Proteins of the MucR/Ros family play a crucial role in bacterial infection or symbiosis with eukaryotic hosts. MucR from Sinorhizobium meliloti plays a regulatory role in establishing symbiosis with the host plant, both dependent and independent of Quorum Sensing. Here, we report the first characterization of MucR isolated from Sinorhizobium meliloti by mass spectrometry and demonstrate that this protein forms higher-order oligomers in its native condition of expression by SEC-MALS. We show that MucR purified from Sinorhizobium meliloti can bind DNA and recognize the region upstream of the ndvA gene in EMSA, revealing that this gene is a direct target of MucR. Although MucR DNA binding activity was already described, a detailed characterization of Sinorhizobium meliloti DNA targets has never been reported. We, thus, analyze sequences recognized by MucR in the rem gene promoter, showing that this protein recognizes AT-rich sequences and does not require a consensus sequence to bind DNA. Furthermore, we investigate the dependence of MucR DNA binding on the length of DNA targets. Taken together, our studies establish MucR from Sinorhizobium meliloti as a member of a new family of Histone-like Nucleoid Structuring (H-NS) proteins, thus explaining the multifaceted role of this protein in many species of alpha-proteobacteria.

The Ros/MucR family is constituted by proteins controlling the expression of genes crucial for the interaction with eukaryotic hosts. Ros/MucR family members were classified as H-NS-like proteins in α-proteobacteria, as they share fundamental features with H-NS proteins playing a pivotal role in controlling gene expression by structuring the bacterial genome. Here, we identified two new Ros/MucR family members in Sinorhizobium meliloti. They differ from classical MucR homologs since MucR2 lacks the circular oligomeric structure typical of other family members and MucR3 shows a concentration-dependent oligomerization ability with a low propensity to form circular particles, as shown by cryogenic electron microscopy. Moreover, MucR2 and MucR3 present a new zinc coordination sphere. The newly identified MucRs bind DNA, but lack the DNA bridging activity, which is crucial for structuring the bacterial genome. Using mass spectrometry, light scattering, NMR, EMSA and bridging assay, our study reports the first identification and characterization of two new MucRs and indicates that Ros/MucR family members control gene expression through distinct mechanisms. These results provide an important framework for future studies aimed at dissecting the interplay among MucR proteins and understanding how they can jointly orchestrate condition-dependent gene expression in bacterial species expressing multiple mucR homologous genes.