Suppressor Analysis of the MotB(D33E) Mutation, a Putative Proton-Binding Residue of the Flagellar Motor in Salmonella

摘要: MotA and MotB form the stator of proton-driven bacterial flagellar motor, which conducts protons couples proton flow to motor rotation. Asp-33 Salmonella Typhimurium MotB, is a putative proton-binding site, critical for torque generation. However, how does protonation Asp could drive conformational changes requiring generation largely unknown.Here, we carried out genetic motility analysis slow motile motB(D33E) mutant its pseudorevertants. We first confirmed that poor neither due protein instability, mislocalization nor impaired interaction with MotA. isolated 17 pseudorevertants identified suppressor mutations in transmembrane helices TM2 TM3 TM periplasmic domain MotB. The stall produced by was about half wild type while it recovered nearly wild-type levels their high-speed rotation under low load still significantly impaired.These results together suggested MotB(D33E) mutation reduced both proton-conducting activity involving stator-rotor interactions coupled protonation/deprotonation Glu-33. Furthermore, second-site recover but not activity.Recently, measure pseudorevertants, developed novel system monitor intracellular pH cells overexpressing MotA/MotB proteins utilizing pH-sensitive GFP (pHluorin). Details these will also be discussed.