The CXXC Motifs Are Essential for the Function of BosR in Borrelia burgdorferi.

Charlotte Mason,Spoorthy Prabhudeva,Xiaoyan Liu,Zhiming Ouyang

doi:10.3389/fcimb.2019.00109

Abstract

BosR, a Fur family member, is essential for the pathogenesis of the Lyme disease pathogen, Borrelia burgdorferi. Unlike typical Fur proteins in which DNA binding represses gene expression, binding of BosR to the rpoS promoter directly activates rpoS transcription in B. burgdorferi. However, virtually nothing is known concerning potential structural features and amino acid residues of BosR that are important for protein function and virulence regulation in B. burgdorferi. Particularly, it remains unknown what structural motifs or residues of BosR coordinate Zn, although previous analyses have indicated that the function of BosR may depend on Zn. To address these information gaps, we herein introduced mutations into four conserved cysteine residues in two putative CXXC motifs of BosR. Our data showed that the ability of BosR to bind Zn was dramatically reduced when the CXXC motifs were mutated. Moreover, we found that the two CXXC motifs contributed to the ability of BosR to form dimers. By using a trans-complementation genetic approach, we additionally demonstrated that both CXXC motifs of BosR were essential for in vivo gene expression regulation. Mutation of any of the four cysteines abolished the transcriptional activation of rpoS. In contrast to wild type BosR, each mutant protein was incapable of binding the rpoS promoter in electrophoretic mobility shift assays. The combined data strongly support that the two CXXC motifs and four cysteines constitute the structural site essential for Zn-coordination, protein dimerization, and the unique regulatory activity of BosR.

Highlights

Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most prevalent vector-borne disease in the United States (Mead, 2015; Rosenberg et al, 2018)
Escherichia coli strains were cultured in lysogeny broth (LB) medium supplemented with appropriate antibiotics at the following concentrations: ampicillin, 100 μg/ml; kanamycin, 50 μg/ml; or spectinomycin, 100 μg/ml
The CXXC motif of Vibrio cholerae Fur protein (VcFur) is not involved in metal binding and gene regulation (Sheikh and Taylor, 2009)

Summary

Introduction

Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most prevalent vector-borne disease in the United States (Mead, 2015; Rosenberg et al, 2018). Like all other bacterial σ54 systems, activation of σ54dependent rpoS transcription in B. burgdorferi requires an AAA+ activator ATPase called Rrp (Yang et al, 2003; Burtnick et al, 2007; Boardman et al, 2008; Ouyang et al, 2008, 2014b; Blevins et al, 2009; Groshong et al, 2012; Yin et al, 2016; Ouyang and Zhou, 2017). Different from other σ54 systems, Rrp alone is not enough for activating rpoS transcription in B. burgdorferi Rather, another activator called BosR is crucially required for σ54-dependent rpoS transcription (Hyde et al, 2009, 2010; Ouyang et al, 2009b, 2011; Katona, 2015). BosR strongly binds to BS1 and BS2 sites in the rpoS promoter region via a novel DNA element termed BosR box (Ouyang et al, 2011, 2014a), supporting that BosR directly binds to the rpoS promoter and activates gene expression

Methods

Results

Conclusion