The Escherichia coli yiiP gene encodes an iron transporter, ferrous iron efflux (FieF), which belongs to the cation diffusion facilitator family (CDF). Transcription of fieF correlated with iron concentration; however, expression appeared to be independent of the ferrous iron uptake regulator Fur. Absence of FieF led to decreased growth of E. coli cells in complex growth medium but only if fur was additionally deleted. The presence of EDTA was partially able to relieve this growth deficiency. Expression of fieF in trans rendered the double deletion strain more tolerant to iron. Furthermore, E. coli cells exhibited reduced accumulation of (55)Fe when FieF was expressed in trans. FieF catalyzed active efflux of Zn(II) in antiport with protons energized by NADH via the transmembrane pH gradient in everted membrane vesicles. Using the iron-sensitive fluorescent indicator PhenGreen-SK encapsulated in proteoliposomes, transmembrane fluxes of iron cations were measured with purified and reconstituted FieF by fluorescence quenching. This suggests that FieF is an iron and zinc efflux system, which would be the first example of iron detoxification by efflux in any organism.

CzcD from Ralstonia metallidurans and ZitB from Escherichia coli are prototypes of bacterial members of the cation diffusion facilitator (CDF) protein family. Expression of the czcD gene in an E. coli mutant strain devoid of zitB and the gene for the zinc-transporting P-type ATPase zntA rendered this strain more zinc resistant and caused decreased accumulation of zinc. CzcD, purified as an amino-terminal streptavidin-tagged protein, . Conserved amino acyl residues that might be involved in binding and transport of zinc were mutated in CzcD and/or ZitB, and the influence on Zn 2؉ resistance was studied. Charged or polar amino acyl residues that were located within or adjacent to membrane-spanning regions of the proteins were essential for the full function of the proteins. Probably, these amino acyl residues constituted a pathway required for export of the heavy metal cations or for import of counter-flowing protons.The cation diffusion facilitators (CDF) (T.C.2.A.4.1.1) (26) are a family of metal transport proteins found in a variety of organisms (16,17,19,23). In contrast to other protein families, such as P-type ATPases or ABC transporters (26), all CDF proteins characterized to date transport only metals, and the majority are involved in Zn 2ϩ transport (3,16,23). One of the first two CDF identified was the CzcD protein (15, 18) from the gram-negative bacterium Ralstonia metallidurans strain CH34 (previously Alcaligenes eutrophus [5]). This transporter is part of a cobalt-zinc-cadmium resistance system (Czc) and decreases the intracellular zinc concentration (1). The CzcD protein is composed of a hydrophobic, membrane-bound domain consisting of about 200 amino acid residues with probably six transmembrane ␣-helices and a 115-amino-acid hydrophilic domain located in the cytoplasm (1).Escherichia coli detoxifies excess Zn 2ϩ by using ZntA, a P-type ATPase, and the CDF protein ZitB (6), which is closely related to CzcD. Previously, site-directed mutagenesis was used to identify amino acyl residues H53, H159, D163, and D186 of ZitB as essential residues (12). These residues were located within predicted transmembrane domains (TMs) of this protein. In this study we compared a variety of additional mutations in ZitB and CzcD to better understand functional aspects of zinc binding and efflux. ZitB-dependent 65 Zn 2ϩ transport into everted membrane vesicles was driven by the proton motive force (PMF). These kinetics suggest that this protein is mainly responsible for zinc homeostasis under most physiological conditions. MATERIALS AND METHODSBacterial strains, growth conditions, and plasmid construction. The media used to cultivate E. coli strains W3110 (wild type) (7), GG48 (⌬zitB::Cm zntA::Km) (6), and GR362 (⌬zntA::Km ⌬zitB ⌬zupT ⌬znuABC ⌬zntB::Cm) (8) were Tris-buffered mineral salts medium (13) containing 2 g of glucose liter Ϫ1 plus 1 g of yeast extract liter Ϫ1 (TGY), the same medium with 2 g of glycerol liter Ϫ1 plus 3 g of Casamino Acids liter Ϫ1 (TGC), and Luria-Bertani broth (LB) (27). Solid Tris-buffered m...

Cation diffusion facilitator (CDF) proteins are a phylogenetically ubiquitous family of intermembrane transporters generally believed to play a role in the homeostasis of a wide range divalent metal cations. CDFs are found in a host of membranes, including the bacterial cell membrane, the vacuolar membrane of both plants and yeast, and the golgi apparatus of animals. As such, they are potentially useful in the engineering of hyperaccumulative phytoremediation systems. While not yet sufficient for reliable biotechnological manipulation, characterization of this family is proceeding briskly. Experimental data suggests that CDFs are generally homodimers that use proton antiport to drive substrate translocation across a membrane. This translocation of both substrate and protons is likely mediated by a combination of histidines, aspartates, and glutamates. Functional data has suggested that CDFs are not limited to metal homeostasis roles, as some appear to be determinants in the operation of high-volume metal resistance systems, and others may facilitate cation-donation as a means of signal transduction. This review seeks to give an overview of the data prompting these conclusions, while presenting additional data whose interpretation is still contentious.

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A flow cytometric method (RAPID-B™) with detection sensitivity of one viable cell of Escherichia coli serotype O157:H7 in fresh spinach (Spinacia oleracea) was developed and evaluated. The major impediment to achieving this performance was mistaking autofluorescing spinach particles for tagged target cells. Following a 5 h non-selective enrichment, artificially inoculated samples were photobleached, using phloxine B as a photosensitizer. Samples were centrifuged at high speed to concentrate target cells, then gradient centrifuged to separate them from matrix debris. In external laboratory experiments, RAPID-B and the reference method both correctly detected E. coli O157:H7 at inoculations of ca. 15 cells. In a follow-up study, after 4 cell inoculations of positives and 6 h enrichment, RAPID-B correctly identified 92% of 25 samples. The RAPID-B method limit of detection (LOD) was one cell in 25 g. It proved superior to the reference method (which incorporated real time-PCR, selective enrichment, and culture plating elements) in accuracy and speed.