Nitrogenase in the archaebacterium Methanosarcina barkeri 227

Abstract

The discovery of nitrogen fixation in the archaebacterium Methanosarcina barkeri 227 raises questions concerning the similarity of archaebacterial nitrogenases to Mo and alternative nitrogenases in eubacteria. A scheme for achieving a 20-to 40-fold partial purification of nitrogenase components from strain 227 was developed by using protamine sulfate precipitation, followed by using a fast protein liquid chromatography apparatus operated inside an anaerobic glove box. As in eubacteria, the nitrogenase activity was resolved into two components. by strain 227 cells. Antiserum against component 2 of Rhodospirillum rubrum nitrogenase was found to cross-react with component 2 from strain 227, and Western immunoblots using this antiserum showed no evidence for covalent modification of component 2. Also, extracts of strain 227 cells prepared before and after switch-off had virtually the same level of nitrogenase activity. In conclusion, the nitrogenase from strain 227 is similar in overall structure to the eubacterial nitrogenases and shows greatest similarity to alternative nitrogenases.In 1984 to 1985, the property of diazotrophy (nitrogen fixation) was reported in the methanogenic archaebacteria Methanosarcina barkeri 227 (19), Methanococcus thermolithotrophicus (2), and M. barkeri Fusaro (3). Sibold et al. (27) reported hybridization between eubacterial nifHDK probes and DNA from four different methanogenic species. These discoveries were the first confirmed examples of diazotrophy among members of the archaebacteria and have been followed by other descriptions of nitrogen-fixing methanogenic archaebacteria (1,9,13,18 There have been several recent studies on the physiology of diazotrophy in methanogens (1,3,16,18,25). In our previous study on M. barkeri 227 (16) we found similarities to the process in eubacteria, including significant decreases in growth yields and rates when N2 was the N source, stimulation of diazotrophic growth by Mo, and switch-off of C2H2-reducing activity in response to added NH4+. Scherer (25) has demonstrated stimulation of diazoatrophic growth in two strains of M. barkeri by V as well as Mo. We and others have found anomalously low in vivo and in vitro nitrogenase activity, as measured by rates of C2H2 reduction to C2H4 in all diazotrophic methanogens studied thus far (2, 3, 16, 18). The rates measured for methanogen cells and extracts are typically 100-to 500-fold lower than those of typical eubacterial diazotrophs with similar growth rates. One hypothesis considered (16) was that C2H2 was a poorer substrate than N2 for methanogen nitrogenases; another is that methanogen nitrogenases are switched off when assayed (18).We report here the partial purification of nitrogenase from strain 227, a description of its subunit composition, and measurements of its activity toward C2H2 and N2. We also describe more detailed studies on NH4' switch-off and its mechanism in strain 227. 6789on May 13, 2018 by guest

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