Riboregulation of photosynthesis genes in the adaptation to different growth conditions in Rhodobacter sphaeroides

在兼性光养α变形杆菌Rhodobacter中，核糖调节对光合基因表达的重要影响在我之前的研究项目中得到了很好的证明。最近的数据显示，RNase E活性的降低强烈影响光营养生长，而不是化学营养生长，单个sRNA （UpsM）的过表达可以终止光营养生长，当细胞离开指数期时，RNase E也有助于光合作用mrna的减少。因此，这一建议将解决光合作用基因的核糖调控在适应不同生长条件中的作用。为此，我们将扩展现有的野生型和一些不同生长条件下的RNase突变体的全球转录组数据集，并包括进一步的RNase突变体和缺乏RNA伴侣Hfq的突变体。所有现有的数据集都来自于在微有氧条件下生长的细胞，我们现在也将在我们的实验中包括有氧和光养条件。为了了解不同生长条件下光合作用基因表达中已知srna的不同加工机制，我们将通过Northern blots分析它们在不同突变菌株中的加工过程并确定它们的半衰期。我们将建立一个体内报告系统，并应用体外降解实验来监测在不同生长条件下mRNA和sRNA加工中起作用的rna酶的活性。此外，我们将应用共免疫沉淀来鉴定在不同生长条件下与不同rna酶相关的蛋白质。最后，我们将阐明反义RNA asPcrK影响光合作用基因表达的机制，以及sRNA UpsM影响光合生长的机制。这些分析将使用过去在我的实验室建立的方法来分析各种srna的作用。我们将测试这些sRNAs的过表达是否会影响光合作用mrna的数量和半衰期，如果是的话，哪些RNases负责改变半衰期。我们希望通过MS2亲和纯化方法确定UpsM的直接RNA靶点和相互作用蛋白，并将测试UpsM对光养生长的影响是否由于其作为Hfq海绵的功能。我们期待在细胞适应变化的生长条件时，RNA加工和sRNAs如何影响光合作用基因的表达方面获得重要的新信息。这些结果也将为核糖调节对细菌适应的贡献提供一般的新见解。

The important impact of riboregulation on the expression of photosynthesis genes in the facultative phototrophic alphaproteobacterium Rhodobacter is well documented by my previous research projects. Recent data revealed that reduced RNase E activity strongly impacts phototrophic but not chemotrophic growth, that overexpression of a single sRNA (UpsM) can abolish phototrophic growth and that RNase E also contributes to the decrease of photosynthesis mRNAs when cells leave exponential phase. This proposal will therefore address the role of riboregulation of photosynthesis genes in the adaptation to different growth conditions. To this end we will extent our existing global transcriptome data sets for wild type and some RNase mutants under different growth conditions and include further RNase mutants and a mutant lacking the RNA chaperone Hfq. All existing data sets stem from cells grown under microaerobic conditions and we will now also include aerobic and phototrophic conditions in our experiments. To understand the mechanisms underlying differential processing of sRNAs with known roles in photosynthesis gene expression under different growth conditions, we will analyze their processing and determine their half-lives in different mutant strains by Northern blots. We will establish an in vivo reporter system and also apply in vitro degradation assays to monitor the activity of RNases with a role in mRNA and sRNA processing under different growth conditions. Furthermore, we will apply co-immunoprecipitation to identify proteins that are associated with the different RNases under different growth conditions. Finally, we will elucidate the mechanisms by which the antisense RNA asPcrK affects expression of photosynthesis genes and by which the sRNA UpsM impacts phototrophic growth. These analyses will use methods that have been established in my lab in the past for analyzing the role of diverse sRNAs. We will test whether overexpression of these sRNAs affects the amount and half-lives of photosynthesis mRNAs and if so, which RNases are responsible for the altered half-lives. We want to identify direct RNA targets of UpsM and interacting proteins by the MS2 affinity purification approach, and will test whether the effect of UpsM on phototrophic growth is due to a function as Hfq sponge. We expect important new information on how RNA processing and sRNAs contribute to altered expression of photosynthesis genes when cells adapt to changing growth conditions. These results will also provide general new insights in the contribution of riboregulation to bacterial adaptation.