Linking the Dynamic Response of the Carbon Dioxide-Concentrating Mechanism to Carbon Assimilation Behavior in Fremyella diplosiphon.

Cyanobacteria use a carbon dioxide (CO2)-concentrating mechanism (CCM) that enhances their carbon fixation efficiency and is regulated by many environmental factors that impact photosynthesis, including carbon availability, light levels, and nutrient access. Efforts to connect the regulation of the CCM by these factors to functional effects on carbon assimilation rates have been complicated by the aqueous nature of cyanobacteria. Here, we describe the use of cyanobacteria in a semiwet state on glass fiber filtration discs-cyanobacterial discs-to establish dynamic carbon assimilation behavior using gas exchange analysis. In combination with quantitative PCR (qPCR) and transmission electron microscopy (TEM) analyses, we linked the regulation of CCM components to corresponding carbon assimilation behavior in the freshwater, filamentous cyanobacterium Fremyella diplosiphon Inorganic carbon (Ci) levels, light quantity, and light quality have all been shown to influence carbon assimilation behavior in F. diplosiphon Our results suggest a biphasic model of cyanobacterial carbon fixation. While behavior at low levels of CO2 is driven mainly by the Ci uptake ability of the cyanobacterium, at higher CO2 levels, carbon assimilation behavior is multifaceted and depends on Ci availability, carboxysome morphology, linear electron flow, and cell shape. Carbon response curves (CRCs) generated via gas exchange analysis enable rapid examination of CO2 assimilation behavior in cyanobacteria and can be used for cells grown under distinct conditions to provide insight into how CO2 assimilation correlates with the regulation of critical cellular functions, such as the environmental control of the CCM and downstream photosynthetic capacity.IMPORTANCE Environmental regulation of photosynthesis in cyanobacteria enhances organismal fitness, light capture, and associated carbon fixation under dynamic conditions. Concentration of carbon dioxide (CO2) near the carbon-fixing enzyme RubisCO occurs via the CO2-concentrating mechanism (CCM). The CCM is also tuned in response to carbon availability, light quality or levels, or nutrient access-cues that also impact photosynthesis. We adapted dynamic gas exchange methods generally used with plants to investigate environmental regulation of the CCM and carbon fixation capacity using glass fiber-filtered cells of the cyanobacterium Fremyella diplosiphon We describe a breakthrough in measuring real-time carbon uptake and associated assimilation capacity for cells grown in distinct conditions (i.e., light quality, light quantity, or carbon status). These measurements demonstrate that the CCM modulates carbon uptake and assimilation under low-Ci conditions and that light-dependent regulation of pigmentation, cell shape, and downstream stages of carbon fixation are critical for tuning carbon uptake and assimilation.

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

Two proteins found in cyanobacteria contain a C-terminal domain with homology to the small subunit of rubisco (RbcS). These small subunit-like domains (SSLDs) are important features of CcmM, a protein involved in the biogenesis of carboxysomes found in all ß-cyanobacteria, and a rubisco activase homolog [activase-like protein of cyanobacteria (ALC)] found in over a third of sequenced cyanobacterial genomes. Interaction with rubisco is crucial to the function of CcmM and is believed to be important to ALC as well. In both cases, the SSLD aggregates rubisco, and this nucleation event may be important in regulating rubisco assembly and activity. Recently, two independent studies supported the conclusion that the SSLD of CcmM binds equatorially to L8S8 holoenzymes of rubisco rather than by displacing an RbcS, as its structural homology would suggest. We use sequence analysis and homology modeling to examine whether the SSLD from the ALC could bind the large subunit of rubisco either via an equatorial interaction or in an RbcS site, if available. We suggest that the SSLD from the ALC of Fremyella diplosiphon could bind either in a vacant RbcS site or equatorially. Our homology modeling takes into account N-terminal residues not represented in available cryo-electron microscopy structures that potentially contribute to the interface between the large subunit of rubisco (RbcL) and RbcS. Here, we suggest the perspective that binding site variability as a means of regulation is plausible and that the dynamic interaction between the RbcL, RbcS, and SSLDs may be important for carboxysome assembly and function.

Cyanobacterial carboxysomes contain an unique rubisco-activase-like protein.

In plants, rubisco activase (Rca) regulates rubisco by removing inhibitory molecules such as ribulose-1,5-bisphosphate (RuBP). In cyanobacteria, a homologous protein (activase-like cyanobacterial protein, ALC), contains a distinctive C-terminal fusion resembling the small-subunit of rubisco. Although cyanobacterial rubisco is believed to be less sensitive to RuBP inhibition, the ALC is widely distributed among diverse cyanobacteria. Using microscopy, biochemistry and molecular biology, the cellular localization of the ALC, its effect on carboxysome/cell ultrastructure in Fremyella diplosiphon, and its function in vitro were studied. Bioinformatic analysis uncovered evolutionary relationships between the ALC and rubisco. ALC localizes to carboxysomes and exhibits ATPase activity. Furthermore, the ALC induces rubisco aggregation in a manner similar to that of another carboxysomal protein, M35, and this activity is affected by ATP. An alc deletion mutant showed modified cell morphology when grown under enriched CO2 and impaired regulation of carboxysome biogenesis, without affecting growth rate. Carbamylation of Fremyella recombinant rubisco was inhibited by RuBP, but this inhibition was not relieved by the ALC. The ALC does not appear to function like a canonical Rca; instead, it exerts an effect on the response to CO2 availability at the level of a metabolic module, the carboxysome, through rubisco network formation, and carboxysome organization.

RcaE-Dependent Regulation of Carboxysome Structural Proteins Has a Central Role in Environmental Determination of Carboxysome Morphology and Abundance in Fremyella diplosiphon.

Carboxysomes are central to the carbon dioxide-concentrating mechanism (CCM) and carbon fixation in cyanobacteria. Although the structure is well understood, roles of environmental cues in the synthesis, positioning, and functional tuning of carboxysomes have not been systematically studied. Fremyella diplosiphon is a model cyanobacterium for assessing impacts of environmental light cues on photosynthetic pigmentation and tuning of photosynthetic efficiency during complementary chromatic acclimation (CCA), which is controlled by the photoreceptor RcaE. Given the central role of carboxysomes in photosynthesis, we investigated roles of light-dependent RcaE signaling in carboxysome structure and function. A ΔrcaE mutant exhibits altered carboxysome size and number, ccm gene expression, and carboxysome protein accumulation relative to the wild-type (WT) strain. Several Ccm proteins, including carboxysome shell proteins and core-nucleating factors, overaccumulate in ΔrcaE cells relative to WT cells. Additionally, levels of carboxysome cargo RuBisCO in the ΔrcaE mutant are lower than or unchanged from those in the WT strain. This shift in the ratios of carboxysome shell and nucleating components to the carboxysome cargo appears to drive carboxysome morphology and abundance dynamics. Carboxysomes are also occasionally mislocalized spatially to the periphery of spherical mutants within thylakoid membranes, suggesting that carboxysome positioning is impacted by cell shape. The RcaE photoreceptor links perception of external light cues to regulating carboxysome structure and function and, thus, to the cellular capacity for carbon fixation. IMPORTANCE Carboxysomes are proteinaceous subcellular compartments, or bacterial organelles, found in cyanobacteria that consist of a protein shell surrounding a core primarily composed of the enzyme ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) that is central to the carbon dioxide-concentrating mechanism (CCM) and carbon fixation. Whereas significant insights have been gained regarding the structure and synthesis of carboxysomes, limited attention has been given to how their size, abundance, and protein composition are regulated to ensure optimal carbon fixation in dynamic environments. Given the centrality of carboxysomes in photosynthesis, we provide an analysis of the role of a photoreceptor, RcaE, which functions in matching photosynthetic pigmentation to the external environment during complementary chromatic acclimation and thereby optimizing photosynthetic efficiency, in regulating carboxysome dynamics. Our data highlight a role for RcaE in perceiving external light cues and regulating carboxysome structure and function and, thus, in the cellular capacity for carbon fixation and organismal fitness.