Fernando Rossine

Intracellular competition shapes plasmid population dynamics Conflicts between levels of biological organization are central to evolution, from populations of multicellular organisms to selfish genetic elements in microbes. Plasmids are extrachromosomal, self-r...

Hi Jack! It's not out yet! Final moments of revision! But the final version should be content wise very similar to the bioarxiv version. www.biorxiv.org/content/10.1...

And a nail in the coffin of Hamilton's rule!

"Fernando, I'm not gonna sugar coat it: those are the worst reads I've ever seen. What are you putting in there?" Then proceeds to troubleshoot and help me solve the issue. Amazing people.

Hey Elizabeth! I use a method to take leaf pics with my students that should work. Illuminate from below! Take a piece of plexiglass, put a piece of white paper on it and then the dish on top of that. Illuminate from below with a flashlight, 1 meter below the plexiglass. Turn off other lights

Hey Jack, thank you so so much! We have a paper in the works that is very related to co-transformations as well! As for the inspiration for this one, I came up with the idea from a theoretical curiosity. It's fun how projects can be born from both theory and experiments!

YaY!!!!! I hope you enjoy it!!!! :)

Thank you so so much!

Sebastian, that makes me so so happy :)

Finally, I'm so so thankful for my collaborators Carlos Sanchez (the best person), Daniel Eaton, and Johan Paulsson. In particular, thanks @baym.lol m for taking a chance on a non-traditional candidate that was proposing a weird project! (21/n)

I hope I convinced you that plasmids are a really cool system to study one of the most defining features of life: multi-scale evolution! (20/n)

Overall, we found out that tradeoffs of within- and between-cell fitness modulate fixation probabilities of plasmid variants, shaping their evolution. Moreover, the dominance curves of plasmid-encoded traits have unintuitive effects on these evolutionary trajectories! (19/n)

We modified our dimer system to release a plasmid that had been chromosomally integrated, creating an invasion-like initial condition. These experiments corroborated theoretical predictions, showing that the high dominance, strong RBS plasmids are favored when invading (18/n)

However, the same model suggested that if the beneficial blue plasmid were initialized with a single copy in each cell, simulating the invasion of a novel type, then a strong RBS should favor invading plasmid fixation. Could we experimentally test this prediction? (17/n)

Once again modelling came to the rescue! Simulations revealed that if a plasmid with a strong RBS has a more dominant trait, then a fitness flatness might actually slow down the fixation of the beneficial plasmid from an equilibrated initial condition. (16/n)

We thought that the big-benefit blue plasmid (rather than the low-benefit) would win faster against the no-benefit red plasmid, but the opposite occurred. I was so surprised that I checked the sequences a million times. Why did the low-benefit plasmid win faster? (15/n)

But here's where another mystery showed up. We had two versions of our blue antibiotic resistance plasmid. One with a strong RBS, giving the cells a big benefit, and one with a weak RBS giving a small benefit. Both had the same promoter and similar within cell fitness. (14/n)

When cells grow under antibiotic pressure, first the non-resistance red plasmid starts winning the within-cell competition, and in a second moment between-cell selection leads to the expansion of blue sectors. Note that without methylation, red wins more! (13/n)

This allows for a fitness conflict between scales of selection! Even though cells carrying antibiotic resistance genes might outgrow cells carrying other plasmids, we show that the transcriptional activity of these genes might impede their fixation! (12/n)

So now we know we can measure within-cell plasmid dynamics, but what features might increase the within-cell fitness of a plasmid? We decided to investigate if transcriptional activity might interfere with replication efficiency reducing within-cell plasmid fitness (11/n)

This discrepancy was due to eclipsing: after a plasmid replicates, hemimethylation prevents it from imediately replicating again, which reduces randomness and increases coexistence. Removing key methylation sites increases within-cell competition and accelerates fixation (10/n)

This finally allowed us to measure how fast was within-cell genetic drift segregating plasmids. However there was a catch: all of our models suggested that this should happen much faster than what we were seeing. We were missing something in our models! (9/n)

Moreover, to isolate within-cell dynamics from between-cell dynamics we used Mother Machines, microfluidic devices that isolate single cell lineages (thanks Carlos!). Look at how each trench first becomes clonal and then dimers are split and plasmid competition begins (8/n)

When we implemented this system on a quasi-neutral pair of plasmids we could see genetic drift occurring first at the within-cell scale (yellow cells give rise to red and blue cells), and then at the between-cell scale (blue and red sectors progressively coarsen) (7/n)

Side quest: We needed precise control on the activation of the recombinase. We hypothesized that the FLP recombinase from the patagonian ancestor of the lager yeast would be thermosensitive (Lagers are cold brewed). It worked on the first attempt! (6/n)

Can we achieve such an initial condition? There's a trick! We create synthetic plasmid dimers, transform them into cells. Then, we activate a recombinase that converts them back to monomers, ensuring an initial condition with equilibrated plasmid composition! (5/n)

Here’s what makes it hard: to measure bacterial fitness we mix two strains in a known ratio and from frequency changes we calculate their relative fitness. However, to do so for plasmids each cell must initially carry a known ratio of both plasmids. (4/n)

Plasmids change the fitness of their host bacteria, but because plasmids are multicopy genetic elements, they also compete within-cell for replication. Plasmid evolution must be driven by both levels of selection, but measuring within-cell competition is very challenging! (3/n)

Here we show that within-cell competition is key to plasmid evolution. Look at this photo of plasmids competing inside cells in a colony!!!

Intracellular competition shapes plasmid population dynamics Conflicts between levels of biological organization are central to evolution, from populations of multicellular organisms to selfish genetic elements in microbes. Plasmids are extrachromosomal, self-r...

I'm so happy that I can finally share the results of my first postdoc paper with @baym.lol!!! Turns out plasmids are an amazing system to study multi-scale evolution and we can track within-cell and between-cell dynamics!
(1/n) www.biorxiv.org/content/earl...