RESUMEN
Cyanobacteria have evolved effective adaptive mechanisms to improve photosynthesis and CO2 fixation. The central CO2-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 CO2-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 ß-carboxysomes within a model cyanobacterium, Synechococcus elongatus PCC7942, in response to light variation. We demonstrated that ß-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 ß-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 ß-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 CO2 fixation.
Asunto(s)
Ciclo del Carbono/fisiología , Fotosíntesis/fisiología , Synechococcus/fisiología , Diurona/farmacología , Transporte de Electrón , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Luz , Microscopía Fluorescente/métodos , Organismos Modificados Genéticamente , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Synechococcus/efectos de los fármacosRESUMEN
The structural dynamics and flexibility of cell membranes play fundamental roles in the functions of the cells, i.e., signaling, energy transduction, and physiological adaptation. The cyanobacterial thylakoid membrane represents a model membrane that can conduct both oxygenic photosynthesis and respiration simultaneously. In this study, we conducted direct visualization of the global organization and mobility of photosynthetic complexes in thylakoid membranes from a model cyanobacterium, Synechococcus elongatus PCC 7942, using high-resolution atomic force, confocal, and total internal reflection fluorescence microscopy. We visualized the native arrangement and dense packing of photosystem I (PSI), photosystem II (PSII), and cytochrome (Cyt) b6f within thylakoid membranes at the molecular level. Furthermore, we functionally tagged PSI, PSII, Cyt b6f, and ATP synthase individually with fluorescent proteins, and revealed the heterogeneous distribution of these four photosynthetic complexes and determined their dynamic features within the crowding membrane environment using live-cell fluorescence imaging. We characterized red light-induced clustering localization and adjustable diffusion of photosynthetic complexes in thylakoid membranes, representative of the reorganization of photosynthetic apparatus in response to environmental changes. Understanding the organization and dynamics of photosynthetic membranes is essential for rational design and construction of artificial photosynthetic systems to underpin bioenergy development. Knowledge of cyanobacterial thylakoid membranes could also be extended to other cell membranes, such as chloroplast and mitochondrial membranes.
Asunto(s)
Cianobacterias/metabolismo , Fotosíntesis/fisiología , Cianobacterias/genética , Microscopía de Fuerza Atómica , Fotosíntesis/genética , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Tilacoides/metabolismoRESUMEN
Carboxysomes are proteinaceous organelles that play essential roles in enhancing carbon fixation in cyanobacteria and some proteobacteria. These self-assembling organelles encapsulate Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase using a protein shell structurally resembling an icosahedral viral capsid. The protein shell serves as a physical barrier to protect enzymes from the cytosol and a selectively permeable membrane to mediate transport of enzyme substrates and products. The structural and mechanical nature of native carboxysomes remain unclear. Here, we isolate functional ß-carboxysomes from the cyanobacterium Synechococcus elongatus PCC7942 and perform the first characterization of the macromolecular architecture and inherent physical mechanics of single ß-carboxysomes using electron microscopy, atomic force microscopy (AFM) and proteomics. Our results illustrate that the intact ß-carboxysome comprises three structural domains, a single-layered icosahedral shell, an inner layer and paracrystalline arrays of interior Rubisco. We also observe the protein organization of the shell and partial ß-carboxysomes that likely serve as the ß-carboxysome assembly intermediates. Furthermore, the topography and intrinsic mechanics of functional ß-carboxysomes are determined in native conditions using AFM and AFM-based nanoindentation, revealing the flexible organization and soft mechanical properties of ß-carboxysomes compared to rigid viruses. Our study provides new insights into the natural characteristics of ß-carboxysome organization and nanomechanics, which can be extended to diverse bacterial microcompartments and are important considerations for the design and engineering of functional carboxysomes in other organisms to supercharge photosynthesis. It offers an approach for inspecting the structural and mechanical features of synthetic metabolic organelles and protein scaffolds in bioengineering.