Enterobactin export in escherichia coli via P43 (ents) and associated components

"December 2006"The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Vita.Thesis (Ph. D.) University of Missouri-Columbia 2006.Ferric iron, critical for the metabolic functions of many microorganisms, is generally insoluble at neutral pH or quickly complexed by host iron storage proteins. To acquire necessary ferric iron against harsh competition in the environment, iron-starved Escherichia coli synthesize, excrete and retrieve an iron-scavenging siderophore molecule termed enterobactin. Despite extensive characterization of the enterobactin system, the export machinery allowing enterobactin secretion to the extracellular environment has only recently been identified. E.coli membrane protein P43 (entS) in the enterobactin gene cluster encodes a Major Facilitator Superfamily (MFS) exporter. A P43 null mutant was unable to efficiently secrete enterobactin to the supernate, but did secrete elevated levels of enterobactin breakdown products as analyzed by TLC, HPLC, and cross-feeding assays. To further evaluate P43 function in enterobactin transport, inverted membrane vesicles were created using French press and incorporated with an iron-binding fluorescent dye, calcein-AM (CA). Differences in siderophore transport were observed between wild-type and the P43-mutant by monitoring CA fluorescence restoration following iron quenching and the addition of enterobactin. Using specific energy poisons in conjunction with this vesicle system, it was determined that proton motive force energy is utilized for this transport. Additional results demonstrate that siderophore transport from the periplasm to the external environment may be due to contributions from several other identified E.coli components, such as the multi-drug export system comprised of the outer membrane protein TolC and the translocase AcrAB. These data all demonstrate P43 provides a critical activity for the E.coli enterobactin secretion machinery and establish a mechanism for cellular release of siderophore.Includes bibliographical reference

Involvement of TolC protein in the export of siderophore enterobactin in Escherichia coli

Abstract only availableTo acquire the necessary iron against harsh competition in the environment, iron starved bacteria synthesize, excrete and retrieve iron scavenging molecules termed siderophores, one of which is enterobactin. TolC protein may play a vital role in the secretion of enterobactin. Enterobactin molecules destined for secretion must cross both the inner (cytoplasmic) and outer membranes and the intervening periplasmic space, believed to be a distance of at least 130Å across. TolC resembles a trans-periplasmic tunnel embedded in the outer membrane of the cell. It is open to the external environment but is closed at its periplasmic entrance. In order for the cell to export enterobactin, TolC is recruited by substrate specific membrane complexes (translocases) in the periplasmic space and inner membrane. When TolC is recruited, the entrance is opened to allow substrate passage through a continuous machinery spanning the entire cell envelope, from the cytosol to the external environment. PCR primers specific for TolC were designed to amplify the TolC gene. The quality of the PCR product was confirmed using agarose gel electrophoresis. The TolC gene was cloned into a pBAD directional TOPO vector containing an N-terminal His-tag and a gene for kanamycin resistance. The recombinant vector was then transformed into One Shot TOP10 competent Escherichia coli cells. Transformants were selected for by plating on LB medium supplemented with kanamycin. Transformed colonies were analyzed using PCR and restriction digestion. Positive transformants were selected and expression was induced with arabinose. SDS-PAGE assay with His-tag In-gel stain revealed TolC expression. Furthermore, analysis of TolC-null mutations using high performance liquid chromatography (HPLC) reveals that the TolC mutant secretes little, if any, enterobactin. However, some levels of breakdown products 2,3-dihydroxybenzoylserine (DHBS) monomer, dimer, and trimer are observed. These data establish that TolC may be a critical component of the E. coli enterobactin secretion machinery and may represent a type of siderophore export mechanism previously undescribed. TolC family proteins are ubiquitous among gram-negative bacteria, and the conserved apertures present a possible chemotherapeutic target in multidrug-resistant pathogens.Molecular Biology Progra