Modifying the genome of P. fluorescens SBW25 is crucial to answering evolutionary questions. Successful strains obtained from evolve-and-resequence experiments can carry multiple mutations. Identifying which of these many mutations underlie the adaptive phenotype requires introduction of each mutation into the genome of the ancestor. We can introduce any mutation into the SBW25 genome using a process called two-step allelic exchange which relies on RecA-dependent homologous recombination. As allelic exchange can be frustrating and time-consuming, we constructed a new suicide vector that is compatible with modern cloning techniques and allows a process that used to take several weeks to be carried out in a matter of days. These improvements mean we can now rapidly screen large numbers of mutations in the ancestral genome.
Once mutations have been introduced into the ancestral genome, we need to determine the consequences of these mutations for bacterial fitness. We can measure fitness by comparing the changes in the frequency of our mutant relative to that of the ancestor over time. Very small differences in fitness can have very large evolutionary consequences, but our ability to detect small differences in fitness is limited by the absolute number of bacteria we can count. With traditional plate assays, this number is typically limited to a few hundred colony forming units. However, counting cells using a flow cytometer lets us count tens of thousands of cells in only a few seconds meaning that we can reliably detect fitness differences as small as 1%. Unfortunately, bacterial cells are so small that they can be difficult to distinguish from dust and debris in the flow cytometer. To tell cells apart from debris, we can tag each mutant and the ancestor with a different fluorescent construct. We are currently using constructs that can be introduced by transposition into a fixed chromosomal location. Using these, we can rapidly quantify the fitness effects of introduced mutations in shaking cultures. Static cultures, where mutants can form large collectives that grow at the air liquid interface, present a different challenge for accurate fitness measurement that we’re currently working to overcome.
An alternative approach to studying the genetics of adaptation is to introduce a pool of thousands of mutants to an environment and monitor how the relative frequencies of the different types change over time. This pool of mutants can be generated by interrupting genes using a randomly-integrating transposable elements marked with unique barcodes that enables the frequency of each type to be monitored by high-throughput sequencing. These barcoded pools of mutants will be invaluable in the rapid identification of genes associated with adaptation to new environments – and provide a starting point to investigating plant-microbe interactions in our new growth chambers.
Genetic barcodes integrated at neutral chromosomal sites offer a further powerful approach to studying evolutionary biology. We can use barcodes to track changes in the frequencies of different bacterial lineages over long periods of time by high-throughput sequencing. Andy Farr is developing a randomly barcoded population of P. fluorescens to track lineages of SBW25 within a microbial community in his investigation of the evolution of antibiotic resistance. Loukas Theodosiou will also use this barcoded population to revisit Rainey & Travisano’s work on adaptive radiation using the power of modern genetic techniques.
Functional characterisation of genes identified during evolution experiments can be very difficult. One approach is to observe the phenotype of a strain with the entire gene knocked out compared to an isogenic strain with the gene intact. A more refined approach requires the ability to control exactly when the gene is expressed which can be accomplished using inducible promoters. The most widely used system for inducible expression in bacteria is the LacI/Ptac negative regulation system that has numerous drawbacks including high background expressions and inducer-associated toxicity. We are testing several positive regulation cassettes have been developed for use in Pseudomonas putida, including a cyclohexanone-inducible system that shows great promise in P. fluorescens.
Our goal is to continue adding new strategies to our genetic toolkit that simplify the process of investigating evolutionary questions in SBW25 and related Pseudomonas strains / species.