Ferric nitrate

M1 defined medium containing 0.02% (w/v) of vitamin-free Casamino Acids and 15 mM lactate was used in all physiological experiments [72 (link)]. Growth of the deletion strain under aerobic or anaerobic conditions was determined by recording growth curves in triplicate with a Bioscreen C microbiology reader (Labsystems Oy, Helsinki, Finland) with MR-1 as the control. For aerobic growth, exponential phase cultures were diluted to approximately ~1 × 10⁵ cells/ml in fresh medium, and 400 μl was transferred to the honeycomb plate wells of the Bioscreen C reader. The cultures were shaken at medium intensity continuously, and the turbidity was measured every 30 min at 600 nm and DO (dissolved oxygen) was recorded every hour with an Accumet XL40 meter (Fisher Scientific). For anaerobic growth, exponential phase cultures grown aerobically were centrifuged, purged in nitrogen and suspended in fresh medium to approximately ~1 × 10⁵ cells/ml in an anaerobic glove box. Electron acceptors tested in this study included fumarate (20 mM), nitrate (2 mM), nitrite (1 mM), thiosulfate (3 mM), TMAO (20 mM), and DMSO (20 mM). For electron acceptors containing metals including MnO₂ (5 mM), ferric citrate (10 mM), and cobalt(III)-EDTA (200 μM), growth was monitored by the color change of the cultures and cell counting under a microscope (Nikon Optiphot, Nikon, Japan).
Survival of MR-1 and the ΔarcA strain during the stationary phase was examined. Cultures were grown from a single colony under aerobic conditions with vigorous shaking. After the onset of stationary phase, the cultures were divided into two parts. One was kept in the incubator with vigorous shaking and the other was kept still. The cultures were serially diluted into LB and plated onto LB plates every 12 h. Plates from dilutions that gave 100 to 250 colony form units (CFU) per plate were used to minimize statistical variation due to small sample sizes. Experiments were done in triplicate.

Legionella pneumophila (L. pneumophila) strain Lp02, is a thymine auxotrophic derivative of Philadelphia-1 [19] (link). The dotA mutant strain Lp03 is defective in the Dot/Icm Type IV secretion system [68] (link). The flaA mutant L. pneumophila was previously described [69] (link). Bacterial strains were supplemented with a plasmid that complements thymine auxotrophy and expresses green fluorescent protein (GFP) at the post-exponential phase (PE) [22] (link),[45] (link). L. pneumophila was cultured as described previously [22] (link),[45] (link) in ACES-yeast extract broth supplemented with ferric nitrate and L-cysteine. All experiments were performed in the absence of ferric nitrate and L-cysteine from the macrophage culture medium, to allow L. pneumophila multiplication only intracellularly. All in vitro infections were performed at an MOI of 0.5 for 30 minutes followed by rinsing of the infected macrophages which allowed the infection of 20–25% of macrophages with usually 1 organism, unless stated otherwise [28] (link). The quantification of the colony-forming units (CFU) in vitro and in vivo was performed as described [28] (link).

Lanthanum nitrate, barium nitrate, zirconium nitrate, ferric nitrate, ammonia water, citric acid and anhydrous ethanol were procured from Shanghai Aladdin Industrial Corporation. All reagents were employed as received without any further purification.

This was measured using trichloroacetic acid (TCA) as the ferric complex, at 460 nm absorbance [4 ]. Milk samples (4.0 mL) were mixed with 2.0 mL of a 20% TCA solution. The mixture was blended well and then allowed to stand for at least 30 min. It was, thereafter, filtered through a suitable filter paper (Whatman No. 40). The clear filtrate (1.5 mL) was then mixed with 1.5 mL of the ferric nitrate reagent (16.0 g Fe (NO₃)). A total of 3.9 mL H₂O was dissolved in 50 mL of 2M HNO₃ and was then diluted with distilled water to 100 mL; the absorbance was measured at 460 nm using a UV spectrophotometer. As a blank, a mixture of 1.5 mL of ferric nitrate solution and 1.5 mL of water was used. The measurement was carried out within 10 min of the addition of the ferric nitrate solution, as the colored complex is not stable for any length of time. The concentration of thiocyanate was then determined by comparison with standard solutions of known thiocyanate concentrations (10, 15, 20, and 30 g/mL of thiocyanate).