Investigating Electron Transfer to Nitrogenase
Additional Funding Sources
The project described was supported through the Department of Biological Sciences at Idaho State University.
Abstract
Nitrogen is critical for all living organisms, yet atmospheric nitrogen (N2) contains a strong triple bond leaving it chemically inert and unusable by most cells. Some microbes have an enzyme called nitrogenase which converts N2 into ammonia (NH3). Small proteins called flavodoxins (Fld) and ferredoxins (Fd) accumulate the low-potential electrons needed for this reduction reaction. The nitrogen-fixing bacterium, Azotobacter vinelandii, contains multiple Fld and Fd proteins, yet those critical for nitrogen fixing remain unclear. To determine which Fld and Fd proteins are relevant to nitrogenase activity, four genes proposed to play a role in electron transfer, nifF, fdxN, fdxA, and fixP, were selected and the coding region was replaced with an antibiotic resistance cassette. Further, double and triple knockout combinations of the above genes will also be completed. The various deletions will be assessed by comparing growth rates of wild type and mutant strains. Additionally, nitrogenase activity will be measured with whole cell acetylene reduction assays using gas chromatography. Single gene deletions are expected to have minimal impact while certain double/triple deletions will result in significant growth defects under nitrogen-fixing conditions. These experiments will ultimately provide insight into the relevance of Fld and Fd proteins for electron donation to nitrogenase.
Investigating Electron Transfer to Nitrogenase
Nitrogen is critical for all living organisms, yet atmospheric nitrogen (N2) contains a strong triple bond leaving it chemically inert and unusable by most cells. Some microbes have an enzyme called nitrogenase which converts N2 into ammonia (NH3). Small proteins called flavodoxins (Fld) and ferredoxins (Fd) accumulate the low-potential electrons needed for this reduction reaction. The nitrogen-fixing bacterium, Azotobacter vinelandii, contains multiple Fld and Fd proteins, yet those critical for nitrogen fixing remain unclear. To determine which Fld and Fd proteins are relevant to nitrogenase activity, four genes proposed to play a role in electron transfer, nifF, fdxN, fdxA, and fixP, were selected and the coding region was replaced with an antibiotic resistance cassette. Further, double and triple knockout combinations of the above genes will also be completed. The various deletions will be assessed by comparing growth rates of wild type and mutant strains. Additionally, nitrogenase activity will be measured with whole cell acetylene reduction assays using gas chromatography. Single gene deletions are expected to have minimal impact while certain double/triple deletions will result in significant growth defects under nitrogen-fixing conditions. These experiments will ultimately provide insight into the relevance of Fld and Fd proteins for electron donation to nitrogenase.
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