Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties.
Alginate-producing (mucoid) strains of Pseudomonas aeruginosa possess a 54-kDa outer membrane (OM) protein (AlgE) which is missing in nonmucoid bacteria. The coding region of the algE gene from mucoid P. aeruginosa CF3/M1 was subcloned in the expression vector pT7-7 and expressed in Escherichia coli. The level of expression of recombinant AlgE was seven times higher than that of the native protein in P. aeruginosa. Recombinant AlgE was found mainly in the OM. A putative precursor protein (56 kDa) of AlgE could be immunologically detected in the cytoplasmic membrane (CM). Surface exposition of AlgE in the OM of E. coli was indicated by labeling lysine residues with N-hydroxysuccinimide-biotin. Secondary-structure analysis suggested that AlgE is anchored in the OM by 18 membrane-spanning beta-strands, probably forming a beta-barrel. Recombinant AlgE was purified, and isoelectric focusing revealed a pI of 4.4. Recombinant AlgE was spontaneously incorporated into planar lipid bilayers, forming ion channels with a single-channel conductance of 0.76 nS in 1 M KCl and a mean lifetime of 0.7 ms. Single-channel current measurements in the presence of other salts as well as reversal potential measurements in salt gradients revealed that the AlgE channel was strongly anion selective. For chloride ions, a weak binding constant (Km = 0.75 M) was calculated, suggesting that AlgE might constitute an ion channel specific for another particular anion, e.g., polymannuronic acid, which is a precursor of alginate. Consistent with this idea, the open-state probability of the channel decreased when GDP-mannuronic acid was added. The AlgE channel was inactivated when membrane voltages higher than +85 mV were applied. The electrophysiological characteristics of AlgE, including its rectifying properties, are quite different from those of typical porins.
Rehm
B H
BH
Lehrstuhl für Biologie der Mikroorganismen, Bochum, Germany.
Boheim
G
G
Tommassen
J
J
Winkler
U K
UK
D000595:Amino Acid Sequence; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002462:Cell Membrane / Q000737:chemistry; D003432:Cross-Linking Reagents; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism*; D007525:Isoelectric Focusing; D008051:Lipid Bilayers; D008239:Lysine / Q000032:analysis; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D003432:Cross-Linking Reagents; D007473:Ion Channels; D008051:Lipid Bilayers; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase; D008239:Lysine