journal club

Exploring Carboxysomes with Imaging Technology

Microscopes and MatLab make for some insightful tools if we want to understand what's going on inside a cell.

Cyanobacteria are photosynthetic prokaryotes – that means that (1) they convert carbon dioxide into sugar, and (2) they are bacteria. If you consider all photosynthetic organisms on the planet today – trees in the Amazon rainforest, algae in the standing water out back, phytoplankton in the ocean – cyanobacteria make up less than 0.2% of the biomass. They are also doing over a third of the photosynthesis.

That’s contributing to a third of the oxygen we breathe, and a third of the carbon fixation taking CO2 out of the atmosphere.

How are these tiny organisms such powerful workhorses in their environments? It turns out that photosynthesis is a fairly inefficient reaction. Remember RuBisCo, the most abundant enzyme on the planet? Its role (in the Dark Reactions) is to fix carbon dioxide into a 6-carbon sugar, but it’s actually not very picky about the carbon dioxide part. It can pick up oxygen just as easily, leading to cell waste instead of cell energy.

Enter carboxysomes. Now cyanobacteria are just that – bacteria. They are prokaryotic, so they don’t have organelles. They do have bacterial microcompartments (BMCs) like carboxysomes, however, which are special protein complexes in the cell. Now, the carboxysome does not “do” photosynthesis. Instead, it only concentrates CO2 near RuBisCo enzymes, which maxes out carboxylation, and minimizes wasteful oxygenation. This is the secret to cyanobacteria’s photosynthetic success.

Carboxysomes, then, are a powerful cellular machine that help to make cyanobacteria the powerful carbon fixers they are. In the paper Life cycle of a cyanobacterial carboxysome, scientists set out to better understand this incredible system. And in doing so, they develop some cool new tools for understanding BMCs in general, by all sorts of different bacteria.

So this paper is all about understanding carboxysomes within cyanobacteria. Previous studies have shown us how carboxysomes are assembled inside the cells. But what happens afterwards?

In this experiment, the scientists set out to better understand the carboxysome life cycle, its function, and its degradation in real time.

Let’s Set Up This Experiment

To observe a BMC inside of a cell, we’re going to use fluorescent microscopy. That means we need to pair it will some strategically placed dyes within the cell.

We’re studying carboxysomes here, so what’s a good protein to tag so we can see them? RuBisCo, of course. The researchers tagged RuBisCo with GFP (green fluorescent protein) so that the carboxysomes would glow green under a microscope.

We also want to study carboxysomes individually. To deal with this, the researchers made cyanobacterial strains with minimal carboxysomes available in each cell. They then grew them in ambient carbon dioxide levels (0.04% CO2). Each time a cell divides, it passes a carboxysome to its daughter cell. Eventually, then, cells will have only one left. Such cells, grown in ambient CO2, will have a growth rate completely dependent on carboxysome activity. In other words, growth rate is a direct read of how well the carboxysome is performing.

Building on this system, the researchers also wanted complete control of carboxysome assembly. It turns out that most genes required for carboxysome activity are on the ccm gene. So they knocked this gene out, and then added it back in on an inducible plasmid. That gives us two strains:

  1. Δccm, a knockout strain that cannot make carboxysomes.
  2. Δccm+, a knockout strain with a plasmid carrying ccm. It can make carboxysomes only if you induce (turn on) the plasmid.

If the cell doesn’t have carboxysomes, can it still survive? Well, kind of. Remember, all carboxysomes do is concentrate CO2 around RuBisCo. With the carboxysomes gone, RuBisCo has to deal with lower levels of CO2, and will be plagued by lots of wasteful oxygenation. So in order to survive, the researchers had to feed it high concentrations of carbon dioxide (a higher percentage than the ambient CO2 levels).

The same is true for Δccm+ strains – if you never turn their plasmids on. But once the plasmid is induced, the cell starts assembling carboxysomes and can survive at ambient (low) CO2.

Why bother putting a cell system like this together? Well, now we have cells with limited carboxysomes, and we can watch them under a microscope. They divide, and as they divide, some daughter cells get carboxysomes, and some don’t. Eventually, all lineages run out of carboxysomes. For cells fed ambient CO2, losing a carboxysome means instant death. We also have a control group: cells fed high CO2 levels so that empty cyanobacteria can keep growing happily.

With this set-up, we can spy on cyanobacteria very closely – on an individual cell, individual carboxysome level. That can provide us with details never observed before! Most biological studies deal with cell cultures, where the data comes from population of millions of cells within a test tube or petri dish colony. Now, we can read the fine print. What is happening on a personal level?

Conclusions

The researchers in this paper were able to get lots of new insight on the life cycle of a carboxysome. How is it assembled? How does it change throughout its lifespan? How does it degrade? This study is the first look we have at organelle (or rather, BMC) degradation.

They also noticed that there are diverse types of carboxysomes; a small percentage were “ultraproductive.” This may hold secrets for future scientists who want to maximize photosynthesis productivity (in, perhaps, agriculture).

And of course, the tools they used are not exclusive to cyanobacteria. The methods here require a fluorescent microscope to take videos of dividing cells. They then used MatLab scripts to track cells, and carboxysome activity, over time. These kinds of tools can be used on other species, too. Perhaps we want to better understand the BMCs of pathogenic bacteria, or of symbionts in the human gut. By better understanding the bacteria around us, we can inform future advances in human medicine, agriculture, and biotechnology.

References

Hill, N. C., Tay, J. W., Altus, S., Bortz, D. M., & Cameron, J. C. (2020). Life cycle of a cyanobacterial carboxysome. Science Advances, 6(19), eaba1269. https://doi.org/10.1126/sciadv.aba1269

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