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, an particularly chromatic acclimaters, at four organizational levels: gene, cell, population and global ocean.

1. At the gene level, our strategy consists of inactivating in the model Synechococcus strain A15-62 the genes present in the CA4-B genomic island that are potentially involved in the CA4-B process, namely the phycobilin lyases mpeW and mpeQ, the regulators fciA and fciB and a gene of yet unknown function (bbaA/ unk10). Then, we will characterize the pigment phenotype of the mutants compared to the wild strain using various biophysical and biochemical techniques.

2. At the cellular level, we try to understand i) why CA4-A and -B occupy different niches in the marine environment despite the same pigment phenotype, and ii) the evolutionary advantage conferred by their ability to change color compared to strains with fixed pigmentation. For this, we carry out comparative studies on mono-cultures of CA4-A and -B in different conditions of light quantity and quality and different temperatures, and continuous co-cultures in blue or green light of a CA4-capable strain and strains with fixed pigmentation, namely one blue light specialist and one green light specialist.

3. At the population level, we are studying the seasonal variations of the distribution of Synechococcus pigment types at two long-term stations, ASTAN in the English Channel and BOUSSOLE in the western Mediterranean Sea. At each station, we took seawater samples to extract DNA and sequence metagenomes, from which we extracted pigment marker genes, in order to estimate the relative abundance of different pigment types. These data are then correlated with the numerous metadata available (temperature, salinity, water color, etc.) in order to determine the environmental factors associated with each pigment type.

4. At the global ocean level, a powerful model developed by MIT simulating the biogeochemistry of the ocean (Darwin; darwinproject.mit.edu) was adapted to simulate the distribution and abundance of different Synechococcus pigment types in the global ocean as well as the effects of global change.

The work carried out so far within the framework of ANR EFFICACY has already enabled us to obtain several important results:

1. We determined the function of two enzymes, MpeW and MpeQ essential for the CA4-B process. We demonstrated that these enzymes compete with each other: in green light, MpeW is activated and binds a green light catching pigment to a particular site of MpeA, one subunit of the main photosynthetic antenna protein of Synechococcus; in blue light, MpeW is inhibited and it is MpeQ, whose expression is constitutive, which fixes a blue-catching pigment at this same position of MpeA (Grébert et al. 2021). The CA4-B process is therefore similar but opposite to that of the previously characterized CA4-A, which results from the competition between the enzyme MpeZ (activated in blue light) and MpeY (constitutively expressed).

2. The structural characterization of MpeQ revealed its ‘question mark’-shaped architecture and made it possible to identify its active site and highlight the structural differences existing within the MpeQWYZ enzyme family (Kumarapperuma et al. 2022).

3. We studied the mechanisms of evolution of the pigment types of Synechococcus and showed that the progressive sophistication of the light-collecting complexes observed in this genus, from that of green specialists to chromatic acclimaters, was accompanied by a parallel complexification of the genomic region bringing together the main genes involved in the biosynthesis of photosynthetic antennae (Grébert et al. 2022).

4. We managed to identify in culture the subtle differences that exist between CA4-A and CA4-B strains: if they behave the same way in blue and green light, they differ when cultivated in a mixture of these two colors, and their acclimation dynamics after changing from blue to green is also different (Dufour et al. 2024).

5. Co-culture experiments of a CA4-B, a blue specialist and a green specialist showed that specialists win the competition in their preferred color at low light, but only at high blue light it is the CA4-B strain which takes over (Dufour et al. 2025).

6. Modeling of the spectral properties of underwater waters revealed that the marine environment encompasses 5 different spectral niches (violet, blue, green, orange and red) depending on the particle load of the waters, and that the remarkable pigment diversity of Synechococcus allows it to colonize them all (Holtrop et al. 2021).

7. Finally, modeling the global distribution of pigment types showed that chromatic acclimators are the most ubiquitous Synechococcus cells in the ocean, and dominate at the interface between coastal zones, where green specialists dominate, and oceanic gyres, where blue specialists dominate (Mattei et al. 2025).

Most of the tasks planned under the EFFICACY program, which is scheduled to end in June 2025, have been completed.

1. Regarding the understanding of the molecular bases of the CA4-B type chromatic acclimation process, we have characterized by a classical mutagenesis approach the genes of the MpeQ and MpeW enzymes in the model Synechococcus strain A15-62 (CA4-B). We also got in this same strain by a CRISPR approach a mutant of the fciB gene, a transcriptional regulator, whose characterization is still in progress. We are still attempting to get CRISPR mutant of the second CA4 regulator (fciA) and, if possible, of the unk10/bbaA gene, which we hypothesize has a role of binding blue light-harvesting chromophores on the two other binding sites that switch during CA4.

2. Mono- and co-culture experiments of different pigment types of Synechococcus are completed. A first article showing the differences between CA4-A and CA4-B type chromatic acclimators was published (Dufour et al. 2024), and a second article describing the results of continuous mono- and co-cultures of the three pigment types (a CA4-B type chromatic acclimator, a blue light specialist and a green light specialist) and the comparison of their growth rate with the predictions of a competition model for light has just been submitted for publication.

3. Regarding the time series, we collected two complete annual cycles of seawater sampling and metadata associated at two long-term stations: ASTAN in the English Channel and BOUSSOLE in the Mediterranean Sea. The analysis of metagenomes allowed us to highlight strong seasonal variations in the relative abundance of pigment types of Synechococcus and how they relate with variations in environmental parameters. The results show that Channel waters are alternately dominated by a green light specialist and a CA4-A type chromatic acclimator, while Mediterranean waters see the alternation of a blue light specialist (in summer) and a type CA4-A chromatic acclimator (in winter). Several other pigment types are also present seasonally but are less abundant. An article describing these results is in preparation.

4. Finally, in collaboration with MIT (Cambridge, USA), we adapted the global Darwin Ocean biogeochemistry model (https://darwinproject.mit.edu/) in order to model the oceanic distribution of the three pigment types of Synechococcus (chromatic acclimator, blue specialist and green specialist) and an article describing the results was published. Modeling the future distribution of these pigment types in the context of global change is currently underway.

It is important to note that the results that we obtained at one organizational scale allowed us to inform and/or to improve those obtained at one or several other scales. For example, we characterized the molecular function of the mpeW gene, and used its sequence and that of two other markers to identify and quantify the different pigment types in natural populations of Synechococcus.

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.