Orthogonal Degron System for Controlled Protein Degradation in Cyanobacteria

Abstract

Synechococcus elongatus PCC 7942 is a model cyanobacterium for study of the circadian clock, photosynthesis, and bioproduction of chemicals, yet nearly 40% of its gene identities and functions remain unknown, in part due to limitations of the existing genetic toolkit. While classical techniques for the study of genes (e.g., deletion or mutagenesis) can yield valuable information about the absence of a gene and its associated protein, there are limits to these approaches, particularly in the study of essential genes. Herein, we developed a tool for inducible degradation of target proteins in S. elongatus by adapting a method using degron tags from the Mesoplasma florum transfer-mRNA (tmRNA) system. We observed that M. florum lon protease can rapidly degrade exogenous and native proteins tagged with the cognate sequence within hours of induction. We used this system to inducibly degrade the essential cell division factor, FtsZ, as well as shell protein components of the carboxysome. Our results have implications for carboxysome biogenesis and the rate of carboxysome turnover during cell growth. Lon protease control of proteins offers an alternative approach for the study of essential proteins and protein dynamics in cyanobacteria.

Investigating Carboxysome Morphology Dynamics with a Rotationally Invariant Variational Autoencoder

“…Cultures were grown under continuous light with GroLux bulbs (Sylvania) at 125 µ mol photons m −2 s −1 , 2 % CO2, 32 °C, and 130 rpm shaking. Carboxysomes were degraded by inserting a protein degradation tag (PDT) at the C-terminus of the shell protein CcmO, which is essential for carboxysome closure, and expression of a non-native Lon protease from Mesoplasma florum [16]. For visualization of changes in carboxysome morphology, a second copy of the small subunit of Rubisco, rbcS, was tagged with a C-terminal fusion of mNeonGreen (mNG).…”

“…For the induced samples, cultures were induced with 30 µM theophylline. More detailed culturing, genetic assembly, and transformation information was previously described [16].…”

“…Towards a better understanding of carboxysome biogenesis, we sought to develop a quantitative method for investigating the temporal dynamics of carboxysome remodelling. In recent work [16], we used a method for protein down regulation based on the Lon protease from Mesoplasma florum (mf-lon) allowing rapid inducible degradation of proteins of interest. This approach offers the advantage that carboxysome components can be specifically targeted in a manner that causes dynamic rearrangement of carboxysome morphology beginning at an experimentally defined time point.…”

Inducible CRISPR/Cas9 allows for multiplexed and rapidly segregated single target genome editing in Synechocystis sp. PCC 6803

“…These theophylline riboswitches have already been evaluated in various cyanobacteria, [31][32][33][34] and used in developing tools such as NOT-gates in e.g. S6803, 33 inducible protein degradation systems in S. elongatus PCC 7942, 35,36 and inducible CRISPR-interference systems in S6803 and Anabaena. 37,38 In this study they were further used to design a tightly controlled CRISPR/Cas9 system for inducible genome editing in S6803.…”

Investigating Carboxysome Morphology Dynamics with a Rotationally Invariant Variational Autoencoder

“…For example, some evidence suggests that once carboxysomes are formed, they are static until they are ultimately degraded as a unit . Several tools and reporter constructs have been developed to track the dynamics of carboxysome positioning and morphological features in living cells, ,,,, yet the variability of carboxysome features in natural populations and resolution limits of fluorescence microscopy complicate quantitative evaluation of changes in carboxysome size, composition, and intracellular positioning.…”