Ecological Fitness of Light Color Acclimation in Marine Cyanobacteria: a Cross-Scale Analysis – EFFICACY

In the framework of EFFICACY, we are studying Synechococcus pigment types at four organizational levels: gene, cell, population and global ocean.
1. Gene level: our strategy is to knock out, in the model strain Synechococcus sp. A15-62, all genes potentially involved in the CA4-B process, namely the phycobilin lyase genes mpeW and mpeQ, the regulators fciA and fciB and a gene of yet unknown function present in the CA4-B genomic island and upregulated in blue light. Then we characterize the pigment phenotype of mutants as compared to the wild type using various biophysical and biochemical techniques.
2. At the cell level, we are trying to understand i) why CA4-A and -B occupy different niches in the marine environment despite having the same pigment phenotype, and ii) the fitness advantage conferred by their ability to chromatically acclimate compared to cells with fixed pigmentation. For this, we are doing i) comparative studies on mono-cultures of CA4-A and -B strains and ii) co-cultures of CA4 strains and strains with fixed pigmentation (blue or green light specialists).
3. At the population level, we are studying the seasonal variations of pigment types distribution at 2 long-term stations: for this we sample twice a month at ASTAN in the English Channel and once a month at BOUSSOLE station lin the Mediterranean Sea. At each station, we sample seawater for metagenomes, from which we will extract marker pigment genes, and collect numerous associated metadata, including a profile of the underwater light spectrum for correlation analyses.
4. At the global ocean level, we will try to model the distribution of Synechococcus pigment types everywhere in the ocean and to simulate the seasonal variations and those caused by the global change over the coming decades. For this, the data acquired in the other tasks as well as previously published data will be used to feed a powerful model simulating the biogeochemistry of the ocean (Darwin; darwinproject.mit.edu/).

The work carried out within the framework of the ANR project EFFICACY has already enabled us to get several important results:
1. We have determined the function of two enzymes, MpeW and MpeQ, essential for the CA4-B process. Indeed, these enzymes are in competition, MpeW fixing a green light-absorbing pigment capturing at a particular site of the main protein of the photosynthetic antenna in green light, while MpeQ fixes a blue-light absorbing pigment at the same position in blue light (Grébert et al. 2021a, PNAS). This study also allowed us to understand why two types of CA4 have occurred during the evolution: the acquisition of a CA4-A genomic island seems to have allowed green light specialists to become chromatic acclimatizers, while blue light specialists have acquired this capability by integrating a CA4-B island.
2. Another enzyme of the same family as mpeW/Q, MpeV, has also been characterized: it fixes a blue pigment by covalent double bond to another site of the same protein, but is not involved in CA4 (Carrigee et al., 2021; J. Biol. Chem.).
3. We have also studied the evolutionary mechanisms leading to pigment diversification in Synechococcus and we have shown that the progressive complexification of antenna complexes observed in this genus, from that of green specialists to chromatic acclimatizers, was accompanied by a parallel complexification of the genomic region gathering the main genes involved in the biosynthesis of photosynthetic antennas. This work is currently being evaluated and is available in a pre-print in BioRxiv (Grébert et al. 2021b).
4. Finally, we have collaborated with Prof. Jef Huisman (VU University Amsterdam) on the development of a model predicting underwater spectral niches as a function of the water particle load. It highlights the influence of vibrations of water molecules on the underwater light spectrum (Holtrop et al. 2021, Nature Ecol. Evol.).

While progress on tasks planned in the framework of the EFFICACY project has been somewhat delayed by the covid-19 pandemic, we have already obtained some promising results detailed above. The following paragraphs provide an overview of the ongoing work.
1. Regarding the understanding of the molecular bases of the type CA4-B chromatic acclimation process, we expect to obtain soon mutants of all remaining genes involved in the CA4-B process (namely the regulators fciA and fciB, and the unknown gene unk10) by a CRISPR approach and the characterization of these mutants will be carried out in the forthcoming months.
2. Comparisons of the growth rate and photosynthetic activity of CA4-A and B strains under different light conditions is also underway and should provide us with elements to understand the differential distribution of these two pigment types in the natural environment. The results of these experiments as well as co-cultures will then be simulated with a competition model, in collaboration with our colleagues from the University of Amsterdam.
3. Regarding the time series, we already have a complete annual cycle of seawater sampling and associated metadata at the two long-term stations ASTAN and BOUSSOLE. When we have collected enough samples, the analysis of metagenomes will allow us to study the seasonal variations in the relative abundance of pigment types and their relationship to variations in environmental parameters.
4. Finally, all of these data will allow us to feed a global distribution model of Synechococcus pigment types, which will allow us to simulate their seasonal variations throughout the ocean and predict longer-term variations in the context of global change.

1. Carrigee L.A., Frick J.P., Karty J.A., Garczarek L., Partensky F. & Schluchter W.M. 2021. MpeV is the lyase isomerase for the doubly-linked phycourobilin on the ß-subunit of phycoerythrin I & II in marine Synechococcus. Journal of Biological Chemistry 296: 100031. DOI: 10.1074/jbc.RA120.015289
2. Grébert T., Nguyen A.A., Pokhrel S., Joseph K.L., Chen B., Ratin M., Dufour L., Haney A.M., Trinidad J., Karty J.A., Garczarek L., Schluchter W.M., Kehoe D.M. & Partensky F. 2021. Molecular basis of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria. Proceedings of the National Academy of Sciences of the USA. 118 (9) e2019715118. DOI: 10.1073/pnas.2019715118
3. Grébert T., Garczarek L., Daubin V., Humily F., Marie D., Ratin M., Devailly A., Farrant G.K., Mary I., Mella-Flores D., Tanguy G., Labadie K., Wincker P., Kehoe D.M. & Partensky F. 2021. Diversity and evolution of pigment types and the phycobilisome rod gene region of marine Synechococcus cyanobacteria. BioRxiv DOI: 10.1101/2021.06.21.449213.
4. Hess, W.R., Garczarek, L. & Partensky, F. 2021. Chapter 3: Marine Cyanobacteria. In: “The marine microbiome”, 2nd Edition, L.J. Stal and M.S. Cretoiu (eds.), Springer International Publishing, Switzerland. In the press.
5.Holtrop T., Huisman J., Stomp M., Biersteker L., Aerts J., Grébert T., Partensky F., Garczarek L & van der Woerd H.J. 2021.Vibrational modes of water predict spectral niches for photosynthesis in lakes and oceans. Nature Ecology and Evolution 5: 55-66. DOI: 10.1038/s41559-020-01330-x.

The ongoing global change is predicted to have numerous consequences on ocean physico-chemical properties, and notably on ‘ocean color’, a signal used by modelers to assess chlorophyll a biomass at global scales. For phytoplankton cells, changes in ocean color are perceived as a modification of their underwater light niches that can trigger competition between species potentially resulting in dramatic changes in community composition. To tackle the question of the respective fitness of phytoplankton species to survive in environments with altered spectral properties, we will focus on the picocyanobacterium Synechococcus, the second most abundant phytoplanktonic organism of the ocean, and the most diversified one with regard to its pigmentation, with at least seven pigment types displaying distinct genetic signatures, making it possible to differentiate them based on three gene markers. We recently showed that chromatic acclimaters (CA4), i.e. cells capable to change their pigment content to match the dominant light color (blue or green) were the most abundant Synechococcus pigment type in the ocean, with about equal abundances of two genetically different types, CA4-A and CA4-B, which exhibit very complementary ecological niches in the field. During the ANR project EFFICACY, we will study the ecological importance and fitness advantage conferred by the CA4 process using a cross-scale approach. We will: i) characterize the function of key genes of the CA4-B genomic island in order to unveil molecular differences between CA4-B and the well-characterized CA4-A process to better understand how and why natural selection has favored these two distinct forms of chromatic acclimation; ii) make competition experiments between CA4 strains and other Synechococcus strains with fixed pigmentation to determine which ones are best fitted in blue or green light and at different irradiances in order to help interpret the spatial and temporal variations of these pigment types; iii) study the seasonal variations of the relative proportions of the different Synechococcus pigment types at two oceanographically distinct sites, the long-term time series stations BOUSSOLE (Mediterranean Sea) and ASTAN (English Channel), using a metagenomic approach, and iv) integrate data derived from the two latter tasks and previous work from the coordinating partner into a powerful global ocean model (Darwin) that will simulate the present global spatial and temporal distribution of Synechococcus pigment types and predict the effect of global change on this population structure over the forthcoming decades. By using cutting-edge technologies and a powerful, state-of-the-art ocean model to study the pigment diversity of an ecologically relevant microorganism at all scales of organization from the genes to the global ocean, including seasonal variations, this ambitious interdisciplinary project should bring unprecedented insights into the field of environmental microbiology and pave the way to refined forecasting of the evolution of phytoplankton communities at large, in the context of global change.