Membrane association of the Escherichia coli enterobactin synthase proteins EntB/G, EntE, and EntF

Abstract

The cytosolic proteins EntE, EntF, and EntB/G, which are Escherichia coli enzymes necessary for the final stage of enterobactin synthesis, are released by osmotic shock. Here, consistent with the idea that cytoplasmic proteins found in shockates have an affinity for membranes, a small fraction of each was found in membrane preparations. Two procedures demonstrated that the enzymes were enriched in a minor membrane fraction of buoyant density intermediate between that of cytoplasmic and outer membranes, providing indirect support for the notion that these proteins have a role in enterobactin excretion as well as synthesis.

FIG. 1

Enterobactin biosynthesis. diDHBA, 2,3 dihydro-2,3,dihydroxy benzoic acid; 4PP, 4′-phosphopantetheine.

FIG. 2

Association of EntB/G, EntE, and EntF with membranes. E. coli AB1515 (23) was grown in TMM (17) to 5 × 10⁸ cells/ml, and the total membranes and the soluble protein fractions were isolated. Fractions from equal numbers of cells were examined by Coomassie blue staining (A) and Western blotting (B) after electrophoresis on SDS–11% polyacrylamide gels. Lanes: 1, soluble proteins; 2, total membranes; 3, total membranes after a second wash with 10 mM sodium phosphate buffer, pH 7; 4, total membranes after three washes with buffer.

FIG. 3

Association of EntB/G, -E, and -F with membranes. Total membranes were isolated from mid-log-phase E. coli AB1515 by the procedure of Osborn et al. (26). Membranes were resuspended in 65% sucrose, layered with decreasing concentrations of sucrose solutions (56, 53, 50, 47, 44, 41, 38, 35, and 28%), and separated as described by Poquet et al. (27). Membrane fractions were pooled, and 20 μg of protein (4) from each pool was analyzed by Coomassie blue staining (A) and Western blotting (B) after electrophoresis on 11% polyacrylamide gels (17). Lanes: 1, inner membrane fractions; 2, intermediate-density fractions; 3, light outer membrane fractions; 4, heavy outer membrane fractions; St, molecular weight standards.

FIG. 4

Presence of EntB/G, -E, and -F in various gradient fractions. Sucrose gradient centrifugation was performed by the method of Osborn et al. (26). Two hundred and fifty-microliter fractions were collected from the top of the gradient and analyzed for protein content, sucrose density, and NADH oxidase activity (units of activity are micromoles of NADH oxidized per minute. (A) Protein content, sucrose density, and NADH oxidase activity of fractions. Equal amounts of protein from various fractions were subjected to SDS-PAGE and analyzed by staining and Western blotting. (B) Coomassie blue-stained gel of the various fractions. St, molecular mass standards. Numbers on the side indicate positions of molecular mass standards in kilodaltons. Numbers at the top indicate fraction numbers from panel A. TM, total membrane fraction; S, soluble fraction; (C) Western blot analyses of EntB/G, -E, and -F, TonB, and PhoA. Numbers at the top indicate fraction numbers from panel A. TM, total membrane fraction; S, soluble fraction.