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

在兼性光养甲型变形杆菌中，核转录调控对光合作用基因表达的重要影响已经在我之前的研究项目中得到了很好的证明。最近的数据表明，RNase E活性降低对光营养生长有强烈的影响，但对化学营养生长没有影响，单一SNA(UpsM)的过度表达可以取消光营养生长，当细胞离开指数期时，RNase E还导致光合作用mRNAs的减少。因此，这项建议将阐述光合作用基因的核调控在适应不同生长条件中的作用。为此，我们将扩展我们现有的全球转录组数据集，包括不同生长条件下野生型和一些RNase突变体，包括进一步的RNase突变体和一个缺失RNA伴侣Hfq的突变体。所有现有的数据集都来自于在微氧条件下生长的细胞，我们现在也将在我们的实验中包括好氧和光营养条件。为了了解在不同生长条件下光合作用基因表达中已知作用的sRNA的差异处理机制，我们将分析它们的处理过程，并用Northern blotts方法测定它们在不同突变株中的半衰期。我们将建立体内报告系统，并应用体外降解实验来监测在不同生长条件下参与mRNA和SRNA加工的RNase的活性。此外，我们将应用免疫共沉淀来鉴定在不同生长条件下与不同核糖核酸酶相关的蛋白质。最后，我们将阐明反义RNA asPcrK影响光合作用基因表达以及sRNA UpsM影响光营养生长的机制。这些分析将使用我过去在实验室建立的方法来分析不同sRNA的作用。我们将测试这些sRNA的过度表达是否影响光合作用mRNAs的数量和半衰期，如果是这样的话，哪些RNase对改变的半衰期负责。我们希望通过MS2亲和纯化的方法来鉴定UpsM和相互作用蛋白的直接RNA靶标，并将测试UpsM对光营养生长的影响是否源于Hfq海绵的功能。我们期待着关于当细胞适应不断变化的生长条件时，RNA处理和sRNAs如何促进光合作用基因表达变化的重要新信息。这些结果还将为核转录调控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.