Regulation of bacterial DNA transposition

转座子是移动的遗传元件，几乎在所有生物体中发现。它们通过介导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，转座酶是介导转座反应中DNA链切割和连接步骤的蛋白质。限制转座子移动减少了转座子对其宿主造成的诱变负担。然而，有充分的证据表明，保持低水平的转座频率可以使宿主受益，因为转座产生的遗传多样性可以帮助生物体适应不断变化的环境条件。此外，还有一些例子，转座子已经被它们的宿主“驯化”，使得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，目的是利用这些知识来操纵基因组用于基因工程目的。这五年的具体目标是：*1。探索art 200在沙门氏菌中对宿主基因表达、生长和感染性的调节。我们将进一步测试由转座子IS 200编码的sRNA art 200调节沙门氏菌细胞基因表达的想法。 在RNA测序实验中鉴定的10个基因的表达，其水平在art 200耗尽时改变超过2倍，将在art 200耗尽或过表达的条件下使用qRT-PCR和北方印迹分析重新评估。我们还将询问操纵art 200水平是否会影响沙门氏菌的生长和传染性。2.开发用于鉴定Hfq调节的转座子的管道。我们将挖掘Hfq-RIP数据集，以获得富含Hfq下拉的转座酶mRNA的示例。我们将进行Hfq-RIP以确认给定的候选转座酶mRNA被Hfq结合，然后询问当Hfq缺失时候选mRNA的表达是否增加，如如果转座酶表达和推测的转座由Hfq负调控所预期的那样。*3。检查Hfq在细菌转座子的应激相关诱导中的作用。我们将测试的想法，Hfq调节转座子可以通过限制Hfq的可用性，通过螯合ChiX，应激诱导的sRNA，具有不寻常的Hfq结合特性激活。为此，我们将询问转座酶表达（Tn 10，Tn 5和IS 200）和转座频率是否在沙门氏菌中从指数期到稳定期的过渡期增加，其中ChiX表达达到其上限。我们还将测试新发现的处于Hfq调控下的转座子（来自目的2）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. ********