Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment

Abstract

The carboxysome is a complex, proteinaceous organelle that plays essential roles in carbon assimilation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that self-assemble in space to form an icosahedral structure. Despite its significance in enhancing CO 2 fixation and potentials in bioengineering applications, the formation of carboxysomes and their structural composition, stoichiometry, and adaptation to cope with environmental changes remain unclear. Here we use live-cell single-molecule fluorescence microscopy, coupled with confocal and electron microscopy, to decipher the absolute protein stoichiometry and organizational variability of single b-carboxysomes in the model cyanobacterium Synechococcus elongatus PCC7942. We determine the physiological abundance of individual building blocks within the icosahedral carboxysome. We further find that the protein stoichiometry, diameter, localization, and mobility patterns of carboxysomes in cells depend sensitively on the microenvironmental levels of CO 2 and light intensity during cell growth, revealing cellular strategies of dynamic regulation. These findings, also applicable to other bacterial microcompartments and macromolecular self-assembling systems, advance our knowledge of the principles that mediate carboxysome formation and structural modulation. It will empower rational design and construction of entire functional metabolic factories in heterologous organisms, for example crop plants, to boost photosynthesis and agricultural productivity.

Binding Options for the Small Subunit-Like Domain of Cyanobacteria to Rubisco

“…In quantifications of protein abundance in cyanobacteria, a shortfall of RbcS relative to RbcL has been reported (Long et al, 2007(Long et al, , 2011Sun et al, 2019). This finding suggests that isoforms other than the L 8 S 8 rubisco holoenzyme may be present in vivo, although further substantiating analyses and investigations across multiple species as well as conditions are needed.…”

The McdAB Carboxysome Positioning System is Widespread Among β-cyanobacteria

“…Therefore, it will be interesting to compare the ATPase activities of the two McdA types, as well as the stimulatory activities of their respective McdB proteins. How Type 1 and Type 2 systems differ mechanistically, and whether these differences correlate to known factors influencing carboxysome positioning such as carboxysome size and quantity (Gonzalez-Esquer et al, 2016; MacCready et al, 2018) , genome copy number and volume (Griese et al, 2011) , redox state of cells (Sun et al, 2016, 2019) , and McdAB stoichiometry (MacCready et al, 2018) , as well as unknown factors including McdB LLPS behavior and/or intracellular salt/pH conditions (see below), is important for understanding the diversity and evolution of the McdAB system among cyanobacteria.…”

“…Since O 2 competes with CO 2 as a substrate for ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the encapsulation of RuBisCO and carbonic anhydrase within a selectively permeable protein shell (the carboxysome) is necessary for generating the high CO 2 environment needed to drive internal RuBisCO reactions towards the Calvin-Benson-Bassham cycle (CO 2 substrate) and away from the wasteful process of photorespiration (O 2 substrate) (Figure 1AB) (Reviewed in: Kerfeld et al, 2018) . Comprised of thousands of proteins (Sun et al, 2019) , carboxysomes were once thought to be completely paracrystalline (Kaneko et al, 2006) . However, recent reports have challenged this idea by showing that carboxysome formation involves the process of Liquid-Liquid Phase Separation (LLPS) (Wang et al, 2019; Oltrogge et al, 2019) ; a process that describes the ability of proteins to spontaneously demix into dilute and dense phases that resemble liquid droplets (Reviewed in: Alberti et al, 2019) .…”

Correlating single-molecule characteristics of the yeast aquaglyceroporin Fps1 with environmental perturbations directly in living cells

“…As photobleaching of mGFP proceeded during Slimfield excitation distinct fluorescent foci could be observed of half width at half maximum 250-300 nm, consistent with the diffraction-limited point spread function of our microscope system, which were tracked and characterized in terms of their stoichiometry and apparent microscopic diffusion coefficient. Distinct fluorescent foci that were detected within the microscope's depth of field could be tracked for up to several hundred ms, to a super-resolved lateral precision ~40 nm 35 using a bespoke single particle tracking software written in MATLAB (MATHWORKS) and adapted from similar live cell single-molecule studies 10,[35][36][37] .…”