In a recent blog post, I claimed that nobody has yet tried to “evolve” Vibrio natriegens in the laboratory to make them grow even faster. But I was totally wrong, and it warrants a correction.
For context, V. natriegens is a microbe that doubles every 9.7 minutes in highly enriched growth media, or every ~30 minutes in more “minimal” media, with just some sugar and salt. In my recent blog, I explained that these cells divide faster than any other known organism because they make a large number of ribosomes quickly. (It has nothing to do with the time required to copy a genome):
“V. natriegens has at least a dozen ribosomal RNA operons, or gene clusters encoding ribosomal RNA molecules, in its genome… [Also,] these ribosome genes are located next to strong promoters, or genetic sequences that recruit RNA polymerase enzymes. In other words, Vibrio devotes more of its genome to ribosomal genes, and has also evolved a stronger start signal for those genes, meaning the cell makes ribosomal RNA much more frequently, and in higher numbers, than other microbes.”
At the end of the blog post, I proposed some experiments to make Vibrio natriegens grow even faster. Perhaps we could make its ribosomes smaller, such that each one takes less time to create. Or, alternatively:
“…we could take a more agnostic approach and just let evolution take its course, albeit in an accelerated way…Perhaps we could run a Richard Lenski-esque experiment, in which V. natriegens’ cells are grown in a robotic bioreactor and flooded with glucose every few hours…If we repeat this lots of times, some microbes may evolve to grow even faster… Or maybe not; V. natriegens may already be quite close to the theoretical cell division time limit. These experiments haven’t been done yet.”
This last claim — that nobody has yet done these experiments — was totally wrong, as Adam Feist, bioengineer at UC San Diego, explained to me by email. Feist is a co-author on two studies that have done basically this exact experiment!
In one study, they used an automated laboratory evolution (ALE) robot to evolve E. coli cells to grow faster. Their goal was to see whether E. coli could grow as fast as V. natriegens. (Answer: No.) In a second study, they did much the same with V. natriegens to see if they could “break” its speed limit. (Answer: No.)
For the first study, the researchers took six different E. coli strains, including BL21, K-12 MG1655, Crooks, and others. They grew each strain in three triplicate flasks, filled with growth media containing just a minimal amount of sugar and salts, and incubated them at 37°C. Every time the cells reached a certain density, they transferred a tiny amount of cells into a fresh flask with the same exact conditions. And they repeated this again and again for 900 generations of cell growth.
The beauty of this experiment is two-fold: First, it’s super simple and easy to replicate. Every time cells are transferred into a fresh flask, microbes evolve to grow faster such that they can take over the nutrients in that flask. So this is artificial selection in a tube. And second, they froze the cells at regular intervals so they could sequence them, measure their gene expression, and so on to really figure out how the cells were evolving over time.
All six strains started with different growth rates. MG1655 doubled every hour, whereas the Crooks strain doubled every 47 minutes. After hundreds of generations, though, all the strains converged to a similar doubling time: About 40 minutes. All six strains also took similar paths toward the faster growth rate; the same genes kept getting mutated again and again across the flasks.
Genes involved in eating and breaking down glucose were mutated to become more efficient. Ribosomal genes were also massively upregulated across strains, as were genes involved in making amino acids and nucleotides. This all makes sense.
To compensate for this faster growth, the cells also “broke” a handful of genes. They mutated stress response genes, for example, because they no longer needed them in their stable, consistent environment. And they shut down motility genes (it’s super “costly” for a cell to make a flagellum, and why would they need one in a shaking flask anyway?)
Even after all this convergent evolution, though, the key thing to remember is that none of the E. coli strains approached the division time of V. natriegens. All of them maxed out around 40 minutes per cell division in this “minimal” growth media.
In the second study, then, Feist & co. wanted to see if they could use the same principles to make V. natriegens grow faster. So they set up 10 identifical flasks, each with some M9 growth media and a bit of salt and sugar, and inoculated the cells. Every time the V. natriegens cells hit a certain density, a small amount were transferred to a fresh tube. This was repeated for 1,000 generations, but there was no increase in cell division rates.
The cells did acquire mutations, though. Nine of ten flasks mutated a stress response gene (much like the E. coli did) and most flasks made mutations to an ion transporter protein. But despite those changes, the cells had a median division time of 28 minutes at the start and end of the experiment.
So what happens now? Well, for starters, I think we need to run more of these experiments, albeit with some different starting parameters, to see if we can make V. natriegens divide faster after all. Feist proposed, in his email, that we should insert more copies of ribosomal genes into V. natriegens and E. coli, say, and then repeat the evolution experiments with both of them. Perhaps the cells just didn’t have enough time to duplicate these genes on their own but, if we added them artificially, their speeds would go higher.
This seems like quite an easy experiment to do. But without running the experiment, we won’t actually know if the abundance of ribosomal genes alone is sufficient to speed up division times, or if we also have to cut down the size of ribosomes themselves to make cells grow faster. Let’s get busy!
