Abstract
Mesorhizobium loti contains ten genes coding for proteins sharing high amino acid sequence identity with members of the Ros/MucR transcription factor family. Five of these Ros/MucR family members from Mesorhizobium loti (Ml proteins) have been recently structurally and functionally characterized demonstrating that Ml proteins are DNA-binding proteins. However, the DNA-binding studies were performed using the Ros DNA-binding site with the Ml proteins. Currently, there is no evidence as to when the Ml proteins are expressed during the Mesorhizobium loti life cycle as well as no information concerning their natural DNA-binding site. In this study, we examine the ml genes expression profile in Mesorhizobium loti and show that ml1, ml2, ml3 and ml5 are expressed during planktonic growth and in biofilms. DNA-binding experiments show that the Ml proteins studied bind a conserved AT-rich site in the promoter region of the exoY gene from Mesorhizobium loti and that the proteins make important contacts with the minor groove of DNA. Moreover, we demonstrate that the Ml proteins studied form higher-order oligomers through their N-terminal region and that the same AT-rich site is recognized by MucR from Brucella abortus using a similar mechanism involving contacts with the minor groove of DNA and oligomerization.
Highlights
The Ros/MucR transcription factor family[1,2,3] includes proteins such as Ros from Agrobacterium tumefaciens[4] and MucR from Brucella abortus[2] responsible for the expression regulation of virulence genes and MucR from Sinorhizobium meliloti involved in the gene expression regulation necessary for the symbiosis process established by Rhizobia with plant[5]
Due to the close phylogenetic relatedness of the α2-proteobacteria, these organisms use common genes and strategies for facilitating their interactions with their specific host, and the gene encoding the transcriptional regulator Ros/MucR is one of the genes conserved in the α2-proteobacteria that is important for host-bacterium interactions[2]
We show that four of the five ml genes belonging to Ros/MucR family, ml[1], ml2, ml[3] and ml[5] are well expressed in M. loti during the stationary phase of planktonic growth whereas their expression level is very low during exponential phase
Summary
Rhizobium spp. and Brucella spp. are members of the α2 subclass of proteobacteria, which includes bacteria that are symbionts and pathogens of plants and mammalian pathogens[2]. From this point of view, it could be that in the complex soil matrix, some particular environmental conditions, which cannot be replicated when culturing M. loti in TYR broth, activate ml[4] gene for a particular biological function In these studies, we show for the first time that the core DNA-target site recognized by Mls is a five base pair AT-rich sequence containing a T-A step and that these proteins make crucial contacts in the minor groove when they bind to DNA. The data present in our study show that four of the five ml genes studied are expressed in M. loti under planktonic condition as well as in biofilms and demonstrate that Ml proteins and MucR are able to recognize an AT-rich sequence of five base pairs containing a T-A step and oligomerize through the N-terminal region
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