An important role for periplasmic storage inPseudomonas aeruginosacopper homeostasis revealed by a combined experimental and computational modeling study

Abstract

Biological systems require precise copper homeostasis enabling metallation of cuproproteins while preventing metal toxicity. In bacteria, sensing, transport, and storage molecules act in coordination to fulfill these roles. However, there is not yet a kinetic schema explaining the system integration. Here, we report a model emerging from experimental and computational approaches that describes the dynamics of copper distribution in Pseudomonas aeruginosa. Based on copper uptake experiments, a minimal kinetic model describes well the copper distribution in the wild-type bacteria but is unable to explain the behavior of the mutant strain lacking CopA1, a key Cu efflux ATPase. The model was expanded through an iterative hypothesis-driven approach, arriving to a mechanism that considers the induction of compartmental pools and the parallel function of CopA and Cus efflux systems. Model simulations support the presence of a periplasmic copper storage with a crucial role under dyshomeostasis conditions in P. aeruginosa. Importantly, the model predicts not only the interplay of periplasmic and cytoplasmic pools but also the existence of a threshold in the concentration of external copper beyond which cells lose their ability to control copper levels.

The interplay of the metallosensor CueR with two distinct CopZ chaperones defines copper homeostasis in Pseudomonas aeruginosa

“…Treatment of cells for 10 min with 0.5 mM CuSO 4 was chosen to examine effects on the overall Cu homeostasis. We have shown that these conditions raise cellular Cu but do not affect cellular growth rates (15,16). Fig.…”

“…8B). In bacteria, there is a strict cellular Cu quota, about 10 4 atoms per cell (12), half or less are expected to be in the cytoplasm (16). These numbers correlate well with the amount of CopZ2 encountered in the cytoplasm during steady state, 40 fmol of CopZ2/g of Cytoplasmic Cu ؉ distribution in P. aeruginosa total protein.…”

“…Genome-wide transcriptomic analysis revealed the presence of cytoplasmic (CueR) and periplasmic (CopS/R) Cu ϩ sensing regulators, their corresponding regulons, multiple metal chaperones, and specific Cu ϩ efflux and influx systems bridging the membranes separating cellular compartments (15). This was later complemented by mathematical simulation of fluxes and compartmental pools describing the experimental metal uptake kinetics (16). These computational models support novel homeostatic elements revealed by the architecture of the CueR and CopS/R regulons; for instance, the participation of the CusCBA system mobilizing cytoplasmic Cu ϩ , or the significant role that periplasmic proteins might play in the response to external Cu 2ϩ .…”

The Cu chaperone CopZ is required for Cu homeostasis in Rhodobacter capsulatus and influences cytochrome cbb3 oxidase assembly

“…Copper homeostasis depends on an intricate network of Cu binding proteins that serve as sensors, chaperones, transporters and dedicated cuproprotein assembly factors (Cobine et al , ; Singleton and Le Brun, ; Arguello et al , ; Stewart et al , ). In combination with Cu storage proteins, initially found only in eukaryotes but now also identified in bacteria (Dennison et al , ; Parmar et al , ), these proteins basically reduce the concentration of free Cu below the detection limit and thus prevent potential toxicity. CopZ‐like Cu‐chaperones have been identified as major determinants for Cu detoxification because they can bind Cu with femtomolar affinity (Xiao et al , ) and can transfer it to P 1B ‐type ATPases for extrusion.…”

“…Copper homeostasis depends on an intricate network of Cu binding proteins that serve as sensors, chaperones, transporters and dedicated cuproprotein assembly factors (Cobine et al, 2006;Singleton and Le Brun, 2007;Arguello et al, 2013;Stewart et al, 2018). In combination with Cu storage proteins, initially found only in eukaryotes but now also identified in bacteria (Dennison et al, 2018;Parmar et al, 2018), these A. The cbb 3 -Cox activity in R. capsulatus wild type and mutant cells was determined by the NADI reaction on MPYE plates.…”