Abstract
Because copper ions are both essential cofactors and cytotoxic agents, the net accumulation of this element in a cell must be carefully balanced. Depending upon the cellular copper status, copper ions must either be imported or ejected. CopA, the principal copper efflux ATPase in Escherichia coli, is induced by elevated copper in the medium, but the copper-sensing regulatory factor is unknown. Inspection of the copA promoter reveals signature elements of promoters controlled by metalloregulatory proteins in the MerR family. These same elements are also present upstream of yacK, which encodes a putative multi-copper oxidase. Homologues of YacK are found in copper resistance determinants that facilitate copper efflux. Here we show by targeted gene deletion and promoter fusion assays that both copA and yacK are regulated in a copper-responsive manner by the MerR homologue, ybbI. We have designated ybbI as cueR for the Cu efflux regulator. This represents the first example of a copper-responsive regulon on the E. coli chromosome and further extends the roles of MerR family members in prokaryotic stress response.
Highlights
Escherichia coli, like most microbes, require a minimal level of copper for insertion into metabolic and respiratory enzymes
Whereas few direct homologues of known copper homeostasis proteins have been found in E. coli, copper export systems have been identified, including the plasmidencoded copper resistance determinants [4]
Numerous attempts have been made to identify the genes involved in controlling copper levels in E. coli; few of these genes have been directly linked to copper metabolism, transport, or regulation [5, 6]
Summary
Plasmid Construction and Media—All polymerase chain reactions (PCR) described used E. coli DH5␣ chromosomal DNA as the template unless otherwise stated. -galactosidase assays were conducted in chemically defined medium consisting of 1ϫ A medium (7.6 mM (NH4)2SO4, 33 mM KH2PO4, 60 mM K2HPO4, and 1.7 mM sodium citrate) [15] supplemented with 40 g/ml of all 20 essential L-amino acids (Sigma), 0.2% glucose, 1 mM MgSO4, and 5 ϫ 10Ϫ5% thiamine. All media components except thiamine and MgSO4 were incubated overnight with 50 g/liter Chelex 100 resin (BioRad) to remove trace metals, mixed and sterile-filtered before use. For construction of the ybbI null strain, alternative media were used at various steps in accordance with the published methods [16]. Primer Extension Analysis—Wild-type DH5␣ and DLG (⌬cusRS) [9] were grown in LB to exponential phase.
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