Periodic Reporting for period 1 - IronFeRhizo (Ironing out Fe homeostasis and ferroproteins metallation in symbiotic Rhizobia)
Periodo di rendicontazione: 2021-06-01 al 2023-05-31
Rhizobia-legume symbiosis is the main route for sustainable nitrogen to contribute to soil fertility. During symbiosis, legumes develop nodules hosting endosymbiotic bacteria expressing nitrogenase, a unique iron and molybdenum metalloenzyme that transforms atmospheric nitrogen into ammonia that can be utilized by plants. This is important for society because would help us to produce nutritive protein resources (pulses) in a sustainable way, this is without using polluting nitrogen fertilizers. Since fertilizers significantly contribute to the rise in food prices observed in recent years, this would have second-derived benefits to our society. Interestingly, iron is not only essential for bacteria but for all organisms on our planet. This includes plants, animals, and ourselves. Probably you or someone closer to you suffer iron deficiency or anemia, as 2 billion people are affected by it worldwide.
The EU-funded IronFeRhizo project has combined biochemical and molecular biology techniques to determine how much iron is required by nitrogen-fixing bacteria, which genes are required in its transport into the cell, and how much is used to metallate the nitrogenase and other metalloproteins in order to increase their capacity to fix atmospheric nitrogen and sustain higher productivities in the field. Moreover, the techniques developed will facilitate research into mineral nutrition in other organisms (bacteria and plants) and would help us to intervene in our food chains to increase the amount of iron that ends up in our plates.
The EU-funded IronFeRhizo project has combined biochemical and molecular biology techniques to determine how much iron is required by nitrogen-fixing bacteria, which genes are required in its transport into the cell, and how much is used to metallate the nitrogenase and other metalloproteins in order to increase their capacity to fix atmospheric nitrogen and sustain higher productivities in the field. Moreover, the techniques developed will facilitate research into mineral nutrition in other organisms (bacteria and plants) and would help us to intervene in our food chains to increase the amount of iron that ends up in our plates.
The IronFeRhizo project had three research objectives:
Objective 1: To quantify iron in rhizobia during their progression in the nodule (WP1).
Objective 2: Identify the genes responsible for iron homeostasis in rhizobia (WP2).
Objective 3: Determine rhizobial Fe partitioning in metalloenzymes required for BNF (WP3).
Within WP1, we have developed a simplified symbiosome cassette able to distinguish endophytic nodule bacteria (within infection threads) from differentiated N-fixing endosymbiotic rhizobia (also known as bacteroids). For all the rhizobia strains tested, partners of different legume hosts, we found that purified nodule bacteria accumulate larger amounts of iron than free-living bacteria. Interestingly, the iron content in bacteroids correlates with their nitrogen capacity, therein reduced in fixing intermediate or fixation null strains. This suggests that nitrogenase expression may be driving iron uptake in rhizobia.
In WP2 we have identified several genes that are important for keeping the iron balance in rhizobia. We found that when mutated, these affect either positively or negatively the nitrogen fixation capacity of that strain. We still need to validate some of those results in field conditions, but we have identified iron content as an important trait to consider in order to develop better inoculants.
In WP3 we tackle a fundamental question in cell biology which is how cells allocate iron to the different enzymes that require this element. This is still a work in progress, but we are developing a targeted approach to study protein metallation for certain proteins of interest.
Last but not least, there is an important aspect of the Marie Sklodowska Curie - Individual Fellowship which is the training of the beneficiary. This project has allowed me to learn different research techniques and transferable skills, manage my first research budget, and supervise and lead my first research team. Equipped with this experience I am better prepared to tackle new research questions and to provide actual solutions to important societal challenges such as the production of nutritious food in a sustainable way.
Objective 1: To quantify iron in rhizobia during their progression in the nodule (WP1).
Objective 2: Identify the genes responsible for iron homeostasis in rhizobia (WP2).
Objective 3: Determine rhizobial Fe partitioning in metalloenzymes required for BNF (WP3).
Within WP1, we have developed a simplified symbiosome cassette able to distinguish endophytic nodule bacteria (within infection threads) from differentiated N-fixing endosymbiotic rhizobia (also known as bacteroids). For all the rhizobia strains tested, partners of different legume hosts, we found that purified nodule bacteria accumulate larger amounts of iron than free-living bacteria. Interestingly, the iron content in bacteroids correlates with their nitrogen capacity, therein reduced in fixing intermediate or fixation null strains. This suggests that nitrogenase expression may be driving iron uptake in rhizobia.
In WP2 we have identified several genes that are important for keeping the iron balance in rhizobia. We found that when mutated, these affect either positively or negatively the nitrogen fixation capacity of that strain. We still need to validate some of those results in field conditions, but we have identified iron content as an important trait to consider in order to develop better inoculants.
In WP3 we tackle a fundamental question in cell biology which is how cells allocate iron to the different enzymes that require this element. This is still a work in progress, but we are developing a targeted approach to study protein metallation for certain proteins of interest.
Last but not least, there is an important aspect of the Marie Sklodowska Curie - Individual Fellowship which is the training of the beneficiary. This project has allowed me to learn different research techniques and transferable skills, manage my first research budget, and supervise and lead my first research team. Equipped with this experience I am better prepared to tackle new research questions and to provide actual solutions to important societal challenges such as the production of nutritious food in a sustainable way.
With the IronFeRhizo project, we have made significant progress in our understanding of the iron required for rhizobia to fix nitrogen and we identified some genes responsible for maintaining iron homeostasis during the symbiosis with legumes. At least one of the mutants we have developed is able to fix more nitrogen in lab conditions so we will test in the future how that strain performs in field conditions. That would be a breakthrough in our capacity to engineer better inoculants and decrease our dependence on nitrogen fertilizers for the production of pulses.