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

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 β-carboxysomes within a model cyanobacterium, Synechococcus elongatus PCC7942, in response to light variation. We demonstrated that β-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 β-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 β-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. Compartmentalization of metabolic pathways in cells provides the fundamental basis for enhancing and modulating the cellular metabolism. Many prokaryotes have evolved specialized metabolic organelles, known as bacterial microcompartments, to sequester key metabolic pathways and thereby improve the efficiency of metabolic activities (for reviews, see Kerfeld et al., 2010; Bobik et al., 2015). Unlike eukaryotic organelles, bacterial microcompartments are assembled entirely by proteins. These organelles consist of interior enzymes that catalyze sequential metabolic reactions (Yeates et al., 2010), surrounded by a single-layer proteinaceous shell (Kerfeld et al., 2005; Tsai et al., 2007; Tanaka et al., 2008; Sutter et al., 2016). The shell facets are composed of hexameric and pentameric proteins, resulting in an overall shell architecture resembling an icosahedral viral capsid (Kinney et al., 2011; Hantke et al., 2014; Kerfeld and Erbilgin, 2015). Interactions between shell proteins are important for the self-assembly of the shell (Sutter et al., 2016). The selectively permeable shell serves to concentrate enzymes and substrates, mediate flux of metabolites, modulate the redox state, and prevent toxic intermediates from diffusing into the cytoplasm (Havemann et al., 2002; Yeates et al., 2008). Carboxysomes were the first bacterial microcompartments to be discovered and are widely distributed among cyanobacteria and some chemoautotrophs as the central machinery for the fixation of CO2 (Shively et al., 1973). Two different types of carboxysomes have been identified (α- and β-carboxysomes), according to the types of the CO2-fixing enzyme, Rubisco (form 1A and form 1B), possessed in cyanobacteria. In most β-cyanobacteria, Rubisco is sequestered in the β-carboxysome lumen by a shell that is composed of shell and shell-associated proteins encoded by a ccmKLMNO operon (Omata et al., 2001; Long et al., 2010; Rae et al., 2012). The carboxysomal carbonic anhydrase is colocalized with Rubisco in the β-carboxysome, serving to create a CO2-rich microenvironment to favor the Rubisco activity. Some cyanobacterial species do not have the carboxysomal β-carbonic anhydrase (CcaA) homologs; instead, the N-terminal domain of CcmM functions as an active γ-carbonic anhydrase (Peña et al., 2010). The shell facets act as a selective barrier that allows the diffusion of HCO3− and retains CO2 in the interior (Dou et al., 2008). Through these mechanisms, carboxysomes elevate the CO2 concentration in the vicinity of Rubisco and thereby enhance the efficiency of carbon fixation. Supported by this nanoscale CO2-fixing machinery, cyanobacteria contribute more than 25% of global carbon fixation (Field et al., 1998; Liu et al., 1999). The efficiency of carboxysomes in enhancing carbon fixation has attracted tremendous interest in engineering the CO2-fixing organelle in other organisms. For example, introducing β-carboxysomes into higher plants that use the ancestral C3 pathway of photosynthesis could potentially enhance photosynthetic carbon fixation and crop production (Lin et al., 2014a, 2014b). However, engineering of functional carboxysomes requires extensive understanding about the principles underlying the formation of β-carboxysomes and the physiological integration of β-carboxysomes into the cellular metabolism. Indeed, cyanobacterial cells have evolved comprehensive systems to regulate the biosynthesis and spatial organization of carboxysomes, allowing them to modulate the capacity for photosynthetic carbon fixation. Recent studies elucidated that the β-carboxysome assembly is initiated from the packing of Rubisco enzymes, followed by the encapsulation of peripheral shell proteins (Cameron et al., 2013; Chen et al., 2013). In the model rod-shaped cyanobacterium Synechococcus elongatus PCC7942 (hereafter Synechococcus), three to four β-carboxysomes were observed to be evenly spaced along the centerline of the longitudinal axis of cells, ensuring the equal segregation of the machinery between daughter cells (Savage et al., 2010). Such specific organization of carboxysomes within cyanobacterial cells is likely to be determined by the interaction between carboxysomes and the cytoskeleton (Savage et al., 2010). Advanced understanding of the functions and assembly of β-carboxysome proteins has recently led to the construction of a chimeric protein that can functionally replace four native proteins (CcmM58, CcmM35, CcaA, and CcmN) required for carboxysome formation (Gonzalez-Esquer et al., 2015). These findings outlined the self-assembly nature and integration of carboxysomes in the cell. However, how β-carboxysome biosynthesis and organization are physiologically regulated in cyanobacteria in response to environmental changes remains poorly understood. Here, using a combination of live-cell confocal fluorescence microscopy and biochemical and physiological approaches, we investigated the formation and spatial positioning of β-carboxysomes in Synechococcus under varying light intensities. Our study provides new insights into the regulation of β-carboxysome biosynthesis by light and the roles of photosynthetic electron flow in the carboxysome assembly. Knowledge obtained from this work is fundamental to the bioengineering and modulation of functional carboxysomes to boost photosynthetic carbon fixation in dynamic and diverse environments

Rubisco accumulation factor 1 (Raf1) plays essential roles in mediating Rubisco assembly and carboxysome biogenesis

Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the primary carbon-fixing enzyme in photosynthesis. The formation of carboxysomes requires hierarchical self-assembly of thousands of protein subunits, initiated from Rubisco assembly and packaging to shell encapsulation. Here we study the role of Rubisco assembly factor 1 (Raf1) in Rubisco assembly and carboxysome formation in a model cyanobacterium, Synechococcus elongatus PCC7942 (Syn7942). Cryo-electron microscopy reveals that Raf1 facilitates Rubisco assembly by mediating RbcL dimer formation and dimer–dimer interactions. Syn7942 cells lacking Raf1 are unable to form canonical intact carboxysomes but generate a large number of intermediate assemblies comprising Rubisco, CcaA, CcmM, and CcmN without shell encapsulation and a low abundance of carboxysome-like structures with reduced dimensions and irregular shell shapes and internal organization. As a consequence, the Raf1-depleted cells exhibit reduced Rubisco content, CO2-fixing activity, and cell growth. Our results provide mechanistic insight into the chaperone-assisted Rubisco assembly and biogenesis of carboxysomes. Advanced understanding of the biogenesis and stepwise formation process of the biogeochemically important organelle may inform strategies for heterologous engineering of functional CO2-fixing modules to improve photosynthesis

Clinical screening of Nocardia in sputum smears based on neural networks

ObjectiveNocardia is clinically rare but highly pathogenic in clinical practice. Due to the lack of Nocardia screening methods, Nocardia is often missed in diagnosis, leading to worsening the condition. Therefore, this paper proposes a Nocardia screening method based on neural networks, aiming at quick Nocardia detection in sputum specimens with low costs and thereby reducing the missed diagnosis rate.MethodsFirstly, sputum specimens were collected from patients who were infected with Nocardia, and a part of the specimens were mixed with new sputum specimens from patients without Nocardia infection to enhance the data diversity. Secondly, the specimens were converted into smears with Gram staining. Images were captured under a microscope and subsequently annotated by experts, creating two datasets. Thirdly, each dataset was divided into three subsets: the training set, the validation set and the test set. The training and validation sets were used for training networks, while the test set was used for evaluating the effeteness of the trained networks. Finally, a neural network model was trained on this dataset, with an image of Gram-stained sputum smear as input, this model determines the presence and locations of Nocardia instances within the image.ResultsAfter training, the detection network was evaluated on two datasets, resulting in classification accuracies of 97.3% and 98.3%, respectively. This network can identify Nocardia instances in about 24 milliseconds per image on a personal computer. The detection metrics of mAP50 on both datasets were 0.780 and 0.841, respectively.ConclusionThe Nocardia screening method can accurately and efficiently determine whether Nocardia exists in the images of Gram-stained sputum smears. Additionally, it can precisely locate the Nocardia instances, assisting doctors in confirming the presence of Nocardia