Genes Involved in Copper Homeostasis inEscherichia coli

CueO is involved in copper tolerance.CueO and CopA are both regulated by CueR (13). To determine the role of CueO in copper tolerance, the cueO gene was disrupted. Chromosomal deletions were performed as described by Datsenko and Wanner (4), and the gene of interest was replaced by a chloramphenicol cassette. The ΔcueO::cm cassette was transduced into E. coli W3110 by P1 transduction. The resulting strain,E. coli GR1 (ΔcueO::cm), was slightly more copper sensitive on complex medium than wild-type strain E. coliW3110 (Table 1; Fig.1). At high copper concentrations thecueO-disrupted strain exhibited a distinctive colony morphology: the colonies were small, colorless, and often mucoid. The copper sensitivity of a cueO deletion-containing strain could be complemented by the presence of the cueO gene on plasmid pTYB2::cueO in trans (Table 1). Other metals tested did not have any effect on acueO-disrupted strain (data not shown).

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Fig. 1.

Effect of several mutations on copper tolerance. Growth curves with different CuCl₂ concentrations are shown. Overnight cultures were diluted 1:500 into fresh Luria-Bertani medium, and after 2 h of growth at 37°C, cells were diluted 1:500 into fresh Luria-Bertani medium with the indicated concentrations of CuCl₂. Cell growth was monitored as the optical density at 600 nm after 15 h of incubation at 37°C with shaking. Strains tested include E. coli GR1 ΔcueO::cm (●), E. coli GR6 ΔcusCFBA::cm(▴), E. coli GR10 ΔcueOΔcusCFBA::cm; (♦), and E. coli W3110 (▪).

The cus determinant encodes a copper efflux system.There are at least two chromosomal copper-responsive determinants responsible for copper homeostasis. One is thecus determinant, regulated by a two-component signal transduction system encoded by the cusRS genes (10). However, deletion of the cus determinant failed to result in decreased copper tolerance (Table 1; Fig. 1). Disruption of both cueO andcusCFBA::cm in the mutant E. coli GR10 (ΔcueOΔcusCFBA::cm) resulted in a substantial decrease in copper tolerance. E. coli GR10 was mucoid even at very low copper concentrations, indicating a stress response, and ceased growth at 1.3 mM CuCl₂, compared with 2.75 mM for GR1 (ΔcueO::cm) and 3.5 mM for the wild-type E. coli W3110, E. coliGR5(ΔcusA::cm), and E. coli GR6(ΔcusCFBA::cm) (Table 1). This effect was observed in the presence of copper but not with cadmium, zinc, or cobalt (data not shown). The structural genescusCFBA resemble genes of proton-cation antiporter complexes such as CzcCBA or SilCFBA involved in heavy metal resistance (8, 11). CusA is a member of the RND superfamily of proteins (18) and the central component of the multicomponent efflux pump. No difference was observed whethercusA or cusCFBA was deleted (Table 1), indicating that cusA is essential for function. These results strongly suggest that the cus determinant encodes a copper efflux system. The copper sensitivity conferred by a copAdisruption is not additive when transferred into a ΔcueOΔcusCFBA::cm double mutant, since the triple mutant E. coli GR16 (ΔcueOΔcusCFBA::cm copA::km) did not exhibit a further decrease in copper tolerance.

CusCFBA may not transport Cu(I) from the cytoplasm.The CzcCBA transenvelope transporter from “Ralstonia metallidurans” CH34 is thought to function as a proton-cation antiporter expelling cations from the cytoplasm across the inner and outer membranes. Two channels in CzcA are proposed to form a charge-relay system, where proton transport generates an electrical field that drives the transport of cations into the periplasm (7). However, thecus determinant probably does not transport cytoplasmic Cu(I), since E. coli GR13 (copA::kmΔcusA::cm) was no more sensitive than strain DW3110 (ΔcopA) (Table 1). If CusCFBA extrudes copper from the cytosol, deletion of the cusdeterminant would be expected to have an additive effect on the phenotype of a copA::km mutant. Other transporters of the RND superfamily, such as AcrB and MexB, can efflux substrates that do not cross the cytoplasmic membrane (12). It was therefore suggested that binding of the substrate might occur on the periplasmic side of the transporter (19). CusA contains multiple methionine residues in the second large periplasmic domain. These residues, which are not present in CzcA, could be involved in copper binding. Furthermore, CusF is a putative periplasmic protein with potential copper binding sites. Thus, it is possible that the CusCFBA transenvelope transporter binds and transports periplasmic copper. Since thecus determinant is also responsible for a small increase in Ag(I) resistance (5), it is likely that the transported copper species is Cu(I).

CopA from “R. metallidurans” can functionally substitute for CueO.CueO is homologous to the putative multicopper oxidases PcoA (E. coli), CopA (“R. metallidurans”; accession no. CAC07979) and CopA (Pseudomonas syringae), which are encoded by genes present in the plasmid-borne copper resistance operon pco inE. coli and the cop operons of “R. metallidurans” and P. syringae, respectively (3, 1). It should be pointed out that the “R. metallidurans” and P. syringae CopA proteins are not P-type ATPases, although PcoA, P. syringae CopA, and probably “R. metallidurans” CopA are essential components of their plasmid-encoded copper resistance determinants. PcoA, CopA (P. syringae), and CopA (“R. metallidurans”) are largely identical to each other and probably have similar functions. The degree of similarity between CueO and PcoA, CopA (P. syringae), and CopA (“R. metallidurans”) is much smaller, suggesting that they are distantly related. However, all four putative multicopper oxidases have leader sequences including a twin-arginine motif for export into the periplasm by the Tat pathway (13). CueO has a methionine-rich region that is also observed in PcoA and the CopA proteins from “R. metallidurans” and P. syringae. CopA (P. syringae) has been shown to be a periplasmic protein (2). CopA from R. metallidurans is functionally similar to CueO, sincecopA on plasmid pTYB2::copA can complement the single mutant E. coli GR1 (ΔcueO::cm) (Table 1) and the double mutantE. coli GR10 (ΔcueOΔcusCFBA::cm) (Table 1). Likewise, the double mutant E. coli GR10 (ΔcueOΔcusCFBA::cm) can also be complemented bycueO on a plasmid (Table 1). By analogy, we suggest that CueO and CopA (“R. metallidurans”) are periplasmic proteins. Possibly, CueO prevents uptake of Cu(I) into the cytoplasm by oxidizing it to Cu(II). Additionally, by oxidizing Cu(I) to Cu(II), CueO might confer copper tolerance by preventing oxidative damage in the periplasm. The functions of PcoA, CopA (P. syringae), and CopA (“R. metallidurans”) might be similar.

Another possibility is that the putative multicopper oxidases are secreted into the periplasm with their copper atoms already incorporated, consistent with the presence of the twin-arginine motifs. The synthesis and secretion of these proteins could result in a net efflux of copper from the cytoplasm, as was measured for thepco system (1). However, E. colistrain GR8, where both copA and cueO were disrupted, exhibited only a slight decrease in copper tolerance compared to strain DW3110, which has only copA disrupted (Table 1). One molecule of the homologous CopA protein (P. syringae) was shown to bind approximately 11 atoms of copper (2). It seems unlikely that the amounts of copper transported by CopA (P. syringae) and possibly by CueO would be sufficient to confer copper resistance.

Conclusions.In this report we show that CueO and CusCFBA are involved in copper tolerance. CueO is homologous to multicopper oxidases such as ceruloplasmin, ascorbate oxidase, PcoA, and CopA (P. syringae) and is probably a periplasmic protein. CueO might oxidize Cu(I) to Cu(II) and can be functionally replaced by CopA from “R. metallidurans”. Thecus determinant encodes a copper efflux system that might transport periplasmic Cu(I) [and Ag(I)] across the outer membrane. This suggests that the two determinants provide alternate fates for periplasmic Cu(I): either oxidation by CueO or transport into the extracellular medium by CusCFBA. These studies are a starting point to further elucidate the molecular mechanisms of copper homeostasis in prokaryotes. Further biochemical studies are necessary to understand the function of CusCFBA, CueO, and other, yet-unidentified components.