Intellectual merit: N2 fixation is effected exclusively by bacteria and archea using the enzyme nitrogenase, which contains Fe and Mo as metal cofactors in its most active form, and Fe and V, or Fe-only in its alternative forms. The aim of this project is to elucidate how the mechanisms and kinetics of bacterial acquisition of Fe, Mo and V from soils may limit or control N2 fixation rates. N2-fixing bacteria produce siderophores (or ?iron carriers?) to bind iron in the external medium and take up the resulting Fe-siderophore complexes. Our recent work has shown that the siderophores are actually ?metallophores? used in the uptake of Mo and V, along with Fe. The biological acquisition of the various nitrogenase metal cofactors thus depends on their binding by bacterial metallophores. This project is organized around two major hypotheses: 1. Free-living N2-fixing bacteria excrete metallophores that are particularly efficient at capturing the metal (Mo, V or Fe) that is limiting N2 fixation; 2. Because of both competition with other metals for metallophore binding and scarcity in soils, Mo is inherently difficult to acquire and free-living N2-fixing bacteria often use alternative nitrogenases, particularly the V-nitrogenase, to fix N2. These hypotheses will be tested through a combination of laboratory and field experiments. Field studies will focus on sites where Mo may be limiting and employ molecular biological techniques to identify alternative nitrogenases.

Broader Impacts: One postdoctoral researcher, one graduate student and several undergraduates will work on this project and be mentored by the PIs. Students from the local junior colleges will participate in the summer field research. The PIs will participate in the Quest summer program for school teachers.

Project Report

Nitrogen fixation, the conversion of biologically unavailable atmospheric nitrogen gas into available ammonia, is a major source of nitrogen to terrestrial systems. The fertility of ecosystems is often limited by the amount of nitrogen available, but the chemical and biological factors that control nitrogen fixation in a given ecosystem are not always known. In the current context of global change, a better understanding of the controls on nitrogen fixation may become critical, as the increase in terrestrial primary production due to increased carbon dioxide in the atmosphere, the so-called "CO2 fertilization effect", has been shown to be eventually limited by the availability of nitrogen. The enzyme nitrogenase, which is responsible for nitrogen fixation, requires iron and, in its most efficient form, molybdenum. When molybdenum is not available, some bacteria fix nitrogen using the alternative vanadium-nitrogenase or iron-only nitrogenase, which have vanadium or iron, respectively, at their active center in place of molybdenum. Previous research has shown molybdenum limitation of nitrogen fixation in tropical forests, indicating that molybdenum limitation may be more important than usually recognized. Alternative nitrogenases may provide a way to relieve molybdenum limitation, but their role and importance in natural and agricultural ecosystems is poorly known, largely because the detection of alternative nitrogenases in-situ remains challenging. Over the course of this project: 1- we confirmed molybdenum limitation of nitrogen fixation in tropical forests, and achieved a better understanding of how molybdenum and phosphorus interact to create conditions of co-limitation; 2- we studied the role of vegetation and atmospheric inputs in the cycling of phosphorus, molybdenum, vanadium and iron in soils to understand how Mo limitation may arise in natural ecosystems; 3- we improved the existing molecular methods for the detection of alternative nitrogenases by developing new primers and increasing the number of nitrogenase gene sequences available, 4- we developed a new assay to detect the activity of alternative nitrogenases; and 5- we showed that alternative nitrogenases have a distinctive nitrogen isotopic signature, which has important consequences for our understanding of the nitrogen cycle in the present and in the past. To disseminate our research, we published our findings in scientific journals; we also developed a laboratory module for college Freshmen on nutrient limitation and nitrogen fixation in the Florida Everglades in the context of global change. We participated in "Science Day" at the local elementary school with activities introducing children to the concept of nutrients and the effect of global change on terrestrial ecosystems.

Agency
National Science Foundation (NSF)
Institute
Division of Earth Sciences (EAR)
Application #
1024553
Program Officer
Enriqueta Barrera
Project Start
Project End
Budget Start
2010-09-01
Budget End
2014-08-31
Support Year
Fiscal Year
2010
Total Cost
$338,809
Indirect Cost
Name
Princeton University
Department
Type
DUNS #
City
Princeton
State
NJ
Country
United States
Zip Code
08544