Cu Homeostasis in Bacteria: The Ins and Outs

Abstract

Copper (Cu) is an essential trace element for all living organisms and used as cofactor in key enzymes of important biological processes, such as aerobic respiration or superoxide dismutation. However, due to its toxicity, cells have developed elaborate mechanisms for Cu homeostasis, which balance Cu supply for cuproprotein biogenesis with the need to remove excess Cu. This review summarizes our current knowledge on bacterial Cu homeostasis with a focus on Gram-negative bacteria and describes the multiple strategies that bacteria use for uptake, storage and export of Cu. We furthermore describe general mechanistic principles that aid the bacterial response to toxic Cu concentrations and illustrate dedicated Cu relay systems that facilitate Cu delivery for cuproenzyme biogenesis. Progress in understanding how bacteria avoid Cu poisoning while maintaining a certain Cu quota for cell proliferation is of particular importance for microbial pathogens because Cu is utilized by the host immune system for attenuating pathogen survival in host cells.

Keywords: P1B-type ATPases; cbb3-type cytochrome c oxidase; copper chaperones; cupric reductase; major facilitator superfamily proteins; metal-sensing transcriptional regulators.

Figure 3

Cu-transport across the outer and inner membranes of bacteria. Bacteria use three established systems for Cu transport across the outer membranes: porins, TonB-dependent transport systems, such as NosA/OprC, and uptake via chalkophores, such as Methanobactin (Mbn), which represent Cu-specific metallophores. Periplasmic Cu is transported into the cytoplasm via the CcoA-like transporter (CalT), which belongs to the major facilitator superfamily. Additional Cu import systems (e.g., YcnJ-like proteins [93]) likely exist in bacteria, but have not been characterized [23]. Mbn is synthesized in the cytoplasm and transported via MbnM into the periplasm and by a so far unknown transporter into the extracellular space. Cu-loaded Mbn is imported back into the periplasm via the TonB-dependent transporter MbnT and bound by the periplasmic protein MbnE. How Cu-Mbn is transported back into the cytosol across the cytoplasmic membrane and how Cu is released from Mbn in the cytosol are unknown. Dashed lines indicate the interaction of the TonB-dependent transporter NosA/OprC and MbnT with the inner membrane ExbB–ExbD–TonB complex. Structures were retrieved from the protein database (PDB) with the following IDs: 2ZFG (OmpF for Porin), 3WDO (YajR for CalT/CcoA), 3j09 (CtaA), 4ZA1 (NosA), 5ZFP (ExbB/ExbD hexameric complex), 5ICQ (MbnE), 1EK9 (OM efflux), 2XJH (Mbn), and depicted using Pymol.

Figure 7

Cryo-electron microscopy structure of the A. fulgidus CopA (PDB 3j09) [239]. (A) CopA contains eight transmembrane helices (H1-H8), four periplasmic loops (PL1-PL4) and three cytoplasmic domains (actuator domain (A-domain), nucleotide-binding domain (N-domain), and phosphorylation-domain (P-domain)). The invariant DKTGT motif of the P-domain is shown in orange. (B) The two transmembrane-metal-binding sites (TM-MBS) of CopA have both trigonal planar coordination geometries and are depicted in magenta (TM-MBS-1; Cys380 (I) and Cys382 (II) in helix 6 and Tyr682 (III) in helix 7)) and blue (TM-MBS-2; Asn 683 (IV) in helix 7, Met711 (V) and Ser715 (VI) in helix 8), respectively. (C) The N-terminus of CopA shows a ferredoxin-like βαββαβ-fold (green) with a typical CxxC motif (N-MBS-2); the two cysteines are highlighted in red. Note, that in the A. fulgidus CopA structure, the N-MBS-1 is not resolved. The structure of CopA was also determined by X-ray crystallography [57].

Figure 1

General view of Cu transport across bacterial membranes. Cu can cross the outer membrane of bacteria via porins and major-facilitator superfamily members, such as CcoA, can import periplasmic Cu into the cytosol. Additional Cu importers likely exist but have not been characterized in detail. Cytosolic Cu is bound by Cu storage proteins, metallothioneines and Cu chaperones, such as CopZ. Cu-chaperones also deliver Cu to P_1B-type ATPases for export into the periplasm. Kinetic differences distinguish CopA1-like ATPases, which are involved in Cu detoxification, and CopA2-like ATPases, which export Cu for cuproenzyme biogenesis. CopA1-like ATPases are the primary interaction partner of CopZ, while the interaction with CopA2-like ATPases is particularly important at low Cu concentrations (dashed arrow). In the periplasm, different types of chaperones transfer Cu either for cuproenzyme biogenesis or for Cu export systems, as with the CusABC system. The structures shown were retrieved from the protein database (PDB) with the following IDs: 2ZFG (OmpF for Porin), 3WDO (YajR for CalT/CcoA), 5NQO (Csp3 for copper storage proteins), 1K0V (CopZ), 3j09 (CopA1, CopA2) 4WBR (ScoI/SenC, chaperones for cuproprotein biogenesis), 2VB2 (CusF, chaperones for Cu detoxification), 3KSS (CusA), 3H94 (CusB), 4K7R (CusC), and are depicted using Pymol.

Figure 2

Examples of Cu binding sites in Cu trafficking proteins and cuproenzymes. Cu-transporting P_1B-type ATPases and Cu chaperones, such as CopZ, contain a linear CxxC Cu binding motif, while in Cu chaperones, e.g., Sco1, a distal histidine residue provides an additional ligand, resulting in a trigonal planar geometry. In the cuproenzyme Cu, Zn superoxide dismutase (SOD), four histidine residues ligate Cu in a square planar geometry. The Cu_A center of cytochrome oxidase displays a tetrahedral geometry with two cysteines, two histidines, one glutamate and one methionine residue. In the CuB center, Cu is ligated by three histidine residues. Chemical structures were generated using ChemDoodle. The figure was adapted and modified from [54].

Figure 4

The major-facilitator-superfamily (MFS)-like Cu importer CcoA from R. capsulatus. (A) Structure of E. coli YajR (PDB 3WDO), which is homologous to R. capsulatus CcoA, but contains a large cytosolic domain, which is absent in CcoA. (B) Schematic representation of the metal-binding site in R. capsulatus CcoA, which is composed of methionine and histidine residues in transmembrane helices 7 and 8. Cysteine residues in the periplasmic loop likely contribute to Cu transport via CcoA.

Figure 5

Schematic depiction of domain-specific copper-reducing enzymes. Eukaryotic cupric reductases are heme-containing, integral membrane proteins, such as Dcytb, or proteins of the Fre- and Steap-families. They contain an extracellular metal-binding site that is primarily used for Fe(III) reduction, but can also reduce Cu(II). Intracellular reductants, such as ascorbic acid or NADPH, serve as electron donors. The bacterial cupric reductase CcoG is specific for Cu(II) and reduces Cu(II) via two iron–sulfur clusters and an unknown physiological electron donor. The only described archaeal cupric reductase is a derivative of the cytosolic CopZ, using an iron–sulfur cluster and an unknown electron donor for reduction.

Figure 6

Cu chaperones and Cu storage proteins. Csp1 and Csp3 are Cu-storage proteins, which mainly differ by the presence of a cleavable Tat-signal sequence in Csp1-type proteins, which are translocated into the periplasm. CupA is a membrane-anchored Cu chaperone that has been identified in several Streptococcus species and which contains two Cu binding sites, S1 and S2. It has been suggested that CupA has also cupric reductase activity. CopZ is the prototype of a cytosolic Cu chaperone and homologous are present in all domains of life. CopZ-like proteins contain a single Cu binding site. SmtA is an example of a bacterial metallothionein and was isolated from Synecococcus sp. SmtA binds multiple Cu or Zn ions via cysteine residue and its structure was solved as Zn-SmtA. Structures were retrieved from the protein database (PDB) with the following IDs: 5FJE (Csp1), 5NQO (Csp3), 4f2e (CupA), 1K0V (CopZ), 1JJD (SmtA), and depicted using Pymol. Cu-binding residues are indicated, and Cu ions are shown as cyan spheres. For details, see the text.

Figure 8

The Post-Albers cycle of P-type ATPases. For details, see text. Cu is shown as cyan sphere.

Figure 9

The CusABC system for Cu export. CusA is an integral membrane protein that binds two CusB copies to form a CusA₃-CusB₆ complex. The methionine triad for Cu/Ag binding in CusA is depicted in the insert. CusC is the outer membrane component that forms a trimer in the outer membrane and interacts with CusB. However, the complete structure of the CusA₃B₆C₃ complex has not been solved yet. The CusA₃B₆C₃ complex can transport Cu from the cytoplasm into the extracellular space but can also receive Cu from the periplasmic Cu chaperone CusF, which binds Cu via methionine and histidine residues (insert). Structures were retrieved from the protein database (PDB) with the following IDs: 3KSS (CusA), 3NE5 (CusBA), 4K7R (CusC), 2VB2 (CusF), and depicted using Pymol.

Figure 10

Periplasmic Cu chaperones and their target cuproproteins. The structures of Cu-chaperones are shown in the left and their corresponding target proteins in the right panels. The respective Cu binding sites are indicated, and Cu ions are shown in cyan. The ScoI-homologue SenC and Cox11 contain a single transmembrane domain of unknown structure indicated by a yellow box. NosZ, which was crystallized as homodimer, contains two Cu binding sites; for the Cu_A center, PccA/PCu_AC and SenC/Sco1 are required, while NosL is involved in the formation of the Cu_Z center. Structures were retrieved from the protein database (PDB) with the following IDs: 4WBR (ScoI/SenC), 2K70 (PCu_AC/PccA), 1SP0 (Cox11), 2HQ3 (NosL), 6WIS (CopG), 4GQZ (CueP), 5DJQ (cbb₃-Cox), 3FYE (aa₃-Cox), 6RZK (NosZ), 3OD3 (CueO), 1EQW (Cu/Zn SOD), and depicted using Pymol.