Metal-induced sensor mobilization turns on affinity to activate regulator for metal detoxification in live bacteria

Abstract

<jats:p>Metal detoxification is essential for bacteria’s survival in adverse environments and their pathogenesis in hosts. Understanding the underlying mechanisms is crucial for devising antibacterial treatments. In the Gram-negative bacterium<jats:italic>Escherichia coli</jats:italic>, membrane-bound sensor CusS and its response regulator CusR together regulate the transcription of the<jats:italic>cus</jats:italic>operon that plays important roles in cells’ resistance to copper/silver, and they belong to the two-component systems (TCSs) that are ubiquitous across various organisms and regulate diverse cellular functions. In vitro protein reconstitution and associated biochemical/physical studies have provided significant insights into the functions and mechanisms of CusS–CusR and related TCSs. Such studies are challenging regarding multidomain membrane proteins like CusS and also lack the physiological environment, particularly the native spatial context of proteins inside a cell. Here, we use stroboscopic single-molecule imaging and tracking to probe the dynamic behaviors of both CusS and CusR in live cells, in combination with protein- or residue-specific genetic manipulations. We find that copper stress leads to a cellular protein concentration increase and a concurrent mobilization of CusS out of clustered states in the membrane. We show that the mobilized CusS has significant interactions with CusR for signal transduction and that CusS’s affinity toward CusR switches on upon sensing copper at the interfacial metal-binding sites in CusS’s periplasmic sensor domains, prior to ATP binding and autophosphorylation at CusS’s cytoplasmic kinase domain(s). The observed CusS mobilization upon stimulation and its surprisingly early interaction with CusR likely ensure an efficient signal transduction by providing proper conformation and avoiding futile cross talks.</jats:p>

A novel copper-sensing two-component system for inducing Dsb gene expression in bacteria

“…In the archetypical Escherichia coli TCS CusRS, Cu + binding to the periplasmic loop of HK CusS promotes its autophosphorylation, and the subsequent phosphorylation of its cognate RR CusR, which then activate the transcription of cusCFBA genes encoding proteins for the Cu + efflux pump CusCFBA to ameliorate copper toxic effects (14)(15)(16)(17)(18)(19). In addition to CusRS, various periplasmic copper-sensing TCSs (e.g., CopRS, PcoRS, and CinRS) have been identified (8,(20)(21)(22)(23), which supports the notion that the bacterial cell envelope is an important target for copper stress (7,24).…”

Mutagenesis and Resistance Development of Bacteria Challenged by Silver Nanoparticles

“…The growth rates were assayed in the absence of AgNPs, normalized by dividing the growth rate of the ancestor. (F) Resistance mechanisms of bacteria upon AgNPs treatment, based on this and previous studies ( 49 – 51 ). P, phosphate group.…”

“…cusS and cusR are known to be associated with silver resistance: the Cus system is the first identified silver resistance system that can transport silver ions into the extracellular space ( 40 , 49 ). ompR is one member of the two-component system EnvZ/OmpR involved in osmoregulation ( 50 ), which may decrease the infiltration of AgNPs into cells.…”

Exploring the mono-/bistability range of positively autoregulated signaling systems in the presence of competing transcription factor binding sites

“…Cellular CusR levels range from ~170 to more than 500 molecules per cell in different measurements [ 29 , 35 , 36 ] while about a dozen CusR binding sites across the genome have been identified [ 37 ]. This suggests that CusR is likely in excess to the binding sites and TFBS competition may not play a significant role in the wild-type system (LG B ≈1).…”

“…This suggests that CusR is likely in excess to the binding sites and TFBS competition may not play a significant role in the wild-type system (LG B �1). The fold change f has been observed to be small with only a modest increase in CusR protein level upon copper exposure [36]. We estimated the value of f to be ~5 based on the PcusR-yfp transcription reporter (S6 Fig) . Considering that the autoregulated cusR promoter contains a single binding site [34], the binding cooperativity h will not likely exceed 2 and LG F will be smaller than 1 given f <9.…”