Exploring the extracellular landscape of cyanobacteria with deep exoproteomics

Extracellular proteins are involved in a remarkable number of fundamental processes in cyanobacteria. Yet, there is limited knowledge regarding the identity and function of these proteins. Here, we introduce a solid-phase enhanced protein aggregation workflow that enables description of the cyanobacterial exoproteome with unprecedented depth. Application to cyanobacteria from three distinct habitats, Synechocystis sp. PCC 6803, Synechcoccus sp. PCC 11901 and Nostoc punctiforme PCC 73102, allowed the identification of up to 62% of all predicted secreted proteins. The approach was then extended to compare the Synechocystis sp. PCC 6803 wild-type secretome with that of a bloom-like aggregated state and a secretion-impaired mutant. Finally, we demonstrate that the workflow can be miniaturized and adapted to a 96-well format for high-throughput secretome analysis. Collectively, these findings challenge the general belief that cyanobacteria lack secretory proteins and point to a functional conservation of the secretome across species from different environments. Our approach can be applied to microbes from a wide range of habitats, with the potential to open new avenues of investigation in microbial exoproteomics.