Bacterial contamination of touch surfaces poses a serious threat for public health. The use of bactericidal surface materials, such as copper and its alloys, might constitute a way to aid the use of antibiotics and disinfectants, thus minimizing the risk of emergence and spread of multiresistant germs. The survival of Escherichia coli on metallic copper surfaces has been studied previously; however, the mechanisms underlying bacterial inactivation on copper surfaces have not been elucidated. Data presented in this study suggest that bacteria are killed rapidly on dry copper surfaces. Several factors, such as copper ion toxicity, copper chelators, cold, osmotic stress, and reactive oxygen species, but not anaerobiosis, influenced killing rates. Strains deleted in copper detoxification systems were slightly more sensitive than was the wild type. Preadaptation to copper enhanced survival rates upon copper surface exposure. This study constitutes a first step toward understanding the reasons for metallic copper surface-mediated killing of bacteria.Copper, both in its metallic and ionic forms, has been exploited empirically since ancient times for medical uses in countless cultures around the globe. Later, the therapeutic powers of this metal were accredited to its antimicrobial properties for the curing of various wound and skin diseases.

The Escherichia coli zupT (formerly ygiE) gene encodes a cytoplasmic membrane protein (ZupT) related to members of the eukaryotic ZIP family of divalent metal ion transporters. Previously, ZupT was shown to be responsible for uptake of zinc. In this study, we show that ZupT is a divalent metal cation transporter of broad substrate specificity. An E. coli strain with a disruption in all known iron uptake systems could grow in the presence of chelators only if zupT was expressed. Heterologous expression of Arabidopsis thaliana ZIP1 could also alleviate iron deficiency in this E. coli strain, as could expression of indigenous mntH or feoABC. Transport studies with intact cells showed that ZupT facilitates uptake of 55 Fe 2؉ similarly to uptake of MntH or Feo. Other divalent cations were also taken up by ZupT, as shown using 57 Co 2؉ . Expression of zupT rendered E. coli cells hypersensitive to Co 2؉ and sensitive to Mn 2؉ . ZupT did not appear to be metal regulated: expression of a ⌽(zupT-lacZ) operon fusion indicated that zupT is expressed constitutively at a low level.

SummaryEscherichia coli possesses multiple routes for iron uptake. Here we present EfeU (YcdN), a novel iron acquisition system of E. coli strain Nissle 1917. Laboratory strains of E. coli such as K12 lack a functional (efeU) ycdN gene caused by a frameshift mutation. EfeU, a member of the oxidase-dependent iron transporters (OFeT), is a homologue of the iron permease Ftr1p from yeast. The ycdN gene is part of the ycdNOB tricistronic operon which is expressed in response to iron deprivation in a Fur-dependent manner. Expression of efeU resulted in improved growth of an E. coli mutant lacking all known ironuptake systems and mediated increased iron uptake into cells. Furthermore, the presence of other divalent metal cations did not impair growth of strains expressing efeU. The EfeU protein functioned as ferrous iron permease in proteoliposomes in vitro. Topology analysis indicated that EfeU is an integral cytoplasmic membrane protein exhibiting seven transmembrane helices. Two REXXE motifs within transmembrane helices of OFeT family members are implicated in iron translocation. Site-directed mutagenesis of each REGLE motif of EfeU diminished iron uptake in vivo and growth yield. In vitro the EfeU variant protein with an altered first REGLE motif was impaired in iron permeation, whereas activity of the EfeU variant with a mutation in the second motif was similar to the wild-type protein.

Escherichia coli excretes the catecholate siderophore enterobactin in response to iron deprivation. While the mechanisms underlying enterobactin biosynthesis and ferric enterobactin uptake and utilization are widely understood, nearly nothing is known about how enterobactin is exported from the cell. Mutant and highperformance liquid chromatography analyses demonstrated that the outer membrane channel tunnel protein TolC but none of the respective seven resistance nodulation cell division (RND) proteins CusA, AcrB, AcrD, AcrF, MdtF (YhiV), or the twin RND MdtBC (YegNO) was essential for enterobactin export across the outer membrane. Mutant E. coli strains with additional deletion of tolC or the major facilitator entS were growth deficient in iron-depleted medium. Strains with deletion of tolC or entS, but not with deletion of genes encoding RND transporters, excreted very little enterobactin into the growth medium. Enterobactin excretion in E. coli is thus probably a two-step process involving the major facilitator EntS and the outer membrane channel tunnel protein TolC. Quantitative reverse transcription-PCR analysis of gene-specific transcripts showed no significant changes in tolC expression upon iron depletion. However, iron starvation led to increased expression of the RND gene mdtF and a decrease in acrD.Enterobactin (33), also known as enterochelin (30), is the catecholate-type siderophore of Escherichia coli and of several other bacteria. Enterobactin, a cyclic triester of 2,3-dihydroxybenzoylserine (DHBS), is one of the most effective ferric iron chelating compounds known (1, 36). While the molecular processes involved in ferric enterobactin uptake by TonB-energized outer membrane receptor proteins such as FepA (recently reviewed in reference 15) were studied exhaustively over the last decades, a mechanism for enterobactin efflux across the cytoplasmic membrane was discovered only recently. EntS (the ybdA gene product) (5), a member of the vast major facilitator superfamily (MFS) of membrane-bound transporters, was shown to be necessary for effective enterobactin export in E. coli (9). Cells with entS deleted excreted very little enterobactin into the surrounding medium, but degradation products of enterobactin were released into supernatants. Since those degradation products are themselves efficient siderophores and were still exported, strains lacking entS suffered no iron depletion (9).Because enterobactin is too big to diffuse freely through the porins of the outer membrane, transport from the periplasm to the outside has to be accomplished by another still unknown transport system. Previously, we and others (18,20,26,27) could demonstrate that transport systems of the resistance nodulation cell division (RND) type (38) may transport their substrates from the periplasm (or from the cytoplasmic membrane in the case of hydrophobic substances) rather than from the cytoplasm to the outside. At least for copper and cobalt, we provided evidence that efflux is probably a two-step process involving a transp...

The ZIP (ZRT-, IRT-like Protein) protein ZupT from Escherichia coli is a transporter with a broad substrate range. Phenotypic and transport analysis showed that ZupT, in addition to Zn(II), Fe(II) and Co(II) uptake, is also involved in transport of Mn(II) and Cd(II). Competition experiments with other substrate cations suggested that ZupT has a slight preference for Zn(II) and kinetic parameters for Zn(II) in comparison to Co(II) and Mn(II) transport support this observation. Metal uptake into cells by ZupT was optimum at near neutral pH and inhibited by ionophores. Bicarbonate or other ions did not influence metal-uptake via ZupT. Amino acid residues of ZupT contributing to substrate specificity were identified by site directed mutagenesis. ZupT with a H89A exchange lost Co(II) and Fe(II) transport activity, while the S117V mutant no longer transported Mn(II). ZupT with E152D was impaired in overall metal uptake but completely lost its ability to transport the substrates Zn(II) and Mn(II). These experimental findings expand our knowledge on the substrate specificity of ZupT and provide further insight into the function of ZupT as a bacterial member of the vastly distributed and important ZIP family.