Mechanisms and consequences of phase variation in bacterial immune systems

相位变异是基因组位点的可逆重排，产生全局基因表达的变化。由于这个过程是随机的，表型异质性在细胞亚群中发展，产生相对适合度的差异。因此，相变化使得细胞比通过经典进化更快地适应环境压力。一些细菌I型限制性修饰（RM）酶，进化以防止噬菌体感染是由阶段变化调节。这些“洗牌子”可以自发地切换限制性内切酶及其同源DNA甲基转移酶的DNA识别序列。一个未经测试的适应性益处是克服由于病毒DNA甲基化而产生的逃逸噬菌体；其他亚群细胞将保留抗性，因为它们的基因组具有替代的RM识别序列。在许多致病菌中也发现了洗牌子，并且伴随着洗牌的基因组甲基化模式的变化与产生有益于种群的多样化有关。这个由Szczelkun和Gorochowski实验室合作的跨学科项目旨在：探索shufflons的分子机制；重组频率及其对细胞适应性的影响；并利用这一现象来合理设计新的基因开关。在洗牌子中，洗牌的基础是编码特异性亚基（HsdS）的基因反转，由酪氨酸重组酶（CreX）催化。我们将首先探索使用纯化的CreX进行体外转换的生化基础，以确定某些重组序列是否有利于hsd变异。为了探索复杂的操纵子重排，我们将使用纳米孔测序。报告分析将被设计用于比较细胞中的重组酶活性，以及用于绘制基因组重排和DNA甲基化的纳米孔测序。为了测试洗牌产生的替代RM酶的活性，我们将在体外测量它们的酶特性。对hsd亚基的结构预测将有助于解释在保持折叠结构的同时如何进行重排。同时，我们将开发数学模型来模拟重组动力学如何影响DNA甲基化和DNA切割，从而影响细胞适应性。随着项目的发展，一个关键目标是解决细胞如何克服洗牌的潜在毒性。第二个关键目标是探索是否可以在合成遗传电路中利用shufflons。

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.