Characteristics of Zinc Transport by Two Bacterial Cation Diffusion Facilitators from Ralstonia metallidurans CH34 and Escherichia coli

ABSTRACT 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, bound Zn2+, Co2+, Cu2+, and Ni2+ but not Mg2+, Mn2+, or Cd2+, as shown by metal affinity chromatography. Histidine residues were involved in the binding of 2 to 3 mol of Zn2+ per mol of CzcD. ZitB transported 65Zn2+ in the presence of NADH into everted membrane vesicles with an apparent Km of 1.4 μM and a V max of 0.57 nmol of Zn2+ min−1 mg of protein−1. 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 Zn2+ 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.

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Topology and Boundaries of the Aerotaxis Receptor Aer in the Membrane of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.188.3.894-901.2006 ◽

2006 ◽

Vol 188 (3) ◽

pp. 894-901 ◽

Cited By ~ 25

Author(s):

Divya N. Amin ◽

Barry L. Taylor ◽

Mark S. Johnson

Keyword(s):

Escherichia Coli ◽

Lateral Diffusion ◽

Membrane Vesicles ◽

Membrane Receptors ◽

Disulfonic Acid ◽

Type I ◽

Membrane Anchor ◽

Whole Cells ◽

Membrane Spanning

ABSTRACT Escherichia coli chemoreceptors are type I membrane receptors that have a periplasmic sensing domain, a cytosolic signaling domain, and two transmembrane segments. The aerotaxis receptor, Aer, is different in that both its sensing and signaling regions are proposed to be cytosolic. This receptor has a 38-residue hydrophobic segment that is thought to form a membrane anchor. Most transmembrane prediction programs predict a single transmembrane-spanning segment, but such a topology is inconsistent with recent studies indicating that there is direct communication between the membrane flanking PAS and HAMP domains. We studied the overall topology and membrane boundaries of the Aer membrane anchor by a cysteine-scanning approach. The proximity of 48 cognate cysteine replacements in Aer dimers was determined in vivo by measuring the rate and extent of disulfide cross-linking after adding the oxidant copper phenanthroline, both at room temperature and to decrease lateral diffusion in the membrane, at 4°C. Membrane boundaries were identified in membrane vesicles using 5-iodoacetamidofluorescein and methoxy polyethylene glycol 5000 (mPEG). To map periplasmic residues, accessible cysteines were blocked in whole cells by pretreatment with 4-acetamido-4′-maleimidylstilbene-2, 2′ disulfonic acid before the cells were lysed in the presence of mPEG. The data were consistent with two membrane-spanning segments, separated by a short periplasmic loop. Although the membrane anchor contains a central proline residue that reaches the periplasm, its position was permissive to several amino acid and peptide replacements.

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A Zn(II)-translocating P-type ATPase from Proteus mirabilis

Biochemistry and Cell Biology ◽

10.1139/o98-071 ◽

1998 ◽

Vol 76 (5) ◽

pp. 787-790 ◽

Cited By ~ 15

Author(s):

Christopher Rensing ◽

Bharati Mitra ◽

Barry P Rosen

Keyword(s):

Escherichia Coli ◽

Proteus Mirabilis ◽

Membrane Vesicles ◽

Coli Strain ◽

E Coli ◽

P Type ◽

Zinc Resistance

A mutant of Proteus mirabilis had been previously isolated as defective in swarming. The mutation had been found to be in a gene related to the Escherichia coli zntA gene, which encodes the ZntA Zn(II)-translocating P-type ATPase. In this study the P. mirabilis genewas expressed in an E. coli strain in which the zntA gene had been disrupted. The P. mirabilis gene complemented the sensitivity to salts of zinc and cadmium. Everted membrane vesicles from the zntA-disrupted strain lost ATP-driven 65Zn(II) uptake. Membranes from the complemented strain had restored 65Zn(II) transport. These results demonstrate that the P. mirabilis homologue of ZntA is a Zn(II)-translocating P-type ATPase.Key words: zinc resistance, P-type ATPase, Proteus mirabilis.

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