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JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 987-995 Vol. 140, No. 3 0021-9193/79/12-0987/09$02.00/0 Manganese, an Essential Trace Element for N2 Fixation By Rhodospirillum rubrum and Rhodopseudomonas capsulata: Role in Nitrogenase Regulation DUANE C. YOCH Department ofBiology, University ofSouth Carolina, Columbia, South Carolina 29208 Received for publication 3 August 1979 Nitrogenase (N2ase) from the photosynthetic bacterium Rhodospirillum rub- rum can exist in two forms, an unregulated form (N2ase A) and a regulatory form (N2ase R), the latter being identified in vitro by its need for activation by a Mn2+_ dependent N2ase activating system. The physiological significance of this Mn2+- dependent N2ase activating system was suggested here by observations that growth of R. rubrum and Rhodopseudomonas capsulata on N2 gas (a condition that produces active N2ase R) required Mn2+, but growth on ammonia or gluta- mate did not. Manganese could not be shown to be required for the biosynthesis of either nitrogenase or glutamine synthetase or for glutamine synthetase turn- over, but it was required for the in vitro activation of N2ases from N2 and glutamate-grown R. rubrum and R. capsulata cells. Chromatium N2ase, in contrast, was always fully active and did not require Mn2+ activation, suggesting that only the purple nonsulfur bacteria are capable of controlling their N2ase activity by this new type of regulatory system. Although R. rubrum could not substitute Fe2+ for Mn2+ in the in vivo N2 fixation process, Fe2+ and, to a lesser extent, Co2, could substitute for Mn2+ in the in vitro activation of N2ase. Electron paramagnetic resonance spectroscopy of buffer-washed R. rubrum chromato- phores showed lines characteristic of Mn2+. Removal of the Mn2+-dependent N2ase activating factor by a salt wash of the chromatophores removed 90% of the Mn2 , which suggested a specific coupling of this metal to the activating factor. The data presented here all indicate that Mn2+ plays an important physiological role in regulating the N2 fixation process by these photosynthetic bacteria. A role for manganese (Mn2") in the nitrogen Both forms of N2ase are interconvertable in vivo fixation process was implicated several years ago under a variety of conditions related to the cells when it was shown that Mn2+ greatly enhanced available nitrogen substrate (or lack of it) (5). nitrogenase (N2ase) activity in extracts from the The relationship between these two forms of photosynthetic bacterium Rhodospirillum rub- N2ase is shown by equation 1: rum (17, 24). Manganese was shown in these studies to act in conjunction with a chromato- Growth on N2 phore-bound or glutamate "activating factor" and ATP to I N2ase A - N2ase R (1) activate the Fe protein of N2ase in extracts pre- N starvation pared from glutamate (18) or N2-grown cells (25). Once activated, the Fe protein could func- Although N2ase A and R appear to be an integral tion normally with MoFe protein to form an part of a system which regulates N2ase activity active N2ase complex (17, 18,24), and Mn2+ was in R. rubrum, a link has not, however, been not required for catalysis. It was subsequently established between N2ase regulation in vivo shown (5) that R. rubrum N2ase could exist in and the in vitro N2ase R activation process (17) two chemically defined forms. One form, N2ase represented by equation 2. A, found only in nitrogen-starved celLs, was fully Activating factor active without the activating cofactors, whereas Mn2+, ATP a second form of the enzyme, whose activity is N2ase Rinactive N2ase Ractive (2) capable of being regulated, was isolated from Spontaneous during N2- or glutamate-grown cells and required a cell disruption Mn2' activating factor system for its activation; For reasons not yet understood, N2ase is always this form of the enzyme was called N2ase R. inactive in extracts prepared from either N2- or 987 988 YOCH J. BBACTERIOL. glutamate-grown cells and must be activated cated, all cells used for the various enzyme prepara- before its activity can be measured. Because tions were cultured in media containing the regular both inactivation of N2ase and its reactivation concentrations of manganese. by the Mn2+-dependent process are observed All glassware used in studies to determine Mn2" only in vitro, it seemed necessary to demonstrate requirements was rinsed four or five times with double- distilled water. Growth media for these studies were that this reaction (equation 2) was also func- also prepared with double-distilled water. These pre- tional at the cellular level if, as we believe, the cautions seemed to be adequate for keeping most activity of N2ase R is really modulated by the contaminating Mn2" out of the media because after cell in response to changes in its nutritional several passages in minus-Mn2" media, N2-fixing cul- environment. Since N2ase R must be active in tures could be shown to have a requirement for this vivo when cells are growing on N2, it seemed trace element. likely that if the Mn2+-dependent N2ase activat- Cultures (except those fixing N2) were grown in 1-, ing system was of physiological significance, it 2-, or 4-liter glass-stoppered Pyrex bottles filled com- might be possible to show an Mn2" requirement pletely to exclude air. Nitrogen-fixing cultures were grown in 250-ml side-arm flasks containing 50 ml of for R. rubrum growing on N2. media. After inoculation, these flask were evacuated This communication describes experiments and refilled at least four times with N2 gas. Illumina- showing that indeed Mn2" is required for growth tion was provided by placing the cultures between two on N2 and that this requirement cannot be ex- banks of four 40-W fluorescent lights (Vita-Lite, Duro- plained by any process related to nitrogen fixa- Test Corp., North Bergen, N.J.). This system provided tion except the activation of N2ase R. N2ase A about 950 footcandles of light at the surface of the and R were also found in Rhodopseudomonas culture bottles. Culture temperatures were maintained capsulata, thus extending this unique N2ase reg- between 25 and 300C. to a second of Extract preparations. All N2ase extracts were ulatory system species photosyn- prepared from freshly harvested cells which were re- thetic bacteria. This regulatory system may, suspended in approximately 2 volumes of argon-satu- however, be limited to the family Rhodospiril- rated 330 mM tricine buffer (pH 8.5) containing 4 mM laceae, since Chromatium vinosum N2ase did sodium dithionite. The resuspended cells before being not respond under any condition to Mn2' and broken were placed in a flask which was closed with a activating factor. serum bottle stopper and evacuated and flushed with argon several times. Cells were disrupted by sonic MATERLALS AND METHODS oscillation with a Sonifier cell disruptor (Heat Systems Inc.) at 65 W output for two 15-s periods. The soni- Bacterial strains. Both R. rubrum strain S-1 and cation vessel was a 60-rnl glass beaker covered with C. vinosum (fornerly Chromatium D) were single- Parafilm with a hole in it just large enough to insert colony (N2-fixing) isolates of the parent strains ob- the sonicator probe. After the probe was inserted, this tained from D. I. Arnon. R. capsulata strain B-10 was closed vessel was purged with argon 5 min before the kindly provided by J. D. Wall (initially from B. Marrs). cell suspension was transferred to it anaerobically by Media and bacterial growth conditions. R. rub- syringe; the gas flow through the vessel was main- rum and R. capsulata were grown photosynthetically tained during the sonication period. The broken cells on the medium of Ormerod et al. (26) supplemented were transferred by syringe to a capped, degassed with 20 mM malate. (For growth of R. capsulata, centrifuge tube and centrifuged at 30,000 x g for 10 thiamine [300 ug/liter] replaced biotin.) The nitrogen min. The pellet was discarded, and the supernatant source for this medium (26) was modified for the extract was either used directly as a source of N2ase various experiments as follows. (i) In experiments to and activating factor or was further fractionated by test the cells' ability to grow in the absence of Mn2" centrifugation at 240,000 x g for 90 min. The super- (Fig. 1 and 2), the nitrogen source was either N2 gas, natant fraction from the high-speed centrifugation 20 mM glutamate, or 10 mM (NH4)2SO4, as indicated. contained the soluble N2ase, and the pellet contained (ii) For testing the cells' ability to derepress N2ase in the chromatophore membranes (to which the N2ase the absence of Mn2+ (Fig. 3) and for cells containing activating factor was bound). high levels of glutamine synthetase (Fig. 4), growth- Enzyme assays. N2ase was assayed and ethylene limiting concentrations of NH4' (2.5 mM) were used was determined as described by Carithers et al. (5). to ensure eventual nitrogen starvation of the culture. Biosynthetic glutamine synthetase activity was as- (iii) To obtain cells which contained predominately sayed as described by Shapiro and Stadtman (29); the N2ase R, the nitrogen source was either growth-limit- procedure of Bender et al. (2) was used for the y- ing concentrations of NH4' (2.5 mM) followed by the glutamyl transferase assay of this enzyme. The gluta- addition of 0.75 mM glutamate after the N2ase was mine synthetase adenylylation number (ii) was deter- completely derepressed (Fig. 4) or, alternatively, grow- mined as previously described (30). Reaction mixture ing the cells on 5 mM glutamate to the point of constituents for these assays are found in the appro- nitrogen starvation (as evidenced by the vigorous pho- priate figure legends. toevolution of H2). C. vinosum was grown photosyn- Protein was determined by the biuret procedure thetically on a medium described by Arnon et al.

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