Small proteins in the soybean symbiont Bradyrhizobium japonicum

大豆共生菌慢生根瘤菌中的小蛋白埃琳娜·埃夫盖尼耶娃-哈肯贝格教授在已测序的细菌基因组中仍然没有识别出小蛋白的基因。本项目旨在揭示细菌大豆共生菌慢生根瘤菌中的小蛋白谱系，并分析一个保守的、在表面生长中起作用的µ蛋白的作用机制。从长远来看，在这个重要的模式生物中，几种微蛋白的功能将被分析。在我们的初步工作中，我们发现了一个编码14个氨基酸微蛋白RreB的保守sORF，它的过度表达在液体培养中没有影响，但导致了菌落大小的异质性。此外，我们还对日本血吸虫USDA 110基因组中长度在30到50个密码子之间的500多个sORF进行了注释。其中只有3个sORF被先前的蛋白质组分析支持，并验证了位于高转录的RRN操纵子中的45个密码子的sORF的翻译。我们的初步数据表明，有必要在全球范围内对日本血吸虫中的微蛋白进行实验检测和功能分析。为了更多地了解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(S)。为了阐明RreB影响表面生长的机制，我们将确定RreB高产菌株的菌落大小是否因为不同的滞后期或不同的生长速度而不同，并将识别与rreB相互作用的表面响应基因。最后，我们将测试RreB对于在包括土壤条件在内的地表生存和/或与大豆共生是否重要。将通过两种互补的方法--多肽分析(Z1-项目)和核糖体图谱(Z2-项目)--对日本血吸虫中的µ-蛋白进行全球检测。我们将使用液体培养，添加和不添加诱导共生相关基因的类黄酮类化合物。这一结果将为注释的µ-蛋白质的翻译提供实验证据，并将发现新的蛋白质，包括小于30aa的µ-蛋白质。将对几种微蛋白进行鉴定，其中包括上述在RRN操纵子中编码的微蛋白。拟议项目的结果将阐明一种与细菌表面生长相关的微蛋白的新机制。此外，日本芽胞杆菌USDA 110的µ蛋白质组将被发现，填补了这一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甲型变形杆菌的基因组注释做出重要贡献。我们还希望我们的研究结果有助于了解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.