Enterobactin

As previous studies demonstrated that Campylobacter used ferric enterobactin as the sole source of iron during growth promotion assays (56 (link)), we measured the growth of C. jejuni strains as previously described (57 (link)). Briefly, a culture grown overnight on MH agar was resuspended in MH broth to an OD₆₀₀ of 0.1. C. jejuni cells were grown in a disposable glass tube to log phase. Deferoxamine mesylate salt (DFO) (Sigma-Aldrich, MO, USA), a chelator, was added to melted MH agar at a final concentration of 20 μM. The cells were mixed with DFO-containing MH agar and adjusted to approximately 10⁷ CFU/mL. Each sample mixture was poured into petri dishes for solidification. A sterile disk containing 25 μL of enterobactin (2 mM) (Sigma-Aldrich, MO, USA) was placed on the surface of the agar in each dish. Autoclaved distilled water was used instead of enterobactin as a negative control.

An overnight culture of the indicator strain ΔentA (1 ml) was pelleted by centrifugation, washed twice with PBS, and resuspended in an equivalent volume of PBS. Melted LB agar containing 200 μM 2,2′-dipyridyl (DIP) was cooled to 55°C, inoculated with 25 μl of the ΔentA mutant, and used to pour plates. After agar plate solidification, agar plugs were removed from the plate using the wide end of a 200-μl pipette tip to create test wells. The resulting plates were used to detect the presence of siderophores in the siderophore extracts from the conditioned medium. Test wells were filled with 50 μl of extracts from the conditioned medium, and plates were incubated at 37°C. Positive (2.5 μM enterobactin [Sigma]) and negative (double-distilled water [ddH₂O]) controls were included in each experiment. The ΔentA indicator strain cannot grow in the presence of the iron chelator DIP unless exogenous siderophores are present in the extracts added to the test well. The presence of a halo of growth around a test well indicated the presence of siderophores in the extract added to the given test well.

An overnight culture of the ΔmacAB mutant grown in LB broth was diluted 1/100 in fresh medium supplemented with 1 mM H₂O_2. Enterobactin (Sigma), Ent-TRI, Ent-DIM, and Ent-MONO (all from EMC Microcollections, Germany) were added to individual tubes at 2.5 μM and incubated at 37°C with aeration. A separate tube containing the ΔmacAB mutant-inoculated LB broth supplemented with 1 mM H₂O₂ was incubated under the same conditions and served as a positive control. Aliquots were collected hourly, serially diluted, and plated on LB agar supplemented with chloramphenicol. Results were expressed as percent survival calculated as [CFU(t_n)/CFU(t₀)] * 100 over time. Each experiment was performed on at least three separate occasions.

The presence of siderophore in the medium extracts was measured using Arnow assay (43 ). Fifty-microliter volumes of extract (from wild-type-conditioned and ΔmacAB mutant-conditioned media) were mixed with an equal volume of 0.5 N HCl, followed by addition of nitrite-molybdate reagent (10 g sodium nitrate and 10 g sodium molybdate in 100 ml of H₂O) and 1 N NaOH. In the presence of catecholate siderophores, this solution appears pink and has an absorption maximum at 510 nm. The concentration of siderophores in the extracts was calculated based on a calibration curve using enterobactin (Sigma) in a range from 0 to 100 μM.

Enterobactin, ferrioxamine E, and ferrichrome were purchased from Sigma-Aldrich, and bacillibactin was purchased from EMC Microcollections GmbH (Tuebingen, Germany).

Enterobactin (ENT) and ferrichrome (FERRI) were obtained from Sigma-Aldrich. Albomycin δ2 (ALBO) was purchased from EMC Microcollection. Pyoverdine (PVD) was purified from P. aeruginosa PAO1 cultures as previously described [28 ]. TCV was synthesized according to a previously published protocol [29 (link)] and TCVL6 was synthesized as described in Supporting information. The protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone) was purchased from Sigma-Aldrich. ⁵⁵FeCl₃ was obtained from Perkin Elmer Life and Analytical Sciences (Billerica, MA, USA), at a concentration of 71.1 mM, with a specific activity of 10.18 Ci/g. RPMI was purchased from Thermo-Fisher.