Small proteins in the soybean symbiont Bradyrhizobium japonicum

大豆共生体缓生根瘤菌中的小蛋白。Elena Evguenieva-Hackenberg教授，Universität gie ßen在测序的细菌基因组中，小蛋白质的基因仍未被识别。本项目旨在揭示大豆细菌共生体日本慢生根瘤菌（bryyyrhizobium japonicum）的小蛋白库，并分析一个保守的微蛋白在表面生长过程中的作用机制。从长远来看，几种微蛋白的功能将在这种重要的模式生物中得到分析。在我们的初步工作中，我们发现了一个保守的sORF编码14aaµ-蛋白RreB，其过表达在液体培养中没有影响，但会导致异质集落大小。此外，我们在日本血吸虫USDA 110基因组中注释了500多个长度在30 ~ 50个密码子之间的sorf。这些sORF中只有三个被先前的蛋白质组学分析所支持，并且在高度转录的rrn操纵子中证实了具有45个密码子的sORF的翻译。我们的初步数据表明，需要对日本芽孢杆菌中微蛋白进行全球实验检测，并对这些小蛋白进行功能分析。为了进一步了解RreB的作用及其分子机制，我们旨在研究其亚细胞定位并确定其相互作用伙伴。为了阐明RreB影响表面生长的机制，我们将确定RreB过量菌株的菌落大小是否因不同的滞后期或不同的生长速率而不同，并将确定与RreB相互作用的表面响应基因。最后，我们将测试RreB是否对包括土壤条件在内的地表生存和/或与大豆的共生重要。日本芽孢杆菌µ-蛋白的全球检测将通过两种互补的方法进行，肽组学（Z1-project）和核糖体分析（Z2-project）。我们将使用液体培养，添加或不添加黄酮类化合物，诱导共生相关基因。该结果将为带注释的µ-蛋白的翻译提供实验证据，并将发现新的µ-蛋白，包括小于30 aa的µ-蛋白。包括上述在rrn操纵子中编码的微蛋白在内的几种微蛋白将被表征。本项目的研究结果将阐明一种与细菌表面生长有关的微蛋白的新机制。此外，日本B. japonicum USDA 110的µ-蛋白质组将被发现，填补了该模式生物基因组注释的最后空白之一。这将为改进相关共生和自由生活的阿尔法变形菌的基因组注释提供重要贡献。我们也期望我们的结果将有助于认识根瘤菌与植物之间的共生关系。

Small proteins in the soybean symbiont Bradyrhizobium japonicumApl. Prof. Dr. Elena Evguenieva-Hackenberg, Universität Gießen Genes for small proteins still remain unrecognized in sequenced bacterial genomes. This project aims to uncover the repertoire of small proteins in the bacterial soybean symbiont Bradyrhizobium japonicum and to analyze the mechanisms of action of a conserved µ-protein with a role in growth on a surface. In the long term, the functions of several µ-proteins will be analyzed in this important model organism.In our preliminary work, we identified a conserved sORF encoding the 14 aa µ-protein RreB, the overexpression of which has no effect in liquid culture but results in heterogeneous colony size. Further, we annotated more than 500 sORFs with lengths between 30 and 50 codons in the genome of B. japonicum USDA 110. Only three of these sORFs were supported by previous proteomic analyses, and translation of a sORF with 45 codons, which is located in the highly transcribed rrn operon, was verified. Our preliminary data show the need for global experimental detection of µ-proteins and for functional analyses of such small proteins in B. japonicum.In order to learn more about the role of RreB and its molecular mechanisms, we aim to study its subcellular localization and to identify its interaction partner(s). To elucidate the mechanisms by which RreB influences growth on surface, we will determine whether the colony sizes of a RreB overproducing strain differ because of different lag phase or different growth rates and will identify surface-responsive genes that interact with rreB. Finally, we will test whether RreB is important for survival on surface including soil conditions and/or for symbiosis with soybean. The global detection of µ-proteins in B. japonicum will be conducted with two complementary approaches, peptidomics (Z1-project) and ribosome profiling (Z2-project). We will use liquid cultures with and without addition of a flavonoid that induces symbiosis-relevant genes. The results will provide experimental evidence for the translation of annotated µ-proteins and will uncover new ones, including µ-proteins smaller than 30 aa. Several µ-proteins including the above mentioned µ-protein encoded in the rrn operon will be characterized. The results from the proposed project will elucidate a new mechanism of a µ-protein related to bacterial growth on surface. Additionally, the µ-proteome of B. japonicum USDA 110 will be uncovered, filling in one of the last gaps in the genome annotation of this model organism. This will provide an essential contribution to improved genome annotations of related symbiotic and free-living Alphaproteobacteria. We also expect that our results will contribute to the knowledge on symbiosis between rhizobia and plants.