Resumo

Sinorhizobium meliloti ( Eisinfer meliloti ) is an alpha-proteobacterium that belongs to the Rhizobiaceae family, being its main characteristic the ability to establish a symbiotic association with legumes and to perform the biological nitrogen fixation process. As well for most microorganisms, iron is an essential nutrient for this bacterium and it possesses different iron acquisition systems. One of these systems involves the usage of heme compounds as nutritional iron sources. We previously demonstrated that the outer membrane heme-binding protein ShmR plays an essential role in heme internalization and iron utilization. We also demonstrated iron and heme responsiveness of the shmR promoter and the involvement of the small protein HmuP on this response. In this work we wanted to understand the mechanisms involved in such regulation. Particularly, we wanted to determine whether HmuP acts as a positive regulator by direct binding to DNA or by inhibiting the action of the negative iron regulator RirA. In order to characterize the shmR promoter region, the transcriptional starting site was identified by 5’ Race. To assess the minimal promoter region necessary for regulation, different length of the upstream region of shmR gene were introduced in front of a promoterless GFP (green fluorescent protein) reporter gene present in a rhizobia replicative plasmid. The constructions were introduce in S. meliloti and assessed for the loss of iron responsiveness by measuring GFP activity. A 176bp sequence was determined as the minimal sequence required for the iron responsive regulation. Deletions in the 5’ or 3’ region of this 176bp segment had different effects on the iron response, suggesting the importance of some specific sequences for this response. Bioinformatics approaches are being held in order to identify putative conserved sequences in the promoter region of shmR . We expect to find the typical RirA box necessary for repression under iron repleted conditions by means of the RirA protein. We also want to know if there is a putative box for HmuP protein binding. Electrophoretic mobility shift assays using the 176bp promoter segment willl enable us to determine the ability of HmuP to bind the DNA. This will give insight in the mechanisms of action of HmuP.