MucR binds multiple target sites in the promoter of its own gene and is a heat‐stable protein: Is MucR a H‐NS‐like protein?

Abstract

The protein MucR from Brucella spp. is involved in the expression regulation of genes necessary for host interaction and infection. MucR is a member of the Ros/MucR family, which comprises prokaryotic zinc‐finger proteins and includes Ros from Agrobacterium tumefaciens and the Ml proteins from Mesorhizobium loti. MucR from Brucella spp. can regulate the expression of virulence genes and repress its own gene expression. Despite the well‐known role played by MucR in the repression of its own gene, no target sequence has yet been identified in the mucR promoter gene. In this study, we provide the first evidence that MucR from Brucella abortus binds more than one target site in the promoter region of its own gene, suggesting a molecular mechanism by which this protein represses its own expression. Furthermore, a circular dichroism analysis reveals that MucR is a heat‐stable protein. Overall, the results of this study suggest that MucR might resemble a H‐NS protein.

Identifying the region responsible for Brucella abortus MucR higher-order oligomer formation and examining its role in gene regulation

“…Here, we identify a hydrophobic region as responsible for the higher-order oligomer formation at the N-terminus of the prokaryotic zinc-finger protein MucR and definitively demonstrate the importance of MucR oligomerization for its regulatory function in Brucella . Based on the results presented here, together with previously published findings 26 , 27 , we propose that the prokaryotic zinc-finger proteins in the Ros/MucR family control gene expression by employing a mechanism similar to that used by the H-NS proteins, rather than working as classical transcriptional regulators.…”

“…To investigate the ability of the mutant protein MucR L36L39I40A to bind DNA, we performed electrophoretic mobility shift assays (EMSAs) using one of the MucR DNA-binding site, named Site1, previously identified in the mucR gene promoter and located at −174 bp from the ATG start codon 27 . Comparing the protein/DNA complexes formed by MucR and MucR L36L39I40A , it is evident that the mutant protein is unable to form the slow mobility MucR/DNA complex indicated by the arrow in Fig.…”

“…The increasing amount of the protein used is indicated on the top of the lanes as well as the name of the proteins. The target sequence Site1 was previously identified in the promoter of mucR gene at −174 bp from the ATG start codon 27 . The arrow indicate the protein/DNA complex formed by MucR with Site1 which does not appear in the EMSAs of MucR L36L39I40A .…”

“…On the other hand, the prokaryotic C 2 H 2 zinc-finger shows some peculiarities such as the second α-helix, the flexibility of the zinc-coordination sphere and the larger hydrophobic core that together with the zinc-finger motif constitutes the DNA-binding domain 1 , 14 – 18 . Our recent studies with full-length Ros/MucR proteins has determined that these proteins form higher-order oligomers 26 , are stable at high temperature and are able to bind multiple sites present in the promoters of their target genes 26 , 27 . These combined experimental findings have led us to propose that the Ros/MucR proteins regulate gene expression in the same manner as the DNA structuring protein H-NS, which is found in many other Gram-negative bacteria 55 .…”

“…Recently, we demonstrated that the AT-rich DNA targets sites for the Mesorhizobium Mls and Brucella MucR contain T-A steps, and that these proteins contact DNA mostly in the minor groove and are able to form higher-order oligomers 26 . Furthermore, we have shown that MucR from Brucella abortus is able to recognize multiple AT-rich sites in the promoter of its own gene and that it is a heat-stable protein with a Tm of 63 °C 27 . The ability to bind AT-rich sites containing T-A steps in the minor groove, the capacity to oligomerize and the heat-stability are also features of another prokaryotic protein family, the histone-like nucleoid structuring proteins, H-NS 28 – 34 .…”