Abstract
We investigated the importance of the production of catecholate siderophores, and the utilization of their iron (III) complexes, to colonization of the mouse intestinal tract by Escherichia coli. First, a ΔtonB strain was completely unable to colonize mice. Next, we compared wild type E. coli MG1655 to its derivatives carrying site-directed mutations of genes for enterobactin synthesis (ΔentA::Cm; strain CAT0), ferric catecholate transport (Δfiu, ΔfepA, Δcir, ΔfecA::Cm; CAT4), or both (Δfiu, ΔfepA, ΔfecA, Δcir, ΔentA::Cm; CAT40) during colonization of the mouse gut. Competitions between wild type and mutant strains over a 2-week period in vivo showed impairment of all the genetically engineered bacteria relative to MG1655. CAT0, CAT4 and CAT40 colonized mice 101-, 105-, and 102-fold less efficiently, respectively, than MG1655. Unexpectedly, the additional inability of CAT40 to synthesize enterobactin resulted in a 1000-fold better colonization efficiency relative to CAT4. Analyses of gut mucus showed that CAT4 hyperexcreted enterobactin in vivo, effectively rendering the catecholate transport-deficient strain iron-starved. The results demonstrate that, contrary to prior reports, iron acquisition via catecholate siderophores plays a fundamental role in bacterial colonization of the murine intestinal tract.
Highlights
For most microorganisms and all animals, iron is indispensable in metabolic processes like catabolism, electron transport, peroxide reduction, and DNA biosynthesis
E. coli CAT0 (DentA::Cm) resulted from a single-step transduction with a P1 lysate grown on E. coli OKN10::Cm
We confirmed the absence of entA, tonB, fiu, fepA, fecA or cir from the chromosome of E. coli strains CAT0, TonB, CAT4 and CAT40 by colony PCR reactions with primers ca. 500 bp upstream and downstream of the target gene
Summary
For most microorganisms and all animals, iron is indispensable in metabolic processes like catabolism, electron transport, peroxide reduction, and DNA biosynthesis. The fact that iron readily oxidizes in aqueous environments poses a problem for its acquisition by living organisms: cells cannot transport the large polymers of insoluble ferric oxyhydroxide that spontaneously form in water. Almost all microorganisms, including commensal and pathogenic bacteria, produce biosynthetic and transport systems to capture the metal from their environments, including their plant and animal hosts. Mammalian body fluids and tissues have low iron availability, in part from its poor solubility at physiological pH (the concentration of free iron in neutral aqueous solutions is ,10218 M [2]), and in part because binding proteins like transferrin, lactoferrin, ferritin and hemoproteins complex Fe+++, reducing the level of free iron to ,10224 M [3,4]. The sequestration of iron from invading pathogens was described as ‘‘nutritional immunity,’’ even though no immune system components are involved in the process [6]
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