Development of a Highly Sensitive Luciferase-Based Reporter System To Study Two-Step Protein Secretion in Cyanobacteria

Abstract

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multi-protein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however little is known regarding their function, regulation and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of non-pilin proteins, we developed a NanoLuc-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. PCC 6803. The NLuc reporter presented a wide dynamic range with at least one order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that the two-step secretion of NLuc is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to explore protein secretion in cyanobacteria further. Importance Protein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here we introduce a NanoLuc (NLuc)-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge on cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

Self-assembly of nanofilaments in cyanobacteria for protein co-localization

“…Gels were then either stained with Oriole Fluorescent Gel Stain (Bio-Rad), Coomassie Brilliant Blue R-250 or used for immunoblotting. For immunoblotting, proteins were transferred onto a 0.2 μm nitrocellulose membrane (Protran BA 83, Amersham) by semi-dry blotting and further processed as described previously 46 . For the detection of PduA*, a customized antibody was generated by Agrisera (Sweden).…”

“…SDS-PAGE was performed as previously described 46 . Gels were then either stained with Oriole Fluorescent Gel Stain (Bio-Rad), Coomassie Brilliant Blue R-250 or used for immunoblotting.…”

Self-Assembly of Nanofilaments in Cyanobacteria for Protein Co-localization

“…For immunoblotting, proteins were transferred onto a 0.2 μm nitrocellulose membrane (Protran BA 83, Amersham) by semidry blotting and further processed as described previously. 57 For the detection of PduA*, a customized antibody was generated by Agrisera (Sweden). This antibody was raised against the synthetic peptide "CPHTDVEKILPK-GI" in rabbits.…”

Cell surface composition, released polysaccharides, and ionic strength mediate fast sedimentation in the cyanobacterium Synechococcus elongatusPCC 7942

“…From the latter, glucose‐tolerant substrains emerged (Williams, 1988) and rapidly became popular hosts due to the availability of a sequenced genome (Kaneko et al, 1996) and their ease of cultivation (e.g., growth on glucose, lack of motility and aggregation). On the other hand, strains of the PCC 6803 lineage have played a key role in unravelling the mechanisms behind motility, phototaxis, and protein secretion (Bhaya et al, 1999; Russo et al, 2022; Schuergers et al, 2016).…”