(Photo)Synthetic Endosymbiosis

Symbiosis is everywhere. From the Greek for "living with," symbiosis is simply a close association between two different species in nature. These relationships can be mutualistic, parasitic, or somewhere in between. Bacterial symbionts live inside bodies, like the bacteria that help us and other animals digest our food, and they live inside cells, like the bacteria that live in plant roots and provide their hosts with nitrogen. They can be metabolic, hygienic, or photosynthetic; ectosymbiotic, on the host surface, or endosymbiotic, inside the host's cells. Back in the 1860's biologists considered each organism to be an individual, autonomous whole, but the Swiss botanist Simon Schwendener observed that lichen is actually an association between two different types of cells, a fungus and a photosynthetic algae. Shortly after that, other botanists began to notice that the chloroplasts, the organelles that provide photosynthetic power to plant cells, resembled free-living photosynthetic bacteria. It took several decades and the rise of molecular biology to learn that these organelles had their own DNA, and to convince scientists that they had started out as bacteria.

Photosynthetic endosymbiosis created plants and still exists in many other organisms and in many forms today. The sea slug Elysia chlorotica is one of the best known examples. It rips the chloroplast organelles out of the algae that it eats and incorporates them into its highly branched digestive system, able to live off of sunlight harvested by these symbionts for several months.
Almost all of the photosynthetic endosymbiotic relationships known show up in invertebrates that don't move a lot and have a very high surface-to-volume ratio, resembling very slow-moving leaves. But all this changed a couple weeks ago with the publication of a really cool paper. Ryan Kerney and his colleagues were studying a species of salamander that is known to be associated with algae during its embryonic stage. What they found was that contrary to what had been reported before, the algae were living inside of the salamander cells!

The relationship between the salamander and the algae is especially interesting because it shows just how wide the range of situations where endosymbiosis can happen. In one extreme, the slug can't survive without the algae and actually becomes photosynthetic, while the salamander can develop just fine without any algae, does not harvest any energy from the algae, and lives most of its adult life underground, away from sunlight.

In my lab, a lot of people study photosynthetic bacteria like the ones that eventually became the plant chloroplast through endosymbiosis. Projects range from basic cell biology and understanding the internal organization of the bacteria to trying to engineer them to produce useful chemicals. A couple years ago, a very awesome and very talented master's student, Henrike Niederholtmeyer (her awesomeness is directly proportional to the number of syllables in her name), was working on engineering these cyanobacteria to produce sugar.

The bacteria typically grow in fresh water, and putting them in salt water can put a lot of pressure one their membranes. To protect themselves, the bacteria produce sucrose (table sugar), which helps to balance out the osmotic pressure. What Henrike did was engineer the bacteria with invertase, the gene that splits sucrose into glucose and fructose, and with a transporter gene that lets the sugar leave the inside of the cell. Now, when the bacteria are put in salt water, they secrete sugars out into environment. It isn't a ton of sugar, but it's enough to create a symbiotic relationship between E. coli (yellow cells, red line) and the cyanobacteria (red cells):

This got Henrike and my advisor, Pam Silver, thinking about how photosynthetic symbiosis is established and how we could re-create it in the lab. At this point, I was fascinated, and convinced Henrike to let me tag along on the experiment. How could we get the cyanobacteria inside of animal cells to establish a synthetic endosymbiosis?

We were lucky to be in a very creative and supportive lab and department and to have one of the best zebrafish labs anywhere right down the hall. Sean Megason's group studies the development of zebrafish embryos and uses powerful microscopes to track every cell as the fish grows. Zebrafish are also relatively easy to microinject (if you are a professional) and clear, letting light into their cells that the cyanobacteria would need to grow. Henrike hooked Ramil Noche, a postdoc in the Megason lab, the same way she hooked me into the project, and his zebrafish skills are unmatched. He injected fresh zebrafish eggs with millions of wild-type cyanobacteria, put them in the incubator, and we waited.

The biggest surprise was that nothing happened. The embryos developed normally into a happy, swimming fish when we injected them with cyanobacteria. Even after 1 hour we could see that something was up, since the cyanobacteria-injected embryo looked normal (panel A, a red dye is used during injections to keep track of which ones are done which is why the embryo looks red), while injecting E. coli had a quite drastic result (panel B).

While the sugar secretors were the inspiration to try this experiment, they don't produce nearly enough sugar to actually support a living animal cell, and all of these pictures show normal, un-engineered cyanobacteria inside of the fish. These fish are not photosynthetic, they just live happily with photosynthetic bacteria inside of their own cells. The zebrafish cell membranes are engineered to be green fluorescent, and the green pigments in the cyanobacteria cells fluoresce red, so they look like little red dots in the images:

Here's one of the amazing images that Ramil took with the confocal microscope, where you can look at just a single plane of the fish, looking into the cells themselves. This image is of the head of a live two-day old embryo, and you can see red dots of where the bacteria are inside its eye and brain:

This result inspired me and Henrike to try and get the cyanobacteria into other cells. Some synthetic biologists are trying to create tumor killing bacteria, that can seek out cancer cells, get inside of them and specifically kill only those cells, leaving healthy cells intact. They engineer these bacteria with a gene called invasin that, as the name implies, allows them to invade mammalian cells. A second gene called listeriolysin is needed for the bacteria to escape the membrane-bound compartment that they get stuck inside, and to get into the cytoplasm. Engineering the cyanobacteria with these two genes allowed them to invade hamster cells in culture at a low but appreciable efficiency.

Listeriolysin also lets the bacteria escape from the digestive forces of macrophages, a type of immune cell that can capture and eat up bacteria. When we had mouse macrophages swallow up the engineered cyanobacteria, we saw them escape digestion and slowly start dividing, a first step for establishing a symbiosis. In the video, taken by Tami Lieberman, you can see the macrophages with the engineered cyanobacteria in them. On the left is the dish in the dark, and the white dots are the bacteria, which slowly die overnight without any light to support them. On the right the light is on, and after a while, one of the red bacteria divides into two:

Again, these mouse cells aren't photosynthetic, but these experiments show how photosynthetic bacteria can develop special relationships with animal cells. Because they aren't pathogens and don't need to steal nutrients from their host cell, these kinds of benign symbiotic associations are possible. The wide range of symbiotic possibilities found in nature can inspire synthetic biologists like us to explore and re-create these kinds of relationships as a way to study the evolution of symbiosis or as a way to design new multi-species biological behaviors greater than the sum of their parts. Endosymbiosis drove the evolution of the eukaryotic kingdom with the mitochondria and the chloroplast, perhaps endosymbiosis will play a role in the evolution of the synthetic kingdom as well.

You can check out our paper in PLoS ONE, out today! Agapakis CM, Niederholtmeyer H, Noche RR, Lieberman TD, Megason SG, Way JC, Silver PA. "Towards a Synthetic Chloroplast."