Regulation of bacterial DNA transposition

转座子是一种可移动的遗传元件，几乎存在于所有生物体中。它们通过调节多种类型的基因重排和影响邻近基因的表达来影响宿主。一般来说，转座子通过影响转座酶表达的系统受到严格的负调控，转座酶是转座反应中介导DNA链切割和连接步骤的蛋白质。限制转座子的动员可以减少转座子对宿主造成的诱变负担。然而，有充分的证据表明，将转座频率保持在低水平可以使宿主受益，因为转座产生的遗传多样性可以帮助有机体适应不断变化的环境条件。此外，还有一些转座子被宿主驯化的例子，转座子编码的蛋白质通过承担重要的细胞功能而对宿主做出贡献。我们的研究计划的目标是探索转座子和它们的宿主之间的互惠关系，目的是利用这一知识来操纵基因组，达到基因工程的目的。这5年的具体目标是：*1.探索第200条S对沙门氏菌宿主基因表达、生长和感染性的调控。我们将进一步测试ART200，一种由转座子IS200编码的sRNA，调节沙门氏菌中细胞基因的表达。在RNA-seq实验中，当art200耗尽时，其水平变化超过2倍的10个基因的表达将在art200耗尽或过度表达的条件下使用qRT-PCR和Northern印迹分析进行重新评估。我们还将询问操纵art200水平是否会影响沙门氏菌的生长和传染性。2.建立一条鉴定Hfq调控转座子的管道。我们将挖掘HfQ-RIP数据集，以寻找富含HfQ下拉列表的转座酶mRNAs的例子。我们将进行Hfq-RIPS来确认给定的候选转座酶mRNA是否与Hfq结合，然后询问当Hfq被删除时，候选mRNA的表达是否像预期的那样增加，如果转座酶的表达和转座可能受到Hfq的负调控。*3.研究Hfq在胁迫相关的细菌转座子诱导中的作用。我们将测试Hfq调节的转座子可以通过Chix的隔离来限制Hfq的可用性的想法，Chix是一种压力诱导的SRNA，具有不寻常的Hfq结合属性。为此，我们将询问转座酶(Tn10、Tn5和IS200)的表达和转座频率是否在沙门氏菌从指数期向稳定期过渡时增加，此时Chix的表达达到上限。我们还将测试新发现的受Hfq调控的转座子(来自Aim 2)，以了解它们对Chix表达的敏感性。********

Transposons are mobile genetic elements found in virtually all organisms. They impact their hosts by mediating numerous types of genetic rearrangements and by influencing the expression of neighboring genes. In general transposons are tightly negatively regulated through systems that impact on the expression of transposase, the protein that mediates DNA strand cutting and joining steps in transposition reactions. Limiting transposon mobilization reduces the mutagenic burden transposons inflict on their hosts. However, there is ample evidence that maintaining transposition frequencies at low levels can benefit the host as the genetic diversity generated by transposition can help an organism adapt to changing environmental conditions. In addition, there are a few examples where transposons have been domesticated' by their hosts such that a transposon-encoded protein contributes to the host by taking on an important cellular function. The goal of our research program is to explore the mutualistic relationship between transposons and their hosts with the intent of using this knowledge to manipulate genomes for genetic engineering purposes. Specific objectives for this 5 year period are:******1. Explore art200's regulation of host gene expression, growth and infectivity in Salmonella. We will further test the idea that art200, an sRNA encoded by the transposon IS200, regulates the expression of cellular genes in Salmonella. The expression of 10 genes identified in an RNA-seq experiment whose levels were altered by more than 2-fold when art200 was depleted will be reevaluated using qRT-PCR and Northern blot analyses under conditions where art200 is depleted or overexpressed. We will also ask if manipulating art200 levels influences Salmonella growth and infectivity.******2. Develop a pipeline for identifying Hfq regulated transposons. We will mine Hfq-RIP data sets for examples of transposase mRNAs that are enriched in Hfq pull-downs. We will perform Hfq-RIPs to confirm that a given candidate transposase mRNA is bound by Hfq and then ask if expression of the candidate mRNA is increased when Hfq is deleted as would be expected if transposase expression and presumably transposition is negatively regulated by Hfq. ***3. Examine the role of Hfq in stress-related induction of bacterial transposons. We will test the idea that Hfq-regulated transposons can be activated by limiting Hfq availability through sequestration by ChiX, a stress-induced sRNA that has unusual Hfq binding properties. Towards this end we will ask if transposase expression (Tn10, Tn5 and IS200) and transposition frequencies increase in Salmonella at the transition from exponential to stationary phase where ChiX expression reaches its upper limit. We will also test transposons newly discovered to be under Hfq regulation (from aim 2) for their sensitivity to ChiX expression. ********