Development of a highly sensitive luciferase-based reporter system to study two-step protein secretion in cyanobacteria
Russo, D.A. et al. (2022) Development of a highly sensitive luciferase-based reporter system to study two-step protein secretion in cyanobacteria. American Society for Microbiology. 204, 2, e00504-21. DOI: https://doi.org/10.1128/jb.00504-21
Cyanobacteria are found in almost every environment and are known for their ability to carry out photosynthesis and produce oxygen—two critical processes for life on Earth. Cyanobacteria, like all organisms, regulate their metabolic activity with the surrounding environment by releasing small molecules. This intricate process is controlled by specific sets of genes known as secretion systems. Understanding regulation and activity of secretion systems in Cyanobacteria has several practical applications. Cyanobacteria have garnered attention for their potential in various biotech applications, particularly in biofuel production and synthesis of valuable compounds. Knowledge of their secretion systems can aid in the engineering of Cyanobacteria to enhance and increase the efficiency of production capabilities.
While extensive work has taken place on understanding secretion systems in model organisms, not much is known about how secretion systems operate in Cyanobacteria. This gap in knowledge is largely due to the lack of quantitative assays for detecting release of small molecules, including proteins, in Cyanobacteria.
This study sought to develop a tractable, high throughput test for secretion system detection in Cyanobacteria via bioluminescent luciferase. They designed mutants containing a known secretion enzyme, LPMO TfAA10A, and fused it with a NanoLuc® luciferase reporter gene (a high intensity luciferase originally isolated from a deep-sea shrimp). At time zero, they combined cyanobacterial cultures with the Promega Nano-Glo® assay buffer, incubated in a 96-well plate and measured luminescence over 24 hours. They found that luciferase activity could be detected quickly (as fast as 2 hours) and in small volumes (1.5μl culture medium), which are key features of a tractable and high throughput system. Furthermore, the researchers could identify the specific location of secreted proteins in cell membranes via immunoblotting. The application of a bioluminescent reporter to track secreted proteins in Cyanobacteria is a powerful concept, important for progress in the field of applied molecular biology and microbiology.
Keywords: luciferase activity, secretion systems, small molecules, cyanobacteria
