title: In vitro analysis of essential binding sites on the promoter of the Serratia marcescens spn operon with the quorum-sensing receptor SpnR.
abstract: The N-acylhomoserine lactone (AHL) receptor SpnR is a LuxR family protein that acts as a negative regulator of AHL-dependent quorum sensing (QS). SpnR binds to DNA in Serratia marcescens AS-1 via the spn box; however, the binding affinity of SpnR with the nucleotides on the spn box has not yet been investigated. In this study, we used an spn-box-modified sensor electrode, and quartz crystal microbalance analysis demonstrated a drastic reduction of the uptake of SpnR. The nucleotides G5 and C16 at the AHL-receptor complex-binding site are conserved in Gram-negative bacteria, including the lux box in Vibrio fischeri, the tra box in Agrobacterium tumefaciens, and the spn box in S. marcescens. Indeed, the affinity of SpnR to DNA was reduced to 8% by G5C substitution of the spn box. The affinity of SpnR tagged with maltose-binding protein to the immobilized gene promoter was reduced in the order of C16G and G5C substitutions, which corresponded with previous reports on the lux box. These results suggest that formation of hydrogen bonds at amino acid residues containing guanine at position 5 on a lux-box-like promoter universally contributes to the stability of the receptor complex, whose interaction initiates a sequential QS process in the LuxR family. Biotechnol. Bioeng. 2016;113: 2513-2517. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Innovation Systems Engineering, Graduate School of Engineering, Utsunomiya University, Utsunomiya, Tochigi, Japan.|CREST, Japan Science and Technology Agency, 7-1-2 Yoto, Utsunomiya, Tochigi, 321-8585, Japan.
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Takayama
authors.affiliation: CREST, Japan Science and Technology Agency, 7-1-2 Yoto, Utsunomiya, Tochigi, 321-8585, Japan. katon@cc.utsunomiya-u.ac.jp.|Department of Material and Environmental Chemistry, Graduate School of Engineering, Utsunomiya University, 7-1-2 Yoto, Utsunomiya, Tochigi, 321-8585, Japan. katon@cc.utsunomiya-u.ac.jp.
authors.forename: Norihiro
authors.initials: N
authors.lastname: Kato
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001665:Binding Sites; D009876:Operon / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D053038:Quorum Sensing / Q000235:genetics*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D012706:Serratia marcescens / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D015534:Trans-Activators
keywords: DNA-protein interaction; LuxR family; biomolecular engineering; quartz crystal microbalance; quorum sensing