; R o o t E n t r y F }>a J C o m p O b j b W o r d D o c u m e n t L O b j e c t P o o l P ` J P ` J ? @ A B C D E F G H I F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; Zinder, Stephen H. MCB-9506330 In 1984, the discovery of nitrogen fixation was reported in methanogenic Archaea (Archaebacteria), the first discovery of this process outside the eubacterial domain. Methanosarcina backeri has two sets of nitrogenase (nif) genes, one which clusters with the nitrogenase from the eubacterium Clostridium pasteurianum, N-terminal sequencing of the purified Fe protein and RNA-blot analysis showed that the latter nitrogenase was expressed in M. barkeri cells growing on Mo-containing medium. Phylogenetic anayses of the nitrogenase sequences have found groupings which are not easily reconciled with the 16S rRNA phylogenetic tree, raising interesting evolutionary questions concerning nitrogenase diversity. Additional nif genes will be cloned and sequenced from M. barkeri. At the present time there are only two nifD sequences for methanogens known, two partial nifK sequences, and no sequences of any other nif genes. The polymerase chain reaction and related cloning techniques are being used to obtain DNA sequences from a wider diversity of Eubacteria and Archaea, includi ng relatives of Methanosarcina and diverse other eubacteria known to fix nitrogen, but which have not had their nif genes sequenced, such as Chlorobium and Heliobacterium. The similarities between methanogenic and clostridial nitrogenases will be examined further in the hope that important gaps can be filled in the phylogenetic tree for nitrogenases and in our understanding of nitrogenase evolution and diversity. %%% Nitrogen is second only to carbon in its importance to animal, plant, and microbial nutrition. Although, we are surrounded by an atmosphere of nitrogen gas, only certain nitrogen-fixing bacteria re able to convert nitrogen to forms which can be used by organisms. A chemical process for fixing nitrogen requires temperatures near 1000 F and pressures over 10,000 pounds per square inch, while bacteria can fix nitrogen at room temperature using the enzyme complex called nitrogenase. This reaction is crucial to nutrient cycling in nearly all ecosystems, including agricultural ones. This laboratory discovered that methane-producing bacteria (or methanogens) can fix nitrogen. Not only are these bacteria of obvious practical significance, but molecular studies have shown that they are members of the Archaebacteria or Archaea, a group completely unrelated to the bacteria previously shown to fix nitrogen (eubacteria), and, in fact, probably more closely related to eucaryotes--cells with nuclei. Studies of the biochemistry and molecular biology of nitrogen fixation in Methanosarcina barkeri were performed. Interestingly, the sequence of amino acids in its nitrogenase resembles that from the eubacterium, Clostridium parteurianum, suggesting that genes were transferred between two distantly related organisms. The differents will be examined further with nitrogenases from a wide variety of cells in order to better understand the evolution and biodiversity of these enzyme complexes. *** ; Oh +' 0 $ H l S u m m a r y I n f o r m a t i o n ( > D h R:WWUSERTEMPLATENORMAL.DOT Zinder, Stephen H. Robert Uffen Robert Uffen @ E J @ @ ? J @ L Microsoft Word 6.0 2 ; e = e L j j j j j j j 1 %
