Uncovering Novel Assembly Factors of the Cyanobacterial Photosynthetic Complexes Through Transcriptomics

In Cyanobacteria, the assembly of the photosynthetic electron transport chain (ETC) within thylakoid membranes (TM) is mediated by assembly factors that guide the formation of photosynthetic complexes through transient interactions with protein subunits and cofactors. While the de novo assembly of photosystem II (PSII) is a highly coordinated spatial and temporal process initiated at the cytoplasmic membrane (CM), the assembly mechanisms of PSI, ATPase, Cyt b6f, and alternative electron transfer complexes remain poorly understood. This project aims to identify novel assembly factors involved in ETC formation by analyzing gene expression during thylakoid membrane regeneration following damage.
A recent protocol enabling de novo TM biogenesis in Synechococcus elongatus PCC 7942 through light modulation will be reproduced and coupled with transcriptomic analysis to uncover early molecular actors of TM biogenesis at the CM and intermembrane space. TM reduction will be induced under high light for 14 days and validated by transmission electron microscopy (TEM). RNA-seq will be performed on control and high-light cultures, across a five days’ time course, to identify differentially expressed genes associated with TM regeneration.
For unannotated proteins, functions will be inferred from phylogenetic profiling, ecological profiling across Cyanobacteria taxonomic distribution, synteny profiling by co-occurrence and genomic co-localization, structural profiling through 3D structure prediction, and homology searches of unknown assembly factors. Selected candidates will be validated by mutagenesis, combined with O₂ measurements and the use of inhibitors to associate specific complexes with their assembly factors.
This approach is expected to identify novel assembly factors involved in the formation of key photosynthetic ETC components, including those participating in alternative electron flow pathways, which remain largely unexplored in cyanobacteria.