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Outline
[Objective] To elucidate the physiological and biochemical mechanisms of iron release from bacterioferritin (Bfr) in Agrobacterium fabrum, identify the physiological reductant that provides electrons for the iron release from Bfr in cells, and determine whether Bfr-associated ferredoxin (Bfd) is required to facilitate electron transfer from the reductant to Bfr, thereby exploring a new approach to inhibit the iron release from Bfr and further disrupt intracellular iron homeostasis as well as impair A. fabrum growth and proliferation. [Methods] Bioinformatics analysis was carried out to identify the gene encoding Bfd-like protein in the A. fabrum genome. Bfr and the predicted Bfd were heterologously expressed and the obtained proteins were purified. In vitro assays were conducted to determine whether the predicted Bfd could assist the physiological reductant in transferring electrons to Bfr. Subsequently, we knocked out the Bfd-encoding gene to assess the impacts of Bfd on the iron release of Bfr and A. fabrum growth at the cellular level. [Results] The A. fabrum gene ATU_RS13485 encoded a (2Fe-2S)-binding protein containing the conserved amino acid motif (…C-X-C…C-X2-C…) existent in Bfd proteins, confirming it as the Bfd of A. fabrum. Purified recombinant Bfd was able to assist FADH2 in delivering electrons to Bfr in vitro, triggering the release of Fe2+ from mineralized A. fabrum Bfr. Upon bfd knockout, intracellular Bfr iron release of A. fabrum was severely impaired, and A. fabrum growth was significantly affected. [Conclusion] ATU_RS13485 encodes the Bfd of A. fabrum. A. fabrum Bfr requires Bfd to utilize electrons from the physiological reductant FADH2 for iron release. The absence of Bfd blocks iron release from Bfr and impairs A. fabrum growth, which suggests that designing inhibitors to disrupt the Bfr-Bfd interaction may offer a promising strategy for controlling crown gall disease caused by A. fabrum.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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