Small proteins in the soybean symbiont Bradyrhizobium japonicum

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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.