TolC Is Involved in Enterobactin Efflux across the Outer Membrane of Escherichia coli

Abstract

Escherichia coli excretes the catecholate siderophore enterobactin in response to iron deprivation. While the mechanisms underlying enterobactin biosynthesis and ferric enterobactin uptake and utilization are widely understood, nearly nothing is known about how enterobactin is exported from the cell. Mutant and highperformance liquid chromatography analyses demonstrated that the outer membrane channel tunnel protein TolC but none of the respective seven resistance nodulation cell division (RND) proteins CusA, AcrB, AcrD, AcrF, MdtF (YhiV), or the twin RND MdtBC (YegNO) was essential for enterobactin export across the outer membrane. Mutant E. coli strains with additional deletion of tolC or the major facilitator entS were growth deficient in iron-depleted medium. Strains with deletion of tolC or entS, but not with deletion of genes encoding RND transporters, excreted very little enterobactin into the growth medium. Enterobactin excretion in E. coli is thus probably a two-step process involving the major facilitator EntS and the outer membrane channel tunnel protein TolC. Quantitative reverse transcription-PCR analysis of gene-specific transcripts showed no significant changes in tolC expression upon iron depletion. However, iron starvation led to increased expression of the RND gene mdtF and a decrease in acrD.Enterobactin (33), also known as enterochelin (30), is the catecholate-type siderophore of Escherichia coli and of several other bacteria. Enterobactin, a cyclic triester of 2,3-dihydroxybenzoylserine (DHBS), is one of the most effective ferric iron chelating compounds known (1, 36). While the molecular processes involved in ferric enterobactin uptake by TonB-energized outer membrane receptor proteins such as FepA (recently reviewed in reference 15) were studied exhaustively over the last decades, a mechanism for enterobactin efflux across the cytoplasmic membrane was discovered only recently. EntS (the ybdA gene product) (5), a member of the vast major facilitator superfamily (MFS) of membrane-bound transporters, was shown to be necessary for effective enterobactin export in E. coli (9). Cells with entS deleted excreted very little enterobactin into the surrounding medium, but degradation products of enterobactin were released into supernatants. Since those degradation products are themselves efficient siderophores and were still exported, strains lacking entS suffered no iron depletion (9).Because enterobactin is too big to diffuse freely through the porins of the outer membrane, transport from the periplasm to the outside has to be accomplished by another still unknown transport system. Previously, we and others (18,20,26,27) could demonstrate that transport systems of the resistance nodulation cell division (RND) type (38) may transport their substrates from the periplasm (or from the cytoplasmic membrane in the case of hydrophobic substances) rather than from the cytoplasm to the outside. At least for copper and cobalt, we provided evidence that efflux is probably a two-step process involving a transp...

Siderophore-Based Iron Acquisition and Pathogen Control

“…As a third alternative, a TolC-dependent but EntS-independent export system(s) that transports enterobactin from both the cytoplasm and the periplasm to the environment might exist. Enterobactin breakdown products, however, were also released through the OM in a tolC mutant (23), suggesting that there are further OM gates for clearing the periplasm from catecholate fragments.…”

“…An E. coli fur tonB double mutant, which is derepressed in enterobactin synthesis but unable to take up Fe-enterobactin, reached only one-third of the dry weight of an enterobactin-deficient fur entC double mutant (23). Another study reported normal growth rates for an E. coli entB mutant and fes entB double mutant, both defective in enterobactin biosynthesis, but very poor growth of fes and fep mutants defective in Fe-enterobactin uptake and hydrolysis, respectively, under the same iron-depleted conditions (327).…”

The TolC-like Protein HgdD of the Cyanobacterium Anabaena sp. PCC 7120 Is Involved in Secondary Metabolite Export and Antibiotic Resistance

“…We observed uptake of ethidium (394 Da) and erythromycin (734 Da; Figs. [3][4][5][6][7][8][9][10][11], and a permeability coefficient for Anabaena sp. (Fig.…”

A small RNA promotes siderophore production through transcriptional and metabolic remodeling

“…The final assembly of DHB and serine into enterobactin depends on the action of EntD, EntB, EntE, and EntF (11). Once enterobactin has been synthesized, it is transported through the inner membrane by EntS (12) and the outer membrane by TolC (13). Outside the cell, enterobactin will bind to ferric Fe (Fe 3+ ) with an extremely high affinity (14).…”