Heterologous production of Ferrigenium straubiae's Fe(II) oxidase Cyc2 for functional and structural studies

Résumé fourni par la source

Neutrophilic Fe(II)-oxidizing bacteria inhabit many ecosystems, where they regulate iron mineralogy, thereby influencing nutrient availability, heavy metal solubility, and greenhouse gas formation, yet the molecular mechanisms of microbial Fe(II) oxidation at neutral pH remain poorly understood. Many representatives encode a homolog of the outer membrane cytochrome Cyc2, which has been proposed as a primary Fe(II) oxidase. In order to facilitate functional and structural studies on Cyc2, we herein address challenges of cyc2 heterologous overexpression, such as correct targeting, limited space in the outer membrane, heme c maturation, and assembly of the holo-protein. Aiming for Cyc2 from Ferrigenium straubiae we identified Escherichia coli strain BL21(DE3) Gold ΔompF and MC4100, carrying the pASK_IBA2 expression system and the pEC86 helper plasmid for cytochrome c maturation, as robust expression systems. Using 2×YL medium, Cyc2 yield was independent of 5-aminolevulinic acid and iron supplementation. By validating Cyc2's outer membrane localization using a urea extraction assay we evidence that F. straubiae β-barrel assembly machinery signal motif was recognized by E. coli. Although semi-native and denatured Cyc2 did not show a heat modifiability shift on an SDS-PAGE, as it is often observed for β-barrels, we found that TMBZ-based heme staining was significantly more prominent for denatured samples. This indicated that heme c was inaccessible for TMBZ in the semi-native state, which is consistent with the predicted localization within the β-barrel of Cyc2. To enable structural studies, we suggest detergent-based solubilization screening overnight and offer a library of 22 amber mutants for photo-crosslinking experiments. Overall, this work established the foundation for heterologous production of Cyc2, enabling downstream structural and biophysical characterization for advancing our understanding of bacterial Fe(II) oxidation.

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