Essential metals for nitrogen fixation in a free-living N 2 -fixing bacterium: chelation, homeostasis and high use efficiency J.-P. Bellenger, 1 * † T. Wichard, 1‡ Y. Xu 1 and A. M. L. Kraepiel 2 1 Department of Geosciences, PEI, Guyot Hall, Princeton University, Princeton, NJ 08544, USA. 2 Chemistry Department, PEI, Guyot Hall, Princeton University, Princeton, NJ 08544, USA. Summary Biological nitrogen fixation, the main source of new nitrogen to the Earth’s ecosystems, is catalysed by the enzyme nitrogenase. There are three nitrogenase isoenzymes: the Mo-nitrogenase, the V-nitrogenase and the Fe-only nitrogenase. All three types require iron, and two of them also require Mo or V. Metal bioavailability has been shown to limit nitrogen fixa- tion in natural and managed ecosystems. Here, we report the results of a study on the metal (Mo, V, Fe) requirements of Azotobacter vinelandii, a common model soil diazotroph. In the growth medium of A. vinelandii, metals are bound to strong complexing agents (metallophores) excreted by the bacterium. The uptake rates of the metallophore complexes are regulated to meet the bacterial metal requirement for diazotrophy. Under metal-replete conditions Mo, but not V or Fe, is stored intracellularly. Under conditions of metal limitation, intracellular metals are used with remarkable efficiency, with essentially all the cellular Mo and V allocated to the nitrogenase enzymes. While the Mo-nitrogenase, which is the most efficient, is used preferentially, all three nitrogenases contribute to N 2 fixation in the same culture under metal limita- tion. We conclude that A. vinelandii is well adapted to fix nitrogen in metal-limited soil environments. Introduction Biological nitrogen fixation, the reaction that converts atmospheric N 2(g) into bioavailable ammonia, is the major natural route of entry for new nitrogen into ecosystems (Galloway et al., 2004). As such, it is a key component of the nitrogen cycle and one of the most important reactions controlling ecosystem fertility (Cleveland et al., 1999; Vitousek et al., 2002). Nitrogen fixation is catalysed by the enzyme nitrogenase, and is carried out exclusively by free-living or symbiotic prokaryotes. The Mo-nitrogenase, which contains Mo at its active site (Einsle et al., 2002), is the most common and most efficient form of the enzyme. Two alternative nitrogenases, the V-nitrogenase and the Fe-only nitrogenase have also been identified (Bishop et al., 1986; Robson et al., 1986; Eady, 1996). In these alternative nitrogenases, which are expressed when Mo is limiting, the Mo atom is replaced by V (in the V-nitrogenase) or Fe (in the Fe-only nitrogenase) (Eady, 2003). A recent study showed that the bioavailability of Mo, which is one of the least abundant essential trace metals in soils (Alloway, 1995), is further reduced by binding to natural organic matter and adsorption to soil particles (Wichard et al., 2009a). This is consistent with other studies showing that Mo is a limiting nutrient for nitrogen fixation in ecosystems ranging from the temper- ate forests of Oregon to the tropical forests of Panama (Silvester, 1989; Gupta, 1997; Barron et al., 2009). This information suggests that the alternative nitrogenases may contribute significantly to nitrogen fixation in selected ecosystems, and possibly worldwide. Indeed, even though only a fraction of N 2-fixing bacteria have the genes encoding for one or both alternative nitrogenases, these genes have been identified in a wide variety of terrestrial environments (Betancourt et al., 2008). The V- nitrogenase is of particular interest because V is 50–100 times more abundant than Mo in soils (Alloway, 1995), suggesting that V could be available when Mo is not. In addition, the V-nitrogenase is more efficient, and expressed preferentially to the Fe-only nitrogenase (Bishop and Joerger, 1990). Recent studies provide a new understanding of the uptake of Mo and V by the soil N 2-fixing bacterium Azo- tobacter vinelandii (Bellenger et al., 2007; 2008a,b; Wichard et al., 2008; 2009b; Kraepiel et al., 2009). They show that strong organic ligands are excreted by the bacteria into their growth medium and form complexes with the available metals (i.e. Fe, Mo, V). These Received 21 May, 2010; accepted 18 January, 2011. *For correspon- dence. E-mail jean-philippe.bellenger@usherbrooke.ca; Tel. (+1) 819-821-7014; Fax (+1) 819-821-8017. Present addresses: † Dépar- tement de Chimie, Université de Sherbrooke, Sherbrooke QC J1K 2R1, Canada; ‡ Institute for Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Lessingtr. 8, 07743 Jena, Germany. Environmental Microbiology (2011) 13(6), 1395–1411 doi:10.1111/j.1462-2920.2011.02440.x © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd