EfaCBA, MntH1 and MntH2 promote growth of <i>E</i>. <i>faecalis</i> in Mn-restricted environments.

(A) Total Fe, Mn and Zn content of BHI medium determined by ICP-OES analysis. Error bars denote SD. (B) Growth of OG1RF (wild-type), Δefa, ΔmntH1, ΔmntH2, ΔefaΔmntH1, ΔefaΔmntH2, ΔmntH1ΔmntH2 and ΔefaΔmntH1ΔmntH2 strains on BHI plates with or without Mn supplementation. Overnight cultures were washed, diluted in PBS and 5 μl aliquots spotted on plates. Plates were incubated for 24 hours before being photographed. (C–F) Growth of OG1RF and its derivatives in Mn-replete (+ Mn, panels C, E) and Mn-depleted (↓ Mn, panels D, F) FMC. Cells were grown to OD600 ~ 0.2 in complete FMC and diluted 1:100 in either complete FMC or FMC depleted for Mn. Growth was monitored using a Bioscreen growth reader monitor. (G-H) Growth of OG1RF on BHI plates (G) or in Mn-depleted FMC broth (H) compared to the triple ΔefaΔmntH1ΔmntH2 mutant strain harboring either the empty plasmid (pTG-empty) or pTG-efa, pTG-mntH1 or pTG-mntH2. Bars show optical density at 600 nm after 14 hours incubation. The graphs show the average and standard deviations of three independent experiments. Statistically significant differences were determined via ordinary one-way ANOVA with Dunnett’s multiple comparison post-test (****p ≤ 0.0001).