Jean-Yves Matroule

Antibiotic (AB) resistance often arises through mutations that alter AB targets, reduce membrane permeability, or increase efflux activity, yet its physiological costs remain poorly understood. In Caulobacter vibrioides, a ΔtipR mutant that constitutively upregulates the RND efflux pump AcrAB₂NodT exhibits heightened sensitivity to copper (Cu), revealing a trade-off linked to excessive pump expression. Deleting acrAB₂nodT or impairing its transport activity restored cell envelope integrity and, ultimately, Cu resistance, uncoupling the effects of pump abundance from active efflux. The ΔtipR mutant also showed increased sensitivity to other metals, including zinc, nickel, and cadmium, indicating a broader vulnerability to metal stress. Mechanistically, pump overexpression depleted the proton motive force and lowered ATP levels, key processes for metal resistance. These findings uncover the physiological cost of efflux upregulation and suggest a potential therapeutic vulnerability: targeting the metabolic burden of resistance mechanisms could improve antibiotic efficacy by exploiting fitness trade-offs.

Copper (Cu) is an essential micronutrient that serves as a cofactor for many enzymes but becomes toxic when present in excess. In most bacteria, CopA-like P1B-type ATPases mediate Cu detoxification by exporting cytoplasmic Cu to the periplasm or extracellular environment. In this study, we show that Caulobacter vibrioides lacks a canonical CopA-like ATPase but encodes a single FixI/CcoI-type Cu-transporting ATPase, previously implicated in Cu delivery to the cbb □-type cytochrome c oxidase (Cox) in species such as Rhodobacter capsulatus . C. vibrioides harbors two terminal cytochrome c oxidases in its cytoplasmic membrane: an aa □-type and a cbb □-type Cox. We also demonstrate that the activity of cbb □-Cox requires the FixI-type Cu transporter and the periplasmic Cu chaperone PccA. In contrast, aa □-Cox activity depends on PccA and the inner membrane-bound protein CtaG. Since the mechanism of Cu acquisition for aa □-Cox remains largely unknown, we conducted a genetic screen and identified a novel outer membrane TonB-dependent receptor (TccA) that is specifically required for aa □-Cox function. We also showed that cbb □-Cox is upregulated under microaerobic conditions, possibly such as those encountered on solid media where O ₂ diffusion is limited. Under normoxic conditions, the expression and the activity of cbb □-Cox decrease, and aa □-Cox becomes the predominant terminal oxidase. These findings demonstrate that C. vibrioides differentially utilizes its Cox enzymes in response to O ₂ availability and relies on a distinct Cu trafficking pathway for their maturation, including an outer membrane component that has not been previously described in bacterial Cu homeostasis.

Bacterial resistance to antibiotics (AB) such as β-lactams, fluoroquinolones, and aminoglycosides often emerges through mutations that alter AB targets, reduce membrane permeability, or increase the activity of AB-modifying enzymes and efflux pumps. Yet the physiological costs associated with AB resistance remain poorly understood. In Caulobacter vibrioides , a Δ tipR mutant that which constitutively upregulates the RND pump AcrAB ₂ nodT, displays heightened sensitivity to copper (Cu), revealing a physiological vulnerability driven by the energetic burden imposed by excessive efflux activity. Deletion of acrAB ₂ nodT in the Δ tipR background restored Cu resistance to wild-type levels, confirming the role of pump overexpression in metal sensitivity. Morphological and microscopy analyses revealed that pump overexpression leads to cell envelope defects and compromised fitness. To disentangle the effects of pump abundance from efflux activity, we engineered an AcrB ₂ transport-impaired mutant. This variant also rescued Cu resistance, demonstrating that high expression and active transport contribute to the observed toxicity. Notably, this sensitivity was not limited to Cu; the Δ tipR mutant also exhibited increased susceptibility to other transition metals, including zinc (Zn), nickel (Ni) and cadmium (Cd), suggesting a broader vulnerability linked to metal stress. Mechanistically, pump overexpression depleted the proton motive force, reduced ATP levels, and impaired motility, all of which are essential for Cu stress adaptation. This physiological tradeoff highlights the importance of precise efflux regulation and reveals a potential therapeutic vulnerability: targeting the cost of pump upregulation could enhance the efficacy of antimicrobial treatments. Importance: While efflux pumps primarily protect bacteria from AB, their excessive activity can impose a fitness cost. This study underscores a crucial tradeoff between resistance and cellular fitness by connecting Cu sensitivity to heightened AcrAB ₂ NodT efflux pump expression in Caulobacter vibrioides . This work challenges the assumption that more efflux always benefits the cell and shows that unregulated pump activity can undermine survival under metal stress. These findings broaden our understanding of bacterial stress physiology and suggest new ways to combat antimicrobial resistance by targeting the hidden costs of resistance mechanisms. Understanding this balance between resistance and fitness opens new perspectives for combination therapy against multidrug-resistant bacteria.

Metals like copper (Cu), zinc, and nickel exhibit dual nature, necessitating a tight regulation of their cellular homeostasis to meet physiological demands while preventing toxicity. In bacteria, metal homeostasis involves inner membrane (IM) P-type ATPases and ABC transporters, envelope-spanning tripartite efflux pumps, and outer membrane (OM) pore-forming proteins. Four decades ago, the OM β-barrel protein PcoB was shown to provide an additional layer of Cu resistance in an Escherichia coli strain isolated from the gut of swine fed with Cu supplements. Interestingly, most PcoB homologs contain a poorly conserved disordered N-terminal domain (NTD) rich in histidine (His) and methionine (Met) residues, which are commonly associated with Cu coordination in cuproproteins. This suggests a potential role for the NTD in PcoB-mediated copper efflux. We previously demonstrated that the free-living bacterium Caulobacter vibroides primarily relies on PcoB for Cu homeostasis. Here, we show that the NTD of C. vibroides PcoB is critical for PcoB function and stability, tolerating the swapping with the poorly conserved E. coli PcoB NTD and significant truncations. Unexpectedly, the predicted signal peptide (SP) was dispensable, challenging traditional concepts of protein translocation mechanisms. Moreover, the PcoB NTD plays a surprising role in stabilizing the periplasmic multicopper oxidase PcoA, encoded within the same operon as PcoB, highlighting a new role for an intrinsically disordered region (IDR). Importance: Bacterial copper (Cu) homeostasis is essential for survival in fluctuating environments, yet the role of single outer membrane proteins in this process remains poorly characterized. Our study reveals that the intrinsically disordered N-terminal region of the β-barrel protein PcoB in Caulobacter vibroides plays a critical role in Cu efflux and PcoB stability. Remarkably, this region harbors an atypical signal peptide and contributes to the stability of the periplasmic multicopper oxidase PcoA, suggesting a novel regulatory function for an intrinsically disordered region. These findings challenge existing paradigms of protein targeting and homeostasis, with broad implications for understanding bacterial adaptation to stress.

Copper (Cu) is potentially threatening for living organisms owing to its toxicity at high concentrations, requiring the onset of diverse detoxification strategies to maintain fitness. We previously showed that the environmental conditions modulate the response of the oligotrophic alphaproteobacterium Caulobacter crescentus to Cu excess. In the present study, we investigated the role of the Fe-importing TonB-dependent receptor (TBDR) CciT and its partner, CciO, a 2-oxoglutarate/Fe ²⁺ -dependent oxygenase, in Cu resistance. CciT is specifically involved in Cu resistance in both rich and poor media. Using inductively coupled plasma optical emission spectrometry, we found that under Cu stress, the cellular Cu content is reduced by overexpression of cciT , while the Fe content increases. Mutations of the three known Fe-importing TBDRs reveal that CciT is the primary Fe importer in these conditions and the only TBDR required for Cu resistance. In addition, the extracellular Fe concentration is positively correlated with the cellular Fe content and negatively correlated with the cellular Cu content, resulting in the protection of the cells against Cu excess. The operon organization of cciT and cciO is highly conserved across bacteria, indicating a functional link between the two proteins. Deletion of cciT , cciO , or both genes leads to similar Cu sensitivity. Catalytic mutations in CciT and CciO also result in Cu sensitivity. While CciO is not required for Cu and Fe transport, its precise function remains unknown. Overall, this study provides new insights into the role of Fe uptake in Cu resistance, emphasizing the critical influence of environmental conditions on bacterial physiology. IMPORTANCE: Copper is an essential metal for many living organisms, as it helps to drive crucial chemical reactions. However, when present in excess, copper turns toxic due to its high reactivity with biological molecules. Bacteria may encounter excess copper in various environments, such as polluted soils, agricultural copper treatments, and within the vacuoles of infected macrophages. In this study, we investigated the copper response in the environmental bacterium Caulobacter crescentus . Our findings reveal that environmental iron levels play a critical role in copper resistance, as increased iron prevents cellular copper accumulation and toxicity. We identified two essential proteins, CciT and CciO, that are involved in iron transport, providing protection against copper excess.

Copper (Cu) is potentially threatening for living organisms owing to its toxicity at high concentrations, requiring the onset of diverse detoxification strategies to maintain fitness. We previously showed that the environmental conditions modulate the response of the oligotrophic alphaproteobacterium Caulobacter crescentus to Cu excess. In the present study, we investigated the role of the Fe-importing TonB-dependent receptor (TBDR) CciT and its partner, CciO, a 2-oxoglutarate/Fe ²⁺ -dependent oxygenase, in Cu resistance. CciT is specifically involved in Cu resistance in both rich and poor media. Using inductively coupled plasma optical emission spectrometry, we found that under Cu stress, the cellular Cu content is reduced by overexpression of cciT , while Fe content increases. Mutations of the three known Fe-importing TBDRs reveal that CciT is the primary Fe importer in these conditions and the only TBDR required for Cu resistance. In addition, the extracellular Fe concentration is positively correlated with the cellular Fe content and negatively correlated with the cellular Cu content, resulting in the protection of the cells against Cu excess. The operon organization of cciT and cciO is highly conserved across bacteria, indicating a functional link between the two proteins. Deletion of cciT , cciO , or both genes leads to similar Cu sensitivity. Catalytic mutations in CciT and CciO also result in Cu sensitivity. While CciO is not required for Cu and Fe transport, its precise function remains unknown. Overall, this study provides new insights into the role of Fe uptake in Cu resistance, emphasizing the critical influence of environmental conditions on bacterial physiology. Importance: Copper is an essential metal for many living organisms, as it helps to drive crucial chemical reactions. However, when present in excess, copper turns toxic due to its high reactivity with biological molecules. Bacteria may encounter excess copper in various environments, such as polluted soils, agricultural copper treatments, and within the vacuoles of infected macrophages. In this study, we investigated the copper response in the environmental bacterium Caulobacter crescentus . Our findings reveal that environmental iron levels play a critical role in copper resistance, as increased iron prevents cellular copper accumulation and toxicity. We identified two essential proteins, CciT and CciO, that are involved in iron transport, providing protection against copper excess.

Chemotaxis is a widespread strategy used by unicellular and multicellular living organisms to maintain their fitness in stressful environments. We previously showed that bacteria can trigger a negative chemotactic response to a copper (Cu)-rich environment. Cu ions toxicity on bacterial cell physiology has been mainly linked to mismetallation events and ROS production, although the precise role of Cu-generated ROS remains largely debated. Here, we found that the cytoplasmic Cu ions content mirrors variations of the extracellular Cu ions concentration and triggers a dose-dependent oxidative stress, which can be abrogated by superoxide dismutase and catalase overexpression. The inhibition of ROS production in the cytoplasm not only improves bacterial growth but also impedes Cu-chemotaxis, indicating that ROS derived from cytoplasmic Cu ions mediate the control of bacterial chemotaxis to Cu. We also identified the Cu chemoreceptor McpR, which binds Cu ions with low affinity, suggesting a labile interaction. In addition, we demonstrate that the cysteine 75 and histidine 99 within the McpR sensor domain are key residues in Cu chemotaxis and Cu coordination. Finally, we discovered that in vitro both Cu(I) and Cu(II) ions modulate McpR conformation in a distinct manner. Overall, our study provides mechanistic insights on a redox-based control of Cu chemotaxis, indicating that the cellular redox status can play a key role in bacterial chemotaxis.