Time-resolved proteomic profiling of Cupriavidus metallidurans CH34 in the copper-induced viable-but-nonculturable state.

Copper-based materials are actively explored for their potential as antimicrobial agents. However, recent studies show that sublethal concentrations of Cu ions can induce the viable-but-nonculturable (VBNC) cell state in certain bacteria, hampering contamination control, and monitoring. In this study we contribute to the unravelling of this largely enigmatic phenomenon by determining the time-resolved proteome of Cu-treated Cupriavidus metallidurans CH34 during VBNC induction and resuscitation. High-throughput quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed at multiple sample time points, revealing the cellular adaptations that trigger VBNC formation and the characteristic spontaneous recovery of culturability. Entry into the VBNC state correlated with a widespread response to oxidative stress as well as downregulated pyruvate metabolism. The expression of specific metal resistance determinants changed with Cu exposure time and culminated in the strong upregulation of proteins linked to periplasmic Cu ion detoxification during the resuscitation phase. We suggest that this delayed induction of Cu resistance proteins is paralleled by the gradual reconstitution of energy reserves through metabolization of intracellular polyhydroxybutyrate, as supported by flow cytometric fluorescence measurements. Furthermore, Cu-treated cells showed upregulation of several motility and chemotaxis proteins, and increased cell motility was observed phenotypically. Our results reveal a highly dynamic proteomic response, provide fundamental insights into the VBNC state and emphasize the advantages of time-resolved proteomic analysis.