Membrane topology of the ZntB efflux system of Salmonella enterica serovar Typhimurium

Abstract

The membrane topology of the ZntB Zn(2+) transport protein of Salmonella enterica serovar Typhimurium was determined by constructing deletion derivatives of the protein and genetically fusing them to blaM or lacZ cassettes. The enzymatic activities of the hybrid proteins indicate that ZntB is a bitopic integral membrane protein consisting largely of two independent domains. The first 266 amino acids form a large, highly charged domain within the cytoplasm, while the remaining 61 residues form a small membrane domain containing two membrane-spanning segments. The overall orientation towards the cytoplasm is consistent with the ability of ZntB to facilitate zinc efflux.

FIG. 1.

Charge distribution and hydropathy profile of the ZntB sequence. (A.) Regions of positive and negative charges were calculated by summing pK values of amino acids within a 15-residue sliding window. Regions with positive charge density are shaded grey, while regions with negative charge are shaded black. (B.) Hydropathy was determined by the algorithm of Kyte and Doolittle using a sliding window of 15 amino acids. Regions predicted to form transmembrane (TM) structures are denoted by open boxes.

FIG. 2.

Model of the membrane topology of ZntB. Individual amino acids are depicted in single-letter code. Positively charged residues are enclosed in grey circles; negatively charged residues are enclosed in black circles. Locations of BlaM and LacZ fusions are labeled. The text following each fusion indicates the phenotype of the strain containing the plasmid encoding the chimera.