplastida genomes. The recipient and donor genomes studied were selected, based on the quality of the genomic resources and the diversity covered, thus generating a pipeline dedicated to each of the main bacterial groups covering the entire diversity of the bacterial domain. Furthermore, with regard to chlamydial LGTs, we have also designed control pipelines relating to the eukaryotic genomes recipients of these LGTs with a focus on amoebae and fungi. Finally, we designed an annotation system aimed at detecting LGTs encoding plastidial transporters in all bacterial groups. .

Major project results
Plastidial endosymbiosis was accompanied not only by the massive transfer of 621 genes of cyanobacterial origin to the nuclear genomes of the lineages derived from them but also by that of 220 genes of various bacterial origins. Among these, genes from intracellular pathogens of the chlamydial group alone account for 8% of cyanobacterial transfers and define the main gene donor among all bacterial groups. The results show a surprising role for chlamydiales in establishing early plastid connectivity.

This work confirmed the existence and robustness of the chlamydial phylogenomic signal as well as its pre-eminence in comparison to all other lateral transfers of bacterial origin. We further showed the selective existence of multiple gene transfers whose topologies agree particularly well with the existence of ancient biotic interactions based on conjugative transfers from the chlamydia genome to the genome of the cyanobacteria ancestor of the plastid. Furthermore, the reality of such conjugative transfers could be demonstrated in the case of menaquinone metabolism in red cyanidial algae. The most striking result of the project, in our eyes, consists of the discovery of an unexpected selective contribution of chlamydia in early plastid connectivity which is unparalleled in other bacteria. Indeed, chlamydia contributed the same number of plastid transporter genes common to the three lineages of Archaeplastida as the cyanobacteria ancestors of the plastid! This suggests that the first metabolic exchanges between the evolving plastid and the cytosol, in the absence of the system for addressing proteins to the plastid, were as much under the control of the ancestral cyanobacteria as under that of a chlamydiales interacting with the latter.

1. Maeno T, Yamakawa Y, Takiyasu Y, Miyauchi H, Nakamura Y, Ono M, Ozaki N, Utsumi Y, Cenci U, Colleoni C, Ball S, Tsuzuki M, Fujiwara S. One of the isoamylase isoforms, CMI294C, is required for semi-amylopectin synthesis in the rhodophyte Cyanidioschyzon merolae. Front Plant Sci. 2022 Aug 16;13:967165. doi: 10.3389/fpls.2022.967165. PMID: 36051298; PMCID: PMC9424615.
2. Oliver D Caspari, Clotilde Garrido, Chris O Law, Yves Choquet, Francis-André Wollman, et al.. Converting antimicrobial into targeting peptides reveals key features governing protein import into mitochondria and chloroplasts. Plant Communications, 2023, 4 (4), pp.100555. ?10.1016/j.xplc.2023.100555?. ?hal-04234816?
3. Ryoma Kamikawa, Takako Mochizuki, Mika Sakamoto, Yasuhiro Tanizawa, Takuro Nakayama, et al.. Genome evolution of a nonparasitic secondary heterotroph, the diatom Nitzschia putrida. Science Advances , 2022, Science Advances, 8 (17), pp.eabi5075. ?10.1126/sciadv.abi5075?. ?hal-03677948?
4. Lea Fermont, Nicolas Szydlowski, Christophe Colleoni. Determination of Glucan Chain Length Distribution of Glycogen using the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Method.. Journal of visualized experiments : JoVE, 2022, Journal of visualized experiments : JoVE, ?10.3791/63392?. ?hal-03678033?
5. Clotilde Garrido, Francis-André Wollman, Ingrid Lafontaine. The Evolutionary History of Peptidases Involved in the Processing of Organelle-Targeting Peptides. Genome Biology and Evolution, 2022, 14 (7), pp.evac101. ?10.1093/gbe/evac101?. ?hal-03766202?
6. Matthieu Colpaert, Derifa Kadouche, Mathieu Ducatez, Trestan Pillonel, Carole Kebbi-Beghdadi, et al.. Conservation of the glycogen metabolism pathway underlines a pivotal function of storage polysaccharides in Chlamydiae. Communications Biology, 2021, 4 (1), ?10.1038/s42003-021-01794-y?. ?hal-03174571?
7. Malika Chabi, Marie Leleu, Lea Fermont, Matthieu Colpaert, Christophe Colleoni, et al.. Retracing Storage Polysaccharide Evolution in Stramenopila. Frontiers in Plant Science, 2021, 12, pp.629045. ?10.3389/fpls.2021.629045?. ?hal-03268608?
8. Clotilde Garrido, Oliver Caspari, Yves Choquet, Francis-André Wollman, Ingrid Lafontaine. Evidence Supporting an Antimicrobial Origin of Targeting Peptides to Endosymbiotic Organelles. Cells, 2020, 9 (8), pp.1795. ?10.3390/cells9081795?. ?hal-03008883?
9. Dana C Price, Ursula W Goodenough, Robyn Roth, Jae-Hyeok Lee, Thamali Kariyawasam, et al.. Analysis of an improved Cyanophora paradoxa genome assembly. DNA Research, 2019, DNA Research, 26 (4), pp.287-299. ?10.1093/dnares/dsz009?. ?hal-03095856?
10. Cenci U, Qiu H, Pillonel T, Cardol P, Remacle C, Colleoni C, Kadouche D, Chabi M, Greub G, Bhattacharya D, Ball S.G. (2018) Host-pathogen biotic interactions shaped vitamin K metabolism in Archaeplastida. Scientific Reports 8:15243

We have made significant progress with regard to plastid origin and have provided strong evidence that the canonical plastid was domesticated due to the direct impact of a now absent player, an environmental chlamydial pathogen (1). This hypothesis called the ménage à trois hypothesis (MATH) specifically addresses the central issue of disconnected supply and demand of carbon at the time of plastid endosymbiosis [2]. Chlamydial cells were central to this process by providing many critical genes, such as glucan transferase and carbohydrate transporters, to the cyanobacterium that was harbored in an inclusion. The MATH hypothesis predicts that genes of chlamydial origin were transferred to the cyanobacterial symbiont genome through conjugation in the common inclusion vesicle. This enabled the biochemical fluxes associated with symbiosis through expression and efficient localization of a set of key transporters absent in cyanobacteria, at a time when the organelle protein targeting system had not yet evolved. In addition, operons or clusters encoding entire pathways of chlamydial origin were donated to modify key fluxes within the cyanobacterium (3,4). If this idea is correct, then the plastid genome became highly chimeric at the early stages of endosymbiosis. Can we find molecular “fossil” evidence that supports the presence of chlamydial genes in extant plastomes to substantiate the chimerism hypothesis? The prospects of finding such evidence are inherently small because plastid genomes have undergone significant genome reduction (i.e., many functions [genes] were transferred to the nucleus via EGT) and primarily retain genes encoding subunits of the photosynthetic electron transport chain and the protein translation machinery required to express them. Such conserved genes are of cyanobacterial origin and were not targeted by chlamydial LGTs. We thus looked at the most-gene rich plastomes currently available. These are found in early-branching unicellular red algae such as Cyanidiophytina, which encode ca. 250 plastid genes, when compared to half that number for most other Archaeplastida (green and red algae glaucophytes and land plants). We have very recently found a 7-gene plastidial cluster of distinctive chlamydial origin, encoding all enzymes of vitamin K synthesis from chorismate , in these early diverging red algae.
In this grant proposal, we aim both to elucidate the evolutionary history of vitamin K metabolism in Gloeobacterales cyanobacteria, eukaryotic algae and plants and to investigate the mono or polyphyletic nature of the chlamydial phylogenomic signal in photosynthetic eukaryotes.
Because Gloeomargarita (5) is presently defined as the closest candidate to the plastid ancestor these experiments will enable us to narrow down the exact timing of the vitamin K cluster transfer. If, as we suspect presently this event occurred post-endosymbiosis but before Archaeplastida diversification, this will provide both a strong cladistic confirmation on the Gloeobacterales origin of the plastid and a proof of concept of the MATH hypothesis.
In addition to this, we have established a collaboration with a deep phylogenetic reconstruction group to probe the nature of the chlamydial phylogenetic signal. We aim at putting to the test the expected monophyletic nature of the shared component of this signal, thereby solidifying or rejecting the MATH hypothesis.
References cited
1. Ball SG et al. (2016) Science (351: 659-660
2. Ball SG, et al. (2013) Plant Cell 25: 7-21,
3. Cenci U., et al. (2016) Front. Cell. Infect. Microbiol 6: 67
4. Cenci U, et al. (2017) Trends in Plant Science 22(4):316-328.
5. Ponce-Toledo RI et al. (2017) Curr. Biol. 27, 386–391