Rhizobia-legume symbiosis (RLS) is the principal entry pathway of sustainable N in agricultural systems. During the symbiosis, plants develop a new organ called the nodule that hosts thousands of endosymbiotic bacteria expressing the nitrogenase, the enzyme responsible to transform the atmospheric nitrogen into ammonia. The nitrogenase unique metallic cofactors (made of Fe and Mo) likely impose an extra demand of these metals to the cell, which already requires a basal amount of Fe to synthesize ‘housekeeping’ metalloproteins. However, the transport systems required by the endosymbiotic rhizobia to satisfy that increased demand remain obscure yet.<br/>In this project, we will combine molecular biology approaches, including cell-labeling, epitope tagging, and loss-of-function genomic approaches coupled with thorough phenotyping and two analytical methods, inductively coupled plasma-mass spectrometry and X-ray absorption spectroscopy, to characterize how much iron does nitrogen-fixing bacteria need, how much is used to metallate the nitrogenase and other metalloproteins, and what genes are required to maintain the Fe homeostasis in the endosymbiotic bacteria.<br/>The methodologies developed within this project will open new avenues for the study of mineral nutrition in other soil bacteria and will offer an insight into how cells partition an element among different metalloproteins. Furthermore, ironing out Fe homeostasis in rhizobia will allow us to maximize the nitrogen fixation capabilities of both natural and synthetic nitrogen-fixing bacteria to be used as inoculants in sustainable agriculture practices.