Diurnal Regulation of In Vivo Localization and CO2-Fixing Activity of Carboxysomes in Synechococcus elongatus PCC 7942

Sun, Yaqi; Huang, Fang; Dykes, Gregory F.; Liu, Luning

doi:10.3390/life10090169

Cited by 9 publications

(8 citation statements)

References 72 publications

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“…coli BL21(DE3) cells and the expression of pHluorin2-S2C alone or pHluorin2-S2C and α-shell-CA (or α-shell) induced by 1 mM arabinose at 25 °C for 16 h, the E. coli cells were prepared on agar plates for confocal microscopy, as described earlier. ,, The E. coli cells were imaged using a Zeiss LSM780 with a 63 × oil-immersion objective with excitation wavelength at 488 nm and emission detection at 500–520 nm.…”

Section: Methodsmentioning

confidence: 99%

Probing the Internal pH and Permeability of a Carboxysome Shell

et al. 2022

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The carboxysome is a protein-based nanoscale organelle in cyanobacteria and many proteobacteria, which encapsulates the key CO 2 -fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase (CA) within a polyhedral protein shell. The intrinsic self-assembly and architectural features of carboxysomes and the semipermeability of the protein shell provide the foundation for the accumulation of CO 2 within carboxysomes and enhanced carboxylation. Here, we develop an approach to determine the interior pH conditions and inorganic carbon accumulation within an α-carboxysome shell derived from a chemoautotrophic proteobacterium Halothiobacillus neapolitanus and evaluate the shell permeability. By incorporating a pH reporter, pHluorin2, within empty α-carboxysome shells produced in Escherichia coli, we probe the interior pH of the protein shells with and without CA. Our in vivo and in vitro results demonstrate a lower interior pH of α-carboxysome shells than the cytoplasmic pH and buffer pH, as well as the modulation of the interior pH in response to changes in external environments, indicating the shell permeability to bicarbonate ions and protons. We further determine the saturated HCO 3 − concentration of 15 mM within α-carboxysome shells and show the CA-mediated increase in the interior CO 2 level. Uncovering the interior physiochemical microenvironment of carboxysomes is crucial for understanding the mechanisms underlying carboxysomal shell permeability and enhancement of Rubisco carboxylation within carboxysomes. Such fundamental knowledge may inform reprogramming carboxysomes to improve metabolism and recruit foreign enzymes for enhanced catalytical performance.

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Section: Methodsmentioning

confidence: 99%

Probing the Internal pH and Permeability of a Carboxysome Shell

et al. 2022

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“…The relative simplicity of these photoautotrophic microbes and their genetic tractability has made them useful subjects for fundamental science and application-driven research. One such organism, Synechococcus elongatus PCC 7942 (hereafter S. elongatus ), is a model for studying photosynthesis, − the circadian clock, − carbon concentrating mechanisms, − and a platform for bioproduction of chemicals. − While many efforts are ongoing to improve the fundamental understanding of S. elongatus in these research areas, there remains much to be learned about its genome, as approximately 40% of gene functions are unknown and 26% of its genes are essential (>700 genes of 2723 total in the genome) . Furthermore, biotechnological applications could benefit from increased refinement of the cyanobacterial genetic toolkit.…”

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confidence: 99%

Orthogonal Degron System for Controlled Protein Degradation in Cyanobacteria

et al. 2021

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Synechococcus elongatus PCC 7942 is a model cyanobacterium for study of the circadian clock, photosynthesis, and bioproduction of chemicals, yet nearly 40% of its gene identities and functions remain unknown, in part due to limitations of the existing genetic toolkit. While classical techniques for the study of genes (e.g., deletion or mutagenesis) can yield valuable information about the absence of a gene and its associated protein, there are limits to these approaches, particularly in the study of essential genes. Herein, we developed a tool for inducible degradation of target proteins in S. elongatus by adapting a method using degron tags from the Mesoplasma florum transfer-mRNA (tmRNA) system. We observed that M. florum lon protease can rapidly degrade exogenous and native proteins tagged with the cognate sequence within hours of induction. We used this system to inducibly degrade the essential cell division factor, FtsZ, as well as shell protein components of the carboxysome. Our results have implications for carboxysome biogenesis and the rate of carboxysome turnover during cell growth. Lon protease control of proteins offers an alternative approach for the study of essential proteins and protein dynamics in cyanobacteria.

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“…Similarly, the circadian clock may regulate McdB condensation on carboxysomes, and this activity may reflect the metabolic status of the carboxysome itself. In line with this proposal, a recent study explored the diurnal regulation of carboxysomes in S. elongatus and found that, in the dark, cells have fewer carboxysomes, and a greater fraction were mislocalized to the cell poles (129). It is possible that this diurnal control of carboxysome positioning is mediated by the McdAB system.…”

Section: The Role Of Liquid-liquid Phase Separation In Carboxysome Positioningmentioning

confidence: 90%

Positioning the Model Bacterial Organelle, the Carboxysome

MacCready

Vecchiarelli

2021

mBio

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Bacterial microcompartments (BMCs) confine a diverse array of metabolic reactions within a selectively permeable protein shell, allowing for specialized biochemistry that would be less efficient or altogether impossible without compartmentalization. BMCs play critical roles in carbon fixation, carbon source utilization, and pathogenesis. Despite their prevalence and importance in bacterial metabolism, little is known about BMC “homeostasis,” a term we use here to encompass BMC assembly, composition, size, copy-number, maintenance, turnover, positioning, and ultimately, function in the cell. The carbon-fixing carboxysome is one of the most well-studied BMCs with regard to mechanisms of self-assembly and subcellular organization. In this minireview, we focus on the only known BMC positioning system to date—the maintenance of carboxysome distribution (Mcd) system, which spatially organizes carboxysomes. We describe the two-component McdAB system and its proposed diffusion-ratchet mechanism for carboxysome positioning. We then discuss the prevalence of McdAB systems among carboxysome-containing bacteria and highlight recent evidence suggesting how liquid-liquid phase separation (LLPS) may play critical roles in carboxysome homeostasis. We end with an outline of future work on the carboxysome distribution system and a perspective on how other BMCs may be spatially regulated. We anticipate that a deeper understanding of BMC organization, including nontraditional homeostasis mechanisms involving LLPS and ATP-driven organization, is on the horizon.

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Diurnal Regulation of In Vivo Localization and CO2-Fixing Activity of Carboxysomes in Synechococcus elongatus PCC 7942

Cited by 9 publications

References 72 publications

Probing the Internal pH and Permeability of a Carboxysome Shell

Probing the Internal pH and Permeability of a Carboxysome Shell

Orthogonal Degron System for Controlled Protein Degradation in Cyanobacteria

Positioning the Model Bacterial Organelle, the Carboxysome

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