Crystal Structure of SmcR, a Quorum-sensing Master Regulator of Vibrio vulnificus, Provides Insight into Its Regulation of Transcription

Abstract

Quorum sensing has been implicated as an important global regulatory system controlling the expression of numerous virulence factors in bacterial pathogens. SmcR, a homologue of Vibrio harveyi LuxR, has been proposed as a quorum-sensing master regulator of Vibrio vulnificus, an opportunistic human pathogen. Previous studies demonstrated that SmcR is essential for the survival and pathogenesis of V. vulnificus, indicating that inhibiting SmcR is an attractive approach to combat infections by the bacteria. Here, we determined the crystal structure of SmcR at 2.1 Å resolution. The protein structure reveals a typical TetR superfamily fold consisting of an N-terminal DNA binding domain and a C-terminal dimerization domain. In vivo and in vitro functional analysis of the dimerization domain suggested that dimerization of SmcR is vital for its biological regulatory function. The N-terminal DNA recognition and binding residues were assigned based on the protein structure and the results of in vivo and in vitro mutagenesis experiments. Furthermore, protein-DNA interaction experiments suggested that SmcR may have a sophisticated mechanism that enables the protein to recognize each of its many target operators with different affinities.

Structural insights into the regulation of sialic acid catabolism by the Vibrio vulnificus transcriptional repressor NanR

“…To assess the importance of the interaction between ManNAc-6P and NanR during the regulation of nan genes, the residues involved in ligand binding were mutated, and their effects on the function of NanR were investigated by using an E. coli dual plasmid system (22). Cells were cotransformed with plasmids that contained a luciferase reporter gene fused to the NanRbinding nanT PSL AR promoter (P nanTp ) and wild-type (WT) or mutant NanR, followed by incubation in the presence of arabinose and in the presence or absence of Neu5Ac.…”

“…3) containing arabinose (0.002%), and incubated at 37°C until the cells reached the early exponential phase. The relative luminescence unit was calculated by dividing the luminescence by the A 600 , as described (22). To screen the ligand-sensing residues, the BSE1201 strain (ΔaraBAD ΔnanE) was used as the host cell instead of DH5α, which ensured that arabinose was not used as a carbon source and that the ManNAc-6P generated from Neu5Ac was accumulated within the cell.…”

“…Site-directed mutations were introduced into this plasmid by using a QuikChange Site-Directed Mutagenesis Kit (Agilent). The WT or mutant nanR genes were then subcloned into the NcoI and XhoI sites of the pBAD-24BS (22) or pHis-parallel1 (33) expression vector to yield the pNB− and pNH− plasmids, respectively. The R57A and H163L mutant nanR genes were subcloned into the SphI and SpeI sites of the pDM4 forms pBS1208 and 1209, respectively.…”

“…Purified His-NanR protein was used to raise a primary polyclonal antibody by immunizing European rabbits (Oryctolagus curiculus) with a primary injection that contained 500 μg of protein, followed by three boosters that contained 200 μg of protein at 2-wk intervals. Western blotting was performed as described (22). The EMSA and DNaseI footprinting assays were performed according to published procedures (20).…”

Distinct Characteristics of Two 2-Cys Peroxiredoxins of Vibrio vulnificus Suggesting Differential Roles in Detoxifying Oxidative Stress

“…The amplified PCR products were cloned into a His 6 tag expression vector, pHIS-parallel1, containing a recombinant tobacco etch virus protease cleavage site (11), resulting in pBANG1105 (for prx1), pBANG1106 (for prx2), pBANG1104 (for trxA), and pBANG1016 (for ahpFNTD) ( Table 1). Proteins were expressed, purified, and His 6 tags were removed from the proteins using the recombinant tobacco etch virus protease as described elsewhere (12).…”