Copper Efflux System Research Articles

Regulation of copper homeostasis in Pseudomonas fluorescens SBW25

Copper is an essential element for life, but too much copper is harmful: copper homeostasis must therefore be carefully regulated. When growing on plant surfaces, the plant growth-promoting bacterium Pseudomonas fluorescens SBW25 activates expression of a copper-transporting P1-type ATPase (CueA). Using a combination of transcriptional gene fusions and site-directed mutants, we show that copper-induced transcription of cueA is controlled by the MerR-type regulator, CueR; CueR is also required for activation of the copper chaperone protein encoded by cueZ (pflu0660). The promoters of cueA and cueZ are also responsive to the metal salts of gold, silver and mercury. In each case, CueR transduces the stimulus. Resistance to exogenously applied copper sulfate shows that cueA and cueR mutants are significantly less resistant than the wild type. This is consistent with the role of CueA as a copper efflux system and a general role for the CueR regulon in copper resistance. A search of the SBW25 genome for orthologues of genes predicted to play additional roles in copper homeostasis identified copCD of the known four-component copABCD system (unusually, copAB are absent from the SBW25 genome). Genetic studies showed that expression of copCD is controlled by copper and mediated by the CopRS two-component regulatory system. Mutants devoid of copCD or copS displayed an increased tolerance to copper and overexpression of copCD caused increased sensitivity. This is consistent with CopCD encoding a copper uptake system. Taken together, we suggest that the Cue and Cop systems are integral to copper homeostasis in P. fluorescens SBW25 with one (Cop) being active at low-copper environments and bringing copper into the cell, and the other (Cue) being active in high-copper environments and serving to export excess copper.

Two MerR homologues that affect copper induction of the Bacillus subtilis copZA operon.

Copper ions induce expression of the Bacillus subtilis copZA operon encoding a metallochaperone, CopZ, and a CPx-type ATPase efflux protein, CopA. The copZA promoter region contains an inverted repeat sequence similar to that recognized by the mercury-sensing MerR protein. To investigate the possible involvement of MerR homologues in copZA regulation, null mutations were engineered affecting each of four putative MerR-type regulators: yyaN, yraB, yfmP and yhdQ. Two of these genes affected copper regulation. Mutation of yhdQ (hereafter renamed cueR) dramatically reduced copper induction of copZA, and purified CueR bound with high affinity to the copZA promoter region. These results suggest that CueR is a direct regulator of copZA transcription that mediates copper induction. Surprisingly, a yfmP mutation also reduced copper induction of copZA. Sequence analysis suggested that yfmP was cotranscribed with yfmO, encoding a putative multidrug efflux protein. The yfmPO operon is autoregulated: a yfmP mutation derepressed the yfmP promoter and purified YfmP bound the yfmP promoter region, but not the copZA promoter region. Since the yfmP mutant strain was predicted to express elevated levels of the YfmO efflux pump, it was hypothesized that copper efflux might be responsible for the reduced copZA induction. Consistent with this model, in a yfmP yfmO double mutant copper induction of copZA was normal. The results demonstrate the direct regulation of the B. subtilis copper efflux system by CueR, and indirect regulation by a putative multidrug efflux system.

Bacterial heavy metal resistance: new surprises.

Bacterial plasmids encode resistance systems for toxic metal ions including Ag+, AsO2-, AsO4(3-), Cd2+, CO2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, Sb3+, TeO3(2-), Tl+, and Zn2+. In addition to understanding of the molecular genetics and environmental roles of these resistances, studies during the last few years have provided surprises and new biochemical mechanisms. Chromosomal determinants of toxic metal resistances are known, and the distinction between plasmid resistances and those from chromosomal genes has blurred, because for some metals (notably mercury and arsenic), the plasmid and chromosomal determinants are basically the same. Other systems, such as copper transport ATPases and metallothionein cation-binding proteins, are only known from chromosomal genes. The largest group of metal resistance systems function by energy-dependent efflux of toxic ions. Some of the efflux systems are ATPases and others are chemiosmotic cation/proton antiporters. The CadA cadmium resistance ATPase of gram-positive bacteria and the CopB copper efflux system of Enterococcus hirae are homologous to P-type ATPases of animals and plants. The CadA ATPase protein has been labeled with 32P from gamma-32P-ATP and drives ATP-dependent Cd2+ uptake by inside-out membrane vesicles. Recently isolated genes defective in the human hereditary diseases of copper metabolism, Menkes syndrome and Wilson's disease, encode P-type ATPases that are more similar to the bacterial CadA and CopB ATPases than to eukaryote ATPases that pump different cations. The arsenic resistance efflux system transports arsenite, using alternatively either a two-component (ArsA and ArsB) ATPase or a single polypeptide (ArsB) functioning as a chemiosmotic transporter. The third gene in the arsenic resistance system, arsC, encodes an enzyme that converts intracellular arsenate [As (V)] to arsenite [As (III)], the substrate of the efflux system. The three-component Czc (Cd2+, Zn2+, and CO2+) chemiosmotic efflux pump of soil microbes consists of inner membrane (CzcA), outer membrane (CzcC), and membrane-spanning (CzcB) proteins that together transport cations from the cytoplasm across the periplasmic space to the outside of the cell. Finally, the first bacterial metallothionein (which by definition is a small protein that binds metal cations by means of numerous cysteine thiolates) has been characterized in cyanobacteria.