Protein II* Influences Ferrichrome-iron Transport in Escherichia coli k12

Abstract

Ferrichrome-promoted iron uptake in Escherichiu coli ~1 2 is strictly dependent upon the tonA gene product, a 'minor' outer membrane protein. By selection for mutants of E. coIi resistant to phages which require 'major' outer membrane proteins as receptors, strains with pronounced protein deficiencies were constructed. Such strains were tested for anomalous behaviour of ferrichrome transport. No significant differences in iron uptake were detected in E. coli K12 strains with markedly reduced amounts of protein I. However, a reduction in the initial velocity (up to 40%) was observed in E. coli deficient in outer membrane protein II*. This difference was only evident when cells were grown under iron-starvation conditions; it was abolished when cells were grown in rich medium. Kinetic parameters for ferrichrome transport were determined for maximum velocity but not for Km; double reciprocal plots showed a biphasic nature, probably attributable to a limited number of outer membrane binding sites and to the multi-component nature of the ferrichrome-iron transport system.

Effect of loop deletions on the binding and transport of ferric enterobactin by FepA

“…1). These alterations revised the FeEnt uptake K m to lower values, and comparable binding studies similarly reduced the adsorption K d , suggesting that previous methodologies for the measurement of these parameters (Coulton and Braun, 1979;Ecker et al, 1986;Newton et al, 1997;Thulasiraman et al, 1998) were subject to substrate depletion artifacts, and therefore inaccurate. These experiments also indicated that it was necessary to customize the transport conditions individually for FepA mutants that manifest either lower affinity or lower transport efficiency.…”

“…Both the binding and transport of ferric siderophores by ligand-gated porins were characterized as multicomponent or co-operative processes (Coulton and Braun, 1979;Locher and Rosenbusch, 1997;Thulasiraman et al, 1998), but the revision of our methodologies essentially eliminated the allostery that occurs during FeEnt binding and uptake in vivo (Fig. 1).…”

“…High‐affinity transport through TonB‐dependent ligand‐gated porins was initially characterized for vitamin B 12 uptake through the protein now known as BtuB (Di Girolamo and Bradbeer, 1971; White et al ., 1973), which showed an initial binding K d of about 0.5 ηM and an overall uptake K m of about 5 ηM for cyanocobalamin. Numerous subsequent estimates of the affinity of OM ferric siderophore binding and transport systems were 20‐ to 5000‐fold lower (Coulton and Braun, 1979; Fiss et al ., 1982; Ecker et al ., 1986; Zhou et al ., 1995; Locher and Rosenbusch, 1997; Newton et al ., 1997; Braun et al ., 1999; Ferguson et al ., 1998; Locher et al ., 1998; Thulasiraman et al ., 1998; Buchanan et al ., 1999), leading to the conclusion that bacterial iron acquisition is much less efficient than vitamin B 12 uptake. However, the current study suggests that existing iron binding and transport methodologies almost uniformly suffer from substrate depletion artifacts that arise from underestimation of the affinities of ferric siderophore–receptor systems.…”

Molecular cloning of the ferrichrome-iron receptor of Escherichia coli K-12

“…Transport of 5Fe3+-ferrichrome. Cells to be tested for the uptake of 55Fe3" from the ferrichrome-iron complex (12) were grown in M9 minimal medium, washed twice with iron uptake medium (10 mM Trishydrochloride, pH 7.2, 0.5% glucose, 1.0 mM MgSO4), and resuspended in iron uptake medium plus 100 ,uM nitrilotriacetate to an absorbance at 578 nm of 0.40, corresponding to 0.38 mg (dry weight)/ml. Bacteria were equilibrated at 37°C for S min, and uptake of (17).…”

Topological analysis of the Escherichia coli ferrichrome-iron receptor by using monoclonal antibodies

“…E. coli K-12 strains PL-6 (aroB thi r cir feuB TuII* r [9]), MC4100 [F-araD ⌬(argF-lac)U169 rspL thi relA flbB deoC pstF rbsR (40)], SG303 (MC4100 aroB [7]), and AW740 (hisG4 thr-1 fhuA31 tsx-78 ⌬ompF zcb::Tn10 ⌬ompC [20]) were used as hosts for protein expression. E. coli CS2529 (F Ϫ thr leuB6 proA argE his thi galK lacY1 trpE non mtl xyl ara-14 rfaK2::⍀Km r [27]) expresses truncated lipopolysaccharide (LPS) and was used to enhance recognition of FhuA on intact cells by MAbs.…”