Measurements of Mo : C and Fe : C ratios in cultured cells of two N 2 -fixing cyanobacteria, Crocosphaera watsonii strain WH8501 and Trichodesmium erythraeum strain IMS101, agree with estimated metal : carbon ratios based on growth rate and the metal use efficiency of the nitrogenase enzyme. Crocosphaera, a single-celled nocturnal N 2 fixer, showed two-to eightfold increases in Mo and Fe cellular concentrations in response to nitrogen fixation activity. Mo required for N 2 assimilation can account for almost the entire Mo pool measured in the cells, implying that Crocosphaera synthesizes its entire nitrogenase pool de novo each night. In contrast, cultures of Trichodesmium, a filamentous, diurnal N 2 -fixing cyanobacterium, did not show diel variations in Mo or Fe carbon ratios or in cellular metal concentrations. Trichodesmium appears to maintain an internal pool of Mo. In Trichodesmium cultures, Mo concentrations were up to 30% higher than needed to support measured N 2 fixation. Trichodesmium colonies collected from the field had Mo : C ratios 10-fold larger than those measured in culture, far in excess of what is needed to fix N 2 at rates normally measured in the field, despite equivalent Fe : C ratios (66 Ϯ 39 [field samples] and 87 Ϯ 64 [cultures] mol mol Ϫ1 ). The average Fe : C ratio measured in N 2 -fixing Crocosphaera (16 Ϯ 11 mol mol Ϫ1 ) was equivalent to theoretical estimates of Fe demand based on nitrogenase requirements (13 Ϯ 5 mol mol Ϫ1 ). These results demonstrate the extremely efficient use of Fe by these organisms and provide support for the use of theoretical estimates of Fe : C ratios to calculate biological Fe demand for N 2 fixation.Nitrogen fixation, the conversion of N 2 gas into bioavailable forms, is an important source of nitrogen for primary production in the oligotrophic ocean (Capone et al. 1997;Karl et al. 1997). Nitrogen fixation is catalyzed by the nitrogenase complex, a metal-rich enzyme containing 2 mol molybdenum (Mo) and 38-50 mol iron (Fe) mol Ϫ1 complex. Recent studies have focused on Fe as a potentially limiting nutrient for both photoautotrophic growth and N 2 fixation in the marine environment (Sunda and Huntsman 1995;Berman-Frank et al. 2001a;Kustka et al. 2002). Although there is an ongoing debate whether the high levels of sulfate (SO ) in seawater can competitively inhibit molybdate 2Ϫ 4 1 To whom correspondence should be addressed. Present address: Department of Geosciences, Princeton University, Princeton, New Jersey 08544 (ctuit@princeton.edu).2 Present address: Department of Geology & Geophysics, SOEST, University of Hawaii, Manoa, 1680 East-West Road, Honolulu, Hawaii 96822.
AcknowledgmentsWe are grateful to Eric Webb for his input throughout this study and for his analysis of the Trichodesmium genome for the putative Mo transporter. We thank James Howard for discussions about nitrogenase and acetylene reduction methods. We also thank Rob Sherrell, Dan McCorkle, and two anonymous reviewers for their comments on the early versions of this manuscript. M...