An aqueous channel for filamentous phage export

Science

Volumn 284, Issue 5419, 1999, Pages 1516-1519

  Marciano, Denise K ^a   Russel, Marjorie ^a   Simon, Sanford M ^a  

^a ROCKEFELLER UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

MEMBRANE PROTEIN; MUTANT PROTEIN; PROTEIN P IV; UNCLASSIFIED DRUG;

ARTICLE; BACTERIOPHAGE F1; CHANNEL GATING; ESCHERICHIA COLI; NONHUMAN; OUTER MEMBRANE; PRIORITY JOURNAL; PROTEIN ISOLATION; PROTEIN TRANSPORT;

ANTI-BACTERIAL AGENTS; BIOLOGICAL TRANSPORT; CARBOHYDRATE METABOLISM; CELL MEMBRANE; CELL MEMBRANE PERMEABILITY; COLIPHAGES; ELECTRIC CONDUCTIVITY; ELECTROPHYSIOLOGY; ESCHERICHIA COLI; INOVIRUS; ION CHANNEL GATING; ION CHANNELS; LIPID BILAYERS; MEMBRANE PROTEINS; MUTATION; POLYSACCHARIDES; PORINS; PROTEOLIPIDS; VANCOMYCIN; VIRAL NONSTRUCTURAL PROTEINS;

EID: 0033612142     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5419.1516     Document Type: Article

References (25)

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11. + culture appeared on plates with tetraose and larger sugars at low frequency. Thus, the maltodextrin growth assay may be useful for selection of pIV mutants with increased permeability as well as for determining in vivo outer membrane permeability.
12. 2+-Sepharose beads. The pIV was eluted in buffer [1% CHAPS (w/v), 400 mM imidazole, 50 mM tris-Cl (pH 8.0), 500 mM NaCl, 2 mM benzamidine]. The fractions containing pIV were pooled, concentrated, and chromatographed on a BioGelA5M column [1% CHAPS (w/v), 25 mM NaHepes (pH 8.0), 500 mM NaCl, 0.5 mM NaEDTA (pH 8.0), 2 mM benzamidine]. Liposomes containing 12 mM lipid (egg phosphatidylcholine and egg phosphatidic acid in a 9:1 ratio) were bath sonicated for 5 min, freezethawed 10 times, and repeatedly extruded through membranes with 200-nm pores (Lipsofast; Avestin Inc.). We added β-octyl glucoside to a concentration of 22.5 mM. Purified pIV was added to liposomes at ∼1:200 (v/v) to give a final pIV concentration of 10 μg/ml in 12 mM lipids. Detergent was removed with Biobeads SM2 (Pharmacia). Planar lipid bilayers were formed by applying a 20 mg/ml lipid solution to a Teflon hole (100 to 250 μm in diameter) separating the cis and trans chambers as described (17). Lipids used were E. coli phosphatidylethanolamine, 1,2-diphytanoyl-sn-glycero-3-priosphocholine (DPPC), and brain phosphatidylserine (PS) in a 5:4:1 ratio; DPPC:PS in a 9:1 ratio; or total E. coli lipids (Avanti Polar Lipids). A bilayer 250 μm in diameter had a typical capacitance of 500 pF and conductance of <5 pS. Proteoliposomes were added to the cis chamber in the presence of a salt gradient. Solutions were stirred until fusion occurred, and then the gradient was dissipated. Data was acquired using an Axopatch 200 (Axon Instruments) and a 16-bit analog data acquisition board (National Instruments). Traces were filtered at 100 Hz. All data acquisition was controlled with software, developed by Yu Chen, written in LabView (National Instruments).
13. 5324G was able to complement the defective phage and allow plaque formation.
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15. 5324G is fully open in this range, actual channel transitions from the closed to the open state could not be measured.
16. 2/4) + (4RLπ/p)]/(2Rπ/p) Resistivity of the solution is p = 77 ohm·cm, pore length is L = 7 nm (measurements from STEM), and pore resistance is R = 1/g where g is conductance. If the pIV multimer is a single channel with two conductance states (O1, O2), then g = 3.4 nS (160 mV, 150 mM KCI) and the estimated diameter is 6 nm. If a single current step (O1) corresponds to a single pIV multimer, then g = 1.7 nS and the estimated diameter is 4 nm. These values are only approximations.
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25. We thank P. Model for continuous advice and helpful discussions. We also thank M. Shiloh and Y. Chen for helpful discussions and support; N. Linderoth, L. Boone, and J. Schmoranzer for preliminary experiments; S. Asheer for technical assistance; O. Anderson, M. Goulian, and R. MacKinnon for critical reading of the manuscript; T. Silhavy and S. Benson for strains; and L Letellier and E. Kanner for suggestions. Supported by NIH Medical Science Training Program grant GM07739 (D.K.M.), NSF grant MCB93-16625 (M.R.), and the Keck Foundation (S.M.S.).