Regulation of bacterial DNA transposition

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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.