SummaryCupriavidus metallidurans CH34 possesses a multitude of metal efflux systems. Here, the function of the novel PIB4-type ATPase CzcP is characterized, which belongs to the plasmid pMOL30-mediated cobaltzinc-cadmium (Czc) resistance system. Contribution of CzcP to transition metal resistance in C. metallidurans was compared with that of three PIB2-type ATPases (CadA, ZntA, PrbA) and to other efflux proteins by construction and characterization of multiple deletion mutants. These data also yielded additional evidence for an export of metal cations from the periplasm to the outside of the cell rather than from the cytoplasm to the outside. Moreover, metal-sensitive Escherichia coli strains were functionally substituted in trans with CzcP and the three PIB2-type ATPases. Metal transport kinetics performed with inside-out vesicles identified the main substrates for these four exporters, the Km values and apparent turn-over numbers. In combination with the mutant data, transport kinetics indicated that CzcP functions as 'resistance enhancer': this PIB4-type ATPase exports transition metals Zn 2+ , Cd 2+ and Co 2+ much more rapidly than the three PIB2-type proteins. However, a basic resistance level has to be provided by the PIB2-type efflux pumps because CzcP may not be able to reach all different speciations of these metals in the cytoplasm.
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...
Cupriavidus metallidurans is adapted to high concentrations of transition metal cations and is a model system for studying metal homeostasis in difficult environments. The elemental composition of C. metallidurans cells cultivated under various conditions was determined, revealing the ability of the bacterium to shield homeostasis of one essential metal from the toxic action of another. The contribution of metal uptake systems to this ability was studied. C. metallidurans contains three CorA members of the metal inorganic transport (MIT) protein family of putative magnesium uptake systems, ZupT of the ZRT/IRT protein, or ZIP, family, and PitA, which imports metal phosphate complexes. Expression of the genes for all these transporters was regulated by zinc availability, as shown by reporter gene fusions. While expression of zupT was upregulated under conditions of zinc starvation, expression of the other genes was downregulated at high zinc concentrations. Only corA 1 expression was influenced by magnesium starvation. Deletion mutants were constructed to characterize the contribution of each system to transition metal import. This identified ZupT as the main zinc uptake system under conditions of low zinc availability, CorA 1 as the main secondary magnesium uptake system, and CorA 2 and CorA 3 as backup systems for metal cation import. PitA may function as a cation-phosphate uptake system, the main supplier of divalent metal cations and phosphate in phosphate-rich environments. Thus, metal homeostasis in C. metallidurans is achieved by highly redundant metal uptake systems, which have only minimal cation selectivity and are in combination with efflux systems that "worry later" about surplus cations.Sophisticated cellular biochemistry needs metals as cofactors. About 40% of all enzymes have them, ranking from Mg (16%) Ͼ Zn (9%) Ͼ Fe (8%) Ͼ Mn (6%) Ͼ Ca (2%) Ͼ Co and Cu (1%) down to K, Na, Ni, V, Mo, W, and only one example of Cd (59). It is an interesting question how the correct metal is allocated to the right protein, a challenge especially for the divalent metal cations Mg 2ϩ , Zn 2ϩ , Fe 2ϩ , Mn 2ϩ , Co 2ϩ , Ni 2ϩ , and Cu 2ϩ . These metals compete with each other for the metal binding sites in enzymes (16). Additionally, Fe 2ϩ/3ϩ and Cu ϩ/2ϩ promote dangerous reactive oxygen species in Fenton and Fenton-like reactions, as described by Haber and Weiss (13).Part of the solution to this problem might be to keep the metal cation bouquet in any cellular compartment in a way that minimizes competition for metal binding sites and the Fenton reaction. This leads to the question of how the cellular metal cation bouquet can be maintained in environments that may contain a single metal in a concentration range from pM to mM. The betaproteobacterium Cupriavidus metallidurans strain CH34 is able to keep its metal homeostasis under a variety of such adverse conditions (19,28,30). The organism can be found in many mesophilic metal-contaminated environments around the globe, such as zinc deserts of Belgium (8). Key to this a...
Background: Metal selectivity is an important feature of the plant cation diffusion facilitator (CDF) transporter family. Results: Mutation of key residues can narrow or broaden metal selectivity. Conclusion: Residues within the cytoplasmic histidine-rich loop and transmembrane domains define metal specificity. Significance: This raises the possibility of engineering transporters for selective biofortification of cereal grains with nutritionally essential metals.
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