The route to rewiring – technical background

It’s quite common for scientific discoveries to be made by chance. Our chance discovery was that we accidentally left experimental samples out for longer than needed and in doing so something remarkable happened. Here we discuss our findings that have recently been published in Science [http://www.sciencemag.org/content/347/6225/1014.short]. Our aim here is to give the reader extra background that we couldn’t fit into the paper, to provide some insight to the historical background of why we were studying the evolution of gene regulation and to also show the step-wise key experimental steps that took place. We have developed two versions of the discussion – a lay version and a technical version.

Our story starts 11 years ago, when Rob Jackson and colleagues were examining the basis of bacterial gene regulation. We were using a common soil bacterium called Pseudomonas fluorescens. When looking for P. fluorescens mutants that could turn on their genes for making the extracellular polysaccharide, cellulose, we found several mutants with an inactivated gene called fleQ. FleQ is a master regulator of the bacterial flagellum, a propeller-like appendage that helps it to move through liquids and gels. The job of FleQ is to bind to DNA at specific enhancer sites in the vicinity of flagellum genes and bind RpoN to activate transcription. When we mutated fleQ the flagellum genes could not be expressed and the cell was thus unable to synthesise a flagellum. As a consequence, we observed [http://www.pnas.org/content/104/46/18247.long] that these mutant bacteria were no longer able to swim, but could still move slowly over agar surfaces, exhibiting an unusual spreading pattern called spidery-spreading. We found that the basis of this spreading was due to a surfactant called viscosin – mutation of the genes for making both viscosin and a flagellum (double mutant strain) resulted in the bacteria being immotile [http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12469/abstract;jsessionid=FACAEA8FFAAA1CBF36F50E1A5291AB85.f01t03].

Abdullah Alsohim was doing an experiment at Reading examining this motility system and he needed some better photos for his PhD thesis. So he inoculated some surface spreading plates up with the double mutants on the Wednesday. He finished his experiment and obtained the photos on the Friday. But he forgot to throw his plates away, leaving them in the incubator. Over the course of the weekend, evolution worked its magic – the mutants were moving, and there looked like there were two variants on the same plate! The photo below shows the slow spreader emerged first with the fast spreader variant seen emerging on the left. Of course, Abdullah was perplexed because he thought he had done something wrong, but we realised that the mutants may have evolved motility – careful purification and re-testing the strains indeed confirmed this to be the case, where we found a slow moving strain emerged first followed by a fast mover. What we felt was remarkable was that FleQ was supposed to be a crucial chokepoint in the regulatory system – there was just no way the bacteria should be able to move again. But we know that in the face of adversity life can overcome these challenges.

Motility evolutionv1

To find out how this had happened we collaborated with David Studholme at Exeter University, who, in 2011, used his bioinformatics skills to analyse the re-sequenced genomes of the slow and fast moving mutants. He found the ntrB mutation in the slow mover and the same ntrB and subsequent ntrC mutation in the fast mover. When we did electron microscopy of the two strains, we were unable to see a flagellum for cells of the slow mover, whereas the fast mover exhibited an atypical “thick” flagellum. Closer examination of the latter strain appears to show multiple flagella intertwined together causing the thick flagellum structure near the cell and giving individual flagella filaments distal to the cell [see photo below].

Flg Evo ThickFlg evo EM

Next, by combining our microbiology know-how with the biochemistry and cell biology skills of Geraldine Mulley, and the evolutionary biology skills of Louise Johnson, Tiff Taylor and Mike Brockhurst we designed and carried out the crucial experiments needed to complete the story. Microarray data from Mike Brockhurst’s lab at York corroborated the motility observations, because we observed partial restoration of flagella gene expression in the slow moving mutant, and full restoration of flagella gene expression to wild-type levels in the fast-moving strain. The array results also showed a very strong up-regulation of genes involved in nitrogen uptake and metabolism in the slow mover, and whilst these genes were also up-regulated in the fast movers it was noticeably less than in the slow movers. This result was the key to understanding what was happening with the mutations in ntrB and ntrC. It has long been established in bacteriology that nitrogen gene expression is controlled by the NtrBC two-component pathway. When the cell needs more nitrogen, the NtrB protein transfers a phosphate group to NtrC, which acts in a similar way to FleQ by binding to enhancer sequences in DNA, recruiting RpoN and activating transcription; for NtrC these sequences are upstream of nitrogen genes. What we now believe is happening in the slow mover is that the ntrB mutation leads to a locked-on, overactive NtrB protein resulting in hyperphosphorylation of NtrC. Liam McGuffin found that this protein shares structural similarities with FleQ [see protein structural video post below] and we believe the level of phosphorylated NtrC becomes so high that it can start activating the flagellum genes (hotwiring the flagellum!) – however, it also activates all the nitrogen uptake and metabolism genes, causing a “toxicity” burden. That is when we see emergence of the fast spreader – an ntrC mutation occurs that we believe alters the DNA targeting specificity of the protein so that it begins to activate flagellum targets more than nitrogen targets. So the fast mover spreads faster on agar plates, either because more cells are making flagella or they are released from the nitrogen toxicity burden – or perhaps both.

Interestingly, we have since used light microscopy to examine cells of the mutants used in the study. The double mutant strain cannot move, the slow mover seems to be a “mixed” population of flagellum producers and non-producers, while the fast movers were swimming around at high speed. Intriguingly, the fast mover also showed a clumping phenotype, although the clumps appeared to be moving. This may be occurring because the cells make multiple flagella and get tangled together.

In parallel to this, our collaboration with Mark Silby and Alex Dills at University of Massachusetts Dartmouth was yielding exciting results, especially when they discovered two alternative mutational routes leading to over activation of NtrC. This was a pivotal moment in the project because it showed that the evolutionary rewiring was rapid and repeatable in two different systems, but could also follow slightly different trajectories in the slow mover, converging on the subsequent mutation of NtrC in the fast mover.

In summary, the bacteria have effectively evolved the ability to swim by hotwiring the nitrogen regulatory system to restart their faulty motility system. To find this out we had to work across traditional disciplines, bridging microbiology, evolution and biochemistry, to build a truly interdisciplinary team. Of course, there are many gaps in our knowledge, one of which is the likelihood of these changes occurring in a real life situation. For Pseudomonas fluorescens real life involves swimming in soil water and moving across plant roots, not agar plates. Nevertheless, we believe our discovery is an amazing tale about life’s resilience, that in the face of catastrophic mutation threatening bacterial extinction, there is still the capacity for innovative changes to rescue the mutants and save the day.

Finally, some thanks to people who have kindly provided encouraging feedback along the way: Prof. Martin Buck, Prof. Allan Downie, Prof. Paul Rainey, Prof. Jim Alfano, Prof. John Mansfield, Prof. Jeff Dangl, Prof. Joyce Loper, Dr Gail Preston, Dr Joerg Schumacher, Dr Xue-Xian Zhang, Prof. Ben Whalley, Prof. Ian Jones.

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