Vol. 59, No. 3 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1993, p. 669-676 0099-2240/93/030669-08$02.00/0 Copyright © 1993, American Society for Microbiology Modification of the Fe Protein of Nitrogenase in Natural Populations of Trichodesmium thiebautii JONATHAN P. ZEHR,lt* MICHAEL WYMAN,2 VERONICA MILLER,3 LINDA DUGUAY,3 AND DOUGLAS G. CAPONE3 Marine Sciences Research Center, State University of New York, Stony Brook, New York 117941; Plymouth Marine Laboratory, Citadel Hill, Plymouth, United Kingdom PL1 2PB2; and Chesapeake Biological Laboratory, University of Maryland, Solomons, Maryland 206883 Received 1 September 1992/Accepted 15 December 1992 The Fe protein of nitrogenase in the marine nonheterocystous cyanobacterium Trichodesmium thiebautii is interconverted between two forms, which is reminiscent of the ADP-ribosylation described in the purple bacterium Rhidospirillum rubrum. In natural populations of T. thiebautii during the day, when nitrogenase activity (NA) is present and while photosynthetic rates are high, a low-molecular-mass form of the Fe protein is present. In the late afternoon, the low-molecular-mass form is partially converted to a higher-molecular-mass form (approximately equal distribution of high- and low-molecular-mass forms of the Fe protein subunits), concurrent with cessation of NA. Some of the higher-molecular-mass form persists through the night until the very early morning, when the lower-molecular-mass form appears. New synthesis of both the Fe and MoFe proteins of nitrogenase appears to occur at this time. The higher-molecular-mass form of the Fe protein is also produced rapidly in response to artificially elevated external 02 levels (40%) during the day. T. thiebautii is capable of recovery of NA in less than 1 h following exposure to 40% 02, which is correlated with the return of the Fe protein to the lower-molecular-mass form. Recovery from exposure to 02 is not dependent upon protein synthesis. The modification of the Fe protein is clearly involved in regulation of NA during the diel cycle of NA in T. thiebautii but may also be involved in protecting the Fe protein during transient 02 concentration increases. The ability to fix atmospheric nitrogen (N2), although clearly beneficial to microorganisms in nature, also poses biochemical and physiological problems for aerobic diaz- otrophs (10). Nitrogenase, the enzyme which catalyzes the reduction of N2 to ammonium, is rapidly inactivated by 02 (10). Although not an insurmountable problem for aerobic heterotrophs, the problem is exacerbated in prokaryotes which depend upon oxygenic photosynthesis for energy and reductant. Despite this paradox, many species of cyanobac- teria possess the ability to fix N2 (33, 34). N2 fixation in the cyanobacteria has been the subject of extensive study in the past 25 years (11, 15). One of the most straightforward and best understood mechanisms in cyanobacteria that permits concurrent N2 fixation and photosynthesis is cellular differentiation of the heterocyst. In heterocystous cyanobacteria, N2 fixation oc- curs primarily in the heterocyst and 02 evolution occurs primarily in vegetative cells (14, 43). However, representa- tives of both filamentous nonheterocystous and unicellular cyanobacterial species also fix N2. Research on various species of nonheterocystous cyanobacteria has not uncov- ered a common mechanism for protecting nitrogenase from 02 (7, 12). Of the species of nonheterocystous cyanobacteria examined, most fix N2 only during the dark phase of a light-dark cycle (13, 18, 21). If forced to fix N2 in the light by growth under continuous light, N2 fixation in these species, in general, is enhanced by reduced 02 or inhibition of photosystem II with the inhibitor dichlorophenyl dimethyl urea (7). In contrast, the marine filamentous nonheterocys- * Corresponding author. t Present address: Biology Department, Rensselaer Polytechnic Institute, Troy, NY 12180. 669 tous cyanobacteria Trichodesmium spp. fix N2 during the day with little change in net 02 evolution rates (28), and do not fix N2 in the dark (2, 25, 36). In these species, then, there are two perplexing issues. (i) How do Trichodesmium spp. perform simultaneous nitrogen fixation and oxygenic photo- synthesis during the day? (ii) How and why does nitrogen fixation occur only during the day, when many other species can store sufficient energy and reductant to fuel nitrogen fixation in the dark? A characteristic of nitrogenase in some heterocystous and nonheterocystous cyanobacteria is a change in the apparent molecular mass of the Fe protein under certain conditions that appears to be correlated with activity (6, 26, 32, 38). In addition, there were initially suggestions of a conformational protection of nitrogenase in response to elevated oxygen in the nonheterocystous cyanobacterium Oscillatoria limosa (39), which may be related to the recently described change in molecular mass of the Fe protein in this species (12, 38). It is possible that the mechanism of change in the apparent molecular mass of the Fe protein in cyanobacteria is similar to the ADP-ribosylation of the Fe protein in Rhodospirillum rubrum (23, 30). Recently, the change in molecular mass of the Fe protein in Tnchodesmium sp. strain NIBB 1067 was shown to be correlated with the diel change in nitrogenase activity (NA) and also was a response to artificially elevated combined nitrogen or 02 concentrations (26, 27, 45). This change in molecular mass may potentially play a central role in the regulation of NA, and since it responds to 02, could be involved in how and why Trichodesmium spp. exclusively maintain NA during the day in situ. We therefore examined natural populations of Tichodesmium thiebautii for the ability to modify the Fe protein of nitrogenase and tried to determine how this modification is involved in the diel cycle of NA and oxygen evolution.