MarR homologs with urate-binding signature

Abstract

Bacteria that associate with living hosts require intricate mechanisms to detect and respond to host defenses. Part of the early host defense against invading bacteria is the production of reactive oxygen species, and xanthine oxidase is one of the main producers of such agents. The end-product of the enzymatic activity of xanthine oxidase, urate, was previously shown to be the natural ligand for Deinococcus radiodurans-encoded HucR and it was shown to attenuate DNA binding by Agrobacterium tumefaciens-encoded PecS and Burkholderia thailandensis-encoded MftR, all members of the multiple antibiotic resistance regulator (MarR) family. We here show that residues involved in binding urate and eliciting the DNA binding antagonism are conserved in a specific subset of MarR homologs. Although HucR controls endogenous urate levels by regulating a uricase gene, almost all other homologs are predicted to respond to exogenous urate levels and to regulate a transmembrane transport protein belonging to either the drug metabolite transporter (DMT) or the major facilitator superfamily (MFS), as further evidenced by the presence of conserved binding sites for the cognate transcription factor within the respective promoter regions. These data suggest the use of orthologous genes for different regulatory purposes. We propose the designation UrtR (urate responsive transcriptional regulator) for this distinct subfamily of MarR homologs based on their common mechanism of urate-mediated attenuation of DNA binding.

Figure 1

Structure of urate-docked HucR. The two monomers are in green and blue and the DNA binding helices are in cartoon representation. The residues identified to be important for ligand binding and attenuated binding to DNA are colored red. Urate is in a mesh rendering.

Figure 2

Economically important pathogenic or symbiotic bacteria that encode an UrtR. Residues involved in urate binding and eliciting the conformational changes are highlighted in red and by asterisk above. E. coli MarR and S. typhimurium SlyA do not conserve these residues and do not contain α1, which harbors the vital tryptophan residue. Secondary structure elements are with reference to HucR (2fbk). Sequences are truncated at the C-termini to generate figure.

Figure 3

Evolutionary relationships of UrtR and other MarR homologs. All UrtR homologs cluster under a single sub-tree whereas blue sub-tree represents the selected MarR homologs. Tree was rooted using E. coli GntR as an out group. Uniprot identification numbers of the proteins are followed by a short description at the termini of each branch. Prototypical UrtR homologs, D. radiodurans HucR (Q9RV71 DEIRA) and A. tumefaciens PecS (Q7D1T4 AGRT5) are highlighted in red.

Figure 4

Consensus UrtR binding site generated with selected intergenic sequences from UrtR regulatory regions. Top sequence was generated with 60 putative binding sequences whereas bottom panel represents the consensus sequence generated after removing overlapping sequences. WebLogo represents the relative frequency of base pairs at each position.

Figure 5

Many UrtR homologs have multiple binding sites in the shared operator region. Overlapping sites generally share 3 bp, resulting in two cognate sites on opposite faces of the duplex.