A popular quip is attributed to Otto von Bismarck: if you like laws or sausages, never watch them being made. Scientific papers, we’d argue, are more like sushi rolls than sausages: you can sometimes appreciate them even more if you see the work taken to put them together. We’re also really keen on communicating science to everyone [http://www.rinkidinks.co.uk/GB/lc/ https://www.kickstarter.com/projects/breadpiginc/great-adaptations-a-childrens-book-about-evolution%5D not just to our colleagues. So, following the release of our paper in the current issue of Science [http://www.sciencemag.org/content/347/6225/1014.short], we’re providing some explanation and backstory via this post.
Microbiologist Rob Jackson has worked on Pseudomonas fluorescens for over 10 years. They’re common bacteria in soil, where they help plants grow and protect them from disease – so they may one day be able to help us grow crops in a more efficient, environmentally friendly way. Together with his then PhD student, Abdullah Alsohim, Rob was trying to understand how P. fluorescens moves around in soil and reaches plant roots. They knew that it could swim using flagella – complex, multifunctional structures that many bacteria use to swim, anchor themselves or sense their environment.
In order to compare the behaviour of bacteria with and without flagella, Rob and Abdullah used a genetically-engineered, flagella-less strain of P. fluorescens. This strain was missing a gene called fleQ, which is a control gene: it acts as a switch, kick-starting all the other genes for parts and assembly of the flagella. Abdullah discovered that the genetically engineered mutants could still move, although differently: on an agar plate, they formed colonies with distinctive tendrils. It turned out that they had a second, backup way to move around, by oozing a lubricant called viscosin and sliding about on it. Abdullah then removed the gene for viscosin as well, creating new, totally immotile bacteria. Placed on an agar plate, his new strain, called AR2, would remain stuck in the middle, unable to spread out to the edges.
Next came a key step in our research: we made a chance discovery. Rather than growing his AR2 bacteria overnight as usual, Abdullah accidentally left some plates growing over a long weekend. By Monday, the bacteria were moving again. Abdullah thought at first that he had done something wrong, but we realised that we could be seeing rapid evolution, and when we repeated the experiment (intentionally this time) the same thing happened – the bacteria started to move, first slowly and then fast. Under an electron microscope, the fast swimmer had clearly regained its flagellum.
Luckily enough, around this time the Jackson lab started a new collaboration with evolutionary biologists. Louise Johnson had recently won a Leverhulme grant, along with Rob and with Mike Brockhurst from the University of York, and they had hired Tiffany Taylor for a separate project involving microbiology and real-time observation of evolution in action in the lab. We had the right team together to recognise and address interesting questions in evolutionary biology – so when Abdullah completed his PhD and moved away, Tiffany was the perfect choice to take over the reins and take the project to a new level by asking how this happened. With the FleQ switch removed, it should have been completely impossible for the flagellum to reappear, let alone so quickly.
To find out how the bacteria had regained flagella, we went straight to their genes. Our collaborator David Studholme at Exeter University analysed the slow- and fast- swimming strains, and found mutations in two new genes: both of them had a mutant NtrB gene, while the fast swimmer had a second mutation in the NtrC gene. These two genes work as a pair to control nitrogen intake: the cell can sense when it’s starving using NtrB, and suck up more nutrients if necessary using NtrC. Like FleQ, NtrC is a control gene, but it normally switches on feeding genes rather than flagella genes.
Because mutation is a random process, it’s often been thought that evolution will tend to work in an unpredictable, meandering way. But when we re-ran the experiment several times, the same genes mutated – sometimes in exactly the same places.
Our next stroke of luck was having Geraldine Mulley in the lab next door. Geraldine had worked on this kind of paired genes before, and she helped us design experiments to test our fast and slow swimmers with different food sources, to see what the two Ntr mutations did. Talking over our results with her, we sketched out our thoughts about what was going on inside our mutants, and then to make sure we’d got the right idea, we measured the activity of all the genes in each mutant with the help of Mike’s colleague Peter Ashton in York.
This scribble from Louise Johnson’s notebook shows how we came to understand the re-evolution. The left hand scribble is the immotile ancestor. Its Ntr genes are doing their usual job, but with fleQ removed, nothing can switch on the flagella genes. In the middle is the slow swimmer: regardless of actual food availability, the mutant NtrB gives out a strong I’m starving signal, resulting in an overload of NtrC. This gives the cell a good hard kick in the biochemistry: it switches on the feeding genes so aggressively that these cells will “overeat” and poison themselves, but it also spills over slightly to trigger the flagella genes. On the right, the fast swimmer’s mutant NtrC has adapted to its new job, and become a multitasker: it doesn’t overactivate the feeding genes so much, but efficiently switches on the flagella.
When Mark Silby and Alex Dills from the University of Massachusetts Dartmouth repeated the experiments in a different variety of P. fluorescens, they found many of the same mutations as we did, but also discovered an alternative first step to the evolutionary pathway: sometimes the first mutation was to one of the feeding genes themselves, so that the cell is actually starving.
Liam McGuffin, a protein bioinformatician, completed the team. His computer programs worked out how the DNA mutations changed the shape of proteins, and showed us the remarkable similarity in shape between the switch that was lost, fleQ, and the switch that was substituted, ntrC – the two are distant molecular cousins [see protein structure video below]. His work joined the dots so that we could trace the process all the way from the DNA, through the proteins and biochemical interactions, to the cell’s behaviour and its evolutionary advantage. We think this is a really nice example of evolution in action: each step is a kludge, and a kludge can bring serious drawbacks – but small steps soon add up to a result that seemed impossible.