Small proteins in the soybean symbiont Bradyrhizobium japonicum

大豆共生菌慢生根瘤菌（Bradyrhizobium japonicum Apl.）Elena Evguenieva-Hackenberg博士教授，吉森大学 在测序的细菌基因组中，小蛋白的基因仍然没有被识别出来。该项目旨在揭示细菌大豆共生体Bradyrhizobium japonicum中的小蛋白的所有组成部分，并分析在表面生长中起作用的保守μ蛋白的作用机制。在我们的前期工作中，我们鉴定了一个保守的sORF，编码14 aa的µ蛋白RreB，其过表达在液体培养中没有影响，但会导致异质性菌落大小。此外，我们在B的基因组中注释了超过500个长度在30到50个密码子之间的sORF。110. casino这些sORF中只有三个得到了先前蛋白质组学分析的支持，并且验证了位于高度转录的rrn操纵子中的具有45个密码子的sORF的翻译。我们的初步数据表明，需要对μ-蛋白进行全球实验检测，并对B中的此类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鉴定其相互作用伴侣。为了阐明RreB影响表面生长的机制，我们将确定RreB过量生产菌株的殖民地大小是否因不同的滞后期或不同的生长速率而不同，并将鉴定与RreB相互作用的表面响应基因。最后，我们将测试RreB是否对在包括土壤条件在内的表面上的生存和/或与大豆的共生是重要的。B中μ-蛋白的全局检测。将采用两种互补的方法，肽组学（Z1-project）和核糖体分析（Z2-project）进行日本血吸虫的基因组学研究。我们将使用添加和不添加诱导共生相关基因的类黄酮的液体培养物。这些结果将为注释的µ-蛋白质的翻译提供实验证据，并将发现新的µ-蛋白质，包括小于30 aa的µ-蛋白质。将表征几种μ-蛋白质，包括在rrn操纵子中编码的上述μ-蛋白质。拟议项目的结果将阐明与表面细菌生长相关的µ-蛋白质的新机制。此外，B.将发现日本大豆USDA 110，填补了该模式生物基因组注释中的最后一个空白。这将为相关共生和自由生活的Alphaproteobacteria的改进基因组注释提供重要贡献。我们也希望我们的研究结果将有助于根瘤菌和植物之间的共生关系的知识。

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.