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.

The Ros/MucR protein family represents a group of prokaryotic zinc-finger transcriptional regulators involved in gene regulation, virulence, and host–microbe interactions. Although structurally characterized in α-proteobacteria, the evolutionary distribution and structural conservation of Ros homologs across broader taxonomic lineages remain unclear. In this study, we performed a large-scale comparative analysis of Ros/MucR homologs across bacterial and eukaryotic taxa using sequence homology searches, multiple sequence alignment, phylogenetic reconstruction, structural modeling, and statistical analysis. A total of 1,840 homologous sequences were identified, with α-proteobacteria showing the highest sequence conservation. Predicted zinc-coordination patterns varied substantially among taxonomic groups, yet structural comparisons indicated that the C-terminal DNA-binding domain remained broadly conserved despite sequence divergence. In contrast, δ-proteobacterial homologs exhibited increased sequence and structural divergence. Analysis of the N-terminal domain revealed that residues associated with oligomerization are strongly conserved in α-proteobacteria but more variable in other lineages, although hydrophobic character at these positions is often retained. Phylogenetic analysis showed a polyphyletic distribution of homologs across taxa, with eukaryotic Ros-like sequences appearing in multiple regions of the tree. Overall, these findings indicate that Ros/MucR proteins combine evolutionary flexibility with structural resilience, maintaining a conserved DNA-binding architecture despite diversification of predicted zinc-coordination residues and oligomerization-related features. This work provides a comparative evolutionary framework for understanding structural conservation and functional diversification within the Ros/MucR protein family.