Light Modulates the Biosynthesis and Organization of Cyanobacterial Carbon Fixation Machinery through Photosynthetic Electron Flow

Abstract

Cyanobacteria have evolved effective adaptive mechanisms to improve photosynthesis and CO 2 fixation. The central CO 2 -fixing machinery is the carboxysome, which is composed of an icosahedral proteinaceous shell encapsulating the key carbon fixation enzyme, Rubisco, in the interior. Controlled biosynthesis and ordered organization of carboxysomes are vital to the CO 2 -fixing activity of cyanobacterial cells. However, little is known about how carboxysome biosynthesis and spatial positioning are physiologically regulated to adjust to dynamic changes in the environment. Here, we used fluorescence tagging and live-cell confocal fluorescence imaging to explore the biosynthesis and subcellular localization of b-carboxysomes within a model cyanobacterium, Synechococcus elongatus PCC7942, in response to light variation. We demonstrated that b-carboxysome biosynthesis is accelerated in response to increasing light intensity, thereby enhancing the carbon fixation activity of the cell. Inhibition of photosynthetic electron flow impairs the accumulation of carboxysomes, indicating a close coordination between b-carboxysome biogenesis and photosynthetic electron transport. Likewise, the spatial organization of carboxysomes in the cell correlates with the redox state of photosynthetic electron transport chain. This study provides essential knowledge for us to modulate the b-carboxysome biosynthesis and function in cyanobacteria. In translational terms, the knowledge is instrumental for design and synthetic engineering of functional carboxysomes into higher plants to improve photosynthesis performance and CO 2 fixation.

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

“…In contrast, RbcL-YFP and CcmK2-YFP strains are only partially segregated, in agreement with previous studies (Savage et al, 2010;Cameron et al, 2013;Sun et al, 2016). Through immunoblot analysis using anti-fluorescence protein, anti-RbcL and anti-CcmK2 antibodies (Supplemental Figure 2B), we estimate that 29.2 ± 7.1 % (mean ± standard deviation (SD), n = 4) of total RbcL and 6.0 ± 0.7 % (n = 3) of total CcmK2 were tagged with YFP in RbcL-YFP and CcmK2-YFP strains.…”

“…We achieved this by identifying the peak value of the foci intensity distribution from each given cell strain obtained from confocal imaging with the peak value of the measured Slimfield foci stoichiometry distribution for the equivalent cell strain under Air/ML. This approach allows us to generate a conversion factor which we then applied to subsequent confocal data acquired under lower light (LL), higher light (HL) and ML with the air supplemented by 3% CO2, and to estimate relative changes in the stoichiometry of carboxysome building components using large numbers of cells, without the need to obtain separate Slimfield datasets for each condition (Figure 2 We find that the number of carboxysomes per cell is dependent on growth conditions: it was reduced under CO2/ML in contrast to Air/ML, whereas HL increases the abundance of β-carboxysomes 7 ( Supplemental Table 3), consistent with previous findings (Whitehead et al, 2014;Sun et al, 2016).…”

“…We constructed ten Syn7942 strains expressing individual β-carboxysome proteins (CcmK3,CcmK4,CcmK2,CcmL,CcmM,CcmN,RbcL,RbcS,CcaA,RbcX) fused with YFP at their C-termini individually (Supplemental Figure 1). Fluorescence tagging at the native chromosomal locus under the control of their native promoters ensures expression of the fluorescently-tagged proteins in context and at physiological levels (Sun et al, 2016). Eight of these strains, in which YFP was fused to CcmK3, CcmK4, CcmL, CcmM, CcmN, RbcS, CcaA, and RbcX respectively, are fully segregated (Supplemental Figures 1C and 2) and exhibit wild-type levels of cell size, growth and carbon fixation within experimental error ( Supplemental Table 1), consistent with previous observations (Savage et al, 2010;Cameron et al, 2013;Sun et al, 2016;Faulkner et al, 2017;Huang et al, 2019).…”

“…Our previous study showed that the content and spatial positioning of β-carboxysomes in Syn7942 are dependent upon light intensity during cell growth, revealing the physiological regulation of carboxysome biosynthesis (Sun et al, 2016). Whether the stoichiometry of different components in the carboxysome structure changes in response to fluctuations in environmental conditions is unknown.…”

“…Moreover, it was revealed that carboxysome biosynthesis in Syn7942 are highly dependent upon environmental conditions during cell growth, such as light intensity (Sun et al, 2016) and CO2 availability (McKay et al, 1993;Harano et al, 2003;Woodger et al, 2003;Whitehead et al, 2014).…”