PREreview of Cyanobacteria form a procarboxysome-like structure in response to high CO₂

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 R_395/470 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 C_i 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 C_i 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.