Cyanobacteria form a procarboxysome-like structure in response to high CO 2

  1. Clair A. Huffine
  2. Catherine Fontana
  3. Anton Avramov
  4. Colin Sempeck
  5. Jeffrey C. Cameron

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Abstract

Fixing 25% of CO 2 globally, cyanobacteria are integral to climate change efforts. The cyanobacterial CO 2 concentrating mechanism (CCM) features the carboxysome, a bacterial microcompartment which houses their CO 2 fixing machinery. The proteinaceous shell of the carboxysome restricts diffusion of CO 2 , both inward and outward. While necessary for CCM function in air (0.04% CO 2 ), when grown in high CO 2 levels (3% CO 2 ) representative of early earth, the shell would harmfully limit CO 2 fixation. To understand how carboxysomes change form and function in response to increased CO 2 conditions, we used a Grx1-roGFP2 redox sensor and single cell timelapse fluorescence microscopy to track subcellular redox states of Synechococcus sp. PCC 7002 grown in air or 3% CO 2 . Comparing different levels of compartmentalization, we targeted the cytosol, a shell-less carboxysomal assembly intermediate called the procarboxysome, and the carboxysome. The carboxysome redox state was dynamic and, under 3% CO 2 , procarboxysome-like structures formed and mirrored cytosolic redox states, indicating that a more permeable shell architecture may be favorable when [CO 2 ] is high. This work represents a step in understanding how cyanobacteria respond to changing CO 2 concentrations and the selective forces driving carboxysome evolution.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/13227085.

    Review of the preprint: Cyanobacteria form a procarboxysome-like structure in the response to high CO2 

    Summary

    Many cyanobacterial species use a carbon concentrating mechanism (CCM) to overcome the inherent inefficiency of their primary carbon fixation enzyme, rubisco. This inefficiency is in part due to a relatively slow carboxylation reaction rate, but is also due O2 outcompeting CO2 in rubisco's active site, frequently resulting in a non-productive oxygenation reaction, especially in atmospheric conditions where the is air is ~20% O2 and ~0.04% CO2. The cyanobacterial CCM sequesters rubisco into a proteinaceous compartment called a carboxysome along with a carbonic anhydrase in order to saturate rubisco's active sites with CO2. The β-carboxysome of cyanobacteria has been well-studied, but there remain many open questions about its biochemistry, especially in vivo. This paper is a step forward in investigating the redox state of carboxysomes at different stages of formation through clever use of a shell knock-out strain (ΔccmO) and a time-resolved fluorescent microscopy experiment workflow. 

    The authors confirmed that the interior of the carboxysome is indeed more oxidized than the cytosol. Unexpectedly, the interior becomes more reduced over the time course of the microscopy experiments, suggesting permeability to some reducing agents in the cell. Furthermore, the carboxysome interiors are reduced upon transitioning the cells to a CO2 replete environment, and stay reduced even when transitioned back to air. They also find that procarboxysome-like structures exist in WT cells when grown in high CO2, and propose that leaving rubisco unencapsulated is preferable under high CO2 conditions where the permeability barrier of the shell would only impede carboxylation. These observations are all useful to the field, but there remain many open questions on the mechanisms of the redox shifts observed in this work. 

    Major Points

    1. The bulk culture redox sensitivity experiments are interesting measurements, but could be used as more than just controls for roGFP sensitivity. 

      1. The authors interpret the result that the carboxysome is reduced upon addition of DTT but isn't further oxidized with the addition of H2O2 to mean that there's differential permeability to DTT and H2O2 (line 143). Another interpretation could be that the carboxysome is already maximally oxidized. In the 2008 Gutscher paper, the highest R395/470 measured is ~0.9, the same ratio the carboxysomes have in WT cells in air. There very well could be differences in permeability, but this isn't an experiment that definitively proves it. 

      2. It could be useful to have the WT strain expressing cytosolic roGFP also undergo the DTT or H2O2 challenge at high 3% CO2. It would be interesting to see if the procarboxysomes still have different permeability to oxidizing or reducing agents than the cytosol as a phase separated droplets within the cell. 

    2. line 206: The experiments showing the formation of procarboxyome-like structures in 3% CO2 could be improved using thin section EM to confirm they aren't just an artifact of the reporter expression. It would confirm whether they form without the reporter.

    3. There could be more discussion on the physiological effects of 3% CO2. Cells grown at 3% CO2 would have an equilibrium cytosolic Ci pool roughly equivalent to the interior of the carboxysome at atmosphere (see Mangan et al. 2014). However, the experiments where the cells are switched from atmosphere to 3% CO2 (or vice versa) are complicated by non-equilibrium conditions.  That complication could throw off our biochemical intuition, so there are a couple statements that should be addressed. 

      1. Has it been shown experimentally that 'the shell would harmfully limit' (line 20) CO2? In the alpha-CB, Flamholz et al. 2019 test the effect of shell gene KOs in a pooled, CO2-dependent experiment and the shell KOs demonstrate a growth defect even in elevated CO2. The citation in line 268, is much more roundabout than the direct KO measurement.  

      2. Some of the discussion around line 239 seems much more complicated than the null hypothesis that changing the Ci pool would likely have effects on global redox metabolism. There could be more evidence provided why the authors favor the more complex model.

      3. The proposal that the cells form procarboxysome-like structures in high CO2 to allow unobstructed diffusion of  CO2 to rubisco (lines 267-273) could be strengthened by a discussion on the relative binding affinity of rubisco for CO2 and O2, since the carboxysome shell is working to keep O2 out as well as CO2 in. It could be helpful to discuss at what concentration CO2 becomes saturating so there's no longer any advantage to the shell to prevent oxygenation

        Minor Points

    1. line 131: 'consistent previous literature" missing 'with'

    2. In line 155, Fig 1c is referenced, but I believe it should be Fig 2C

    3. There is discussion in lines 153- 160 about how the carboxysomes of cells grown at 3% CO2 are more reduced, but it is established later in the paper that there are more likely to be procarboxysome-like structures. To avoid confusion it could help to change the wording of the observations in Figure 2C.

    4. In lines 281-283, I agree that there could be some structural shifts in rubisco prior to or required for carboxysome formation, (eg. see Metskas  nat comm 2022), but I'm skeptical that rubisco carbamylation is responsible for  the shift since carbamylation is reversible and fast relative to this hours timescale. 

    5. line 289: pioneering?

    6. It could be informative to have a supplemental figure where a still image from one of the supplemental movies is shown with the different populations of cells labeled to make it more obvious how the single cell fluorescence microscopy data were collected. 

    Competing interests

    The authors declare that they have no competing interests.

  2. Version published to 10.1101/2024.06.28.601118v1 on bioRxiv