Fig. 3: DtHtaA in mMVs participates in heme binding and delivery.

A. Purified DtHtaA bound heme in vitro. DtHtaA was incubated in 10 μM heme solution for 30 min. Excess heme was washed out and the protein solution was scanned from 350-500 nm. DtHtaA without heme addition was used for comparison. One peak characteristic of heme was detected at 410 nm. B. DtHtaA residing inside mMVs is responsible for heme binding and delivery. One-hundred micrograms of heme-incubated mMVs from wild type (WT), ΔDthtaA, and ΔDthtaA::DthtaA were added to DQ12-45-1b culture, and the maximum OD₆₀₀ was recorded. PC positive control, DQ12-45-1b cultured with 2 μM heme as sole iron source (calibrated as 100%). NC negative control, DQ12-45-1b cultured without iron. C. mMVs capture and deliver heme from environmental hemoproteins. Hemoproteins were incubated with 100 μg mMVs at 30 °C for 2 h. mMVs were then washed and added to DQ12-45-1b culture as sole iron source, and the maximal OD₆₀₀ was recorded (black). The growth of DQ12-45-1b using hemoprotein as sole iron source was recorded (gray). PC positive control, DQ12-45-1b cultured with 2 μM heme as sole iron source (calibrated as 100%). NC negative control, DQ12-45-1b cultured without iron. D. Involvement of DtHtaA and DtHmuUV in free heme and heme-incubated mMVs related heme utilization. The strain DQ12-45-1b, DQ12-45-1b ΔDthtaA, and DQ12-45-1b ΔDthmuUV were cultured in defined minimal medium amended with 2 μM heme, 100 μg no-heme-incubated mMVs and 100 μg heme-incubated mMVs, respectively. After cultured for 72 h at 30 °C, the strains’ maximal growth at OD₆₀₀ were recorded. The growth of DQ12-45-1b in free heme was calibrated as 100%. All mMVs used in this experiment were isolated from DQ12-45-1b cultures at iron-limiting conditions. Error bar represents three independent replicates.