Mechanisms and consequences of phase variation in bacterial immune systems

Phase variation is the reversible rearrangement of a genomic locus, producing changes in global gene expression. Because the process is stochastic, phenotypic heterogeneity develops across cell sub-populations producing differences in relative fitness. Phase variation thus allows cells to adapt to environmental stresses more rapidly than through classical evolution. Some bacterial Type I Restriction-Modification (RM) enzymes that evolved to prevent phage infection are modulated by phase variation. These "shufflons" can spontaneously switch the DNA recognition sequence of the restriction endonuclease and its cognate DNA methyltransferase. An untested fitness benefit is to overcome escape phages that arise due to viral DNA methylation; other sub-populations of cells will retain resistance due to their genomes having an alternative RM recognition sequence. Shufflons are also found in many pathogenic bacteria and changes in genome methylation patterns that accompany shuffling have been implicated in producing diversification that benefits the population. This interdisciplinary project between the Szczelkun and Gorochowski labs aims to: explore the molecular mechanism of the shufflons; the frequency of recombination and its consequences for cell fitness; and exploit the phenomenon for the rational design of new genetic switches. In shufflons, the basis for shuffling is inversion of genes that encode for the specificity subunit (HsdS), catalysed by a tyrosine recombinase (CreX). We will first explore the biochemical basis for switching in vitro using purified CreX to address whether certain recombination sequences, and thus certain HsdS variants, are favoured. To explore complex operon rearrangements, we will use nanopore sequencing. Reporter assays will be designed to compare the recombinase activity in cells, and nanopore sequencing used to map genome rearrangements and DNA methylation. To test the activities of the alternative RM enzymes generated by shuffling, we will measure their enzyme properties in vitro. Structural predictions of the HsdS subunits will help explain how the rearrangements can be accommodated while maintaining a folded structure. In parallel, we will develop mathematical models to simulate how recombination kinetics influences DNA methylation and DNA cleavage, and thus cell fitness. As the project develops, a key aim is to address how cells overcome the potential toxicity of the shuffling. A second key aim is to explore whether the shufflons can be exploited in synthetic genetic circuits.