Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB.

The periplasmic protein FepB of Escherichia coli is a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay the K(d) of the ferric enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (K(d) = 30 nM) and ferric enantioenterobactin (K(d) = 15 nM), the left-handed stereoisomer of the natural E. coli siderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (K(d) = 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.

Selectivity of ferric enterobactin binding and cooperativity of transport in gram-negative bacteria.

The ligand-gated outer membrane porin FepA serves Escherichia coli as the receptor for the siderophore ferric enterobactin. We characterized the ability of seven analogs of enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their 59Fe complexes. The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein. Among the compounds we tested, only ferric enantioenterobactin, the synthetic, left-handed isomer of natural enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (Kd approximately 20 nM). Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2-HOPO; K3MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA. We also compared the binding and uptake of ferric enterobactin by homologs of FepA from Bordetella bronchisepticus, Pseudomonas aeruginosa, and Salmonella typhimurium in the native organisms and as plasmid-mediated clones expressed in E. coli. All the transport proteins bound ferric enterobactin with high affinity (Kd /=50 pmol/min/10(9) cells) in their own particular membrane environments. However, the FepA and IroN proteins of S. typhimurium failed to efficiently function in E. coli. For E. coli, S. typhimurium, and P. aeruginosa, the rate of ferric enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA.

Laboratory and field studies on the effects of the antibiotic tylosin on honey bee Apis mellifera L. (Hymenoptera: Apidae) development and prevention of American foulbrood disease.

Laboratory and field studies were conducted to determine the effectiveness of the antibiotic tylosin in preventing and controlling infections of American foulbrood disease (AFB) of honey bees. Studies conducted on immature worker bees maintained in the laboratory revealed that honey bee larvae could tolerate quite a range of doses of antibiotic in their diet. Intermediate doses of tylosin protected very young larvae from becoming infected by Bacillus larvae at a concentration of 1.5 x 10(8) spores/ml of diet. Antibiotic treatment had no measurable effects on larval or pupal developmental rates until the dose reached a lethal level. Bees in field colonies readily consumed tylosin in powered sugar, up to a level of 800 mg/7 g sugar. No negative colony effects were noted at any dosage rates. Protection against infection by American foulbrood was compared to results obtained with 200 mg Terramycin, the standard dose of the only substance currently registered for foulbrood control. Both 200 mg Terramycin and 100 mg tylosin protected the colonies for up to 3 weeks. A 200-mg dose of tylosin protected the colony for an additional week. Doses of 100 mg or more of tylosin were adequate to eliminate signs of AFB infection in overtly diseased colonies.