Nitrogenase in a Methanogenic Archaeon

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 o L P`J P`J？@A B C D E F G H F Microsoft Word 6.0 Document MSWordDoc Word.Document.6；Zinder，Stephen H.MCB-9506330 1984年，报道了在产甲烷古细菌(古细菌)中发现固氮作用，这是在真细菌领域之外首次发现这一过程。该菌含有两套固氮酶基因，一套与巴氏梭菌的固氮酶基因聚在一起，对纯化的铁蛋白进行N末端测序和RNA印迹分析表明，后者在生长在含钼培养基上的巴氏杆菌细胞中得到表达。对固氮酶序列的系统发育分析发现，与16S rRNA系统发育树不太一致的类群，提出了关于固氮酶多样性的有趣的进化问题。更多的nif基因将从巴克分枝杆菌中克隆和测序。目前已知的产甲烷菌只有两个nifD序列，两个部分nifK序列，没有任何其他nif基因的序列。聚合酶链式反应和相关的克隆技术正被用于从更广泛的真细菌和古生菌中获得DNA序列，包括甲烷八叠球菌的近亲和各种已知的固氮真细菌，但它们的nif基因尚未测序，如氯杆菌和幽门螺杆菌。将进一步研究产甲烷固氮酶和梭状芽孢杆菌固氮酶之间的相似性，以期填补固氮酶系统发育树中的重要空白，以及我们对固氮酶进化和多样性的理解。氮对动物、植物和微生物营养的重要性仅次于碳。虽然我们周围都是氮气，但只有某些固氮菌能够将氮转化为生物体可以利用的形式。固定氮的化学过程需要接近1000华氏度的温度和每平方英寸超过10,000磅的压力，而细菌可以在室温下使用一种名为固氮酶的酶复合体固定氮。这种反应对几乎所有生态系统中的养分循环至关重要，包括农业生态系统。该实验室发现，产甲烷细菌(或产甲烷菌)可以固定氮。这些细菌不仅具有明显的实际意义，而且分子研究表明，它们是古细菌或古细菌的成员，这一组与以前被证明固定氮的细菌(真细菌)完全不相关，事实上，它们可能与真核细胞--有核的细胞--关系更近。对巴氏甲烷链霉菌的固氮生物化学和分子生物学进行了研究。有趣的是，它的固氮酶中的氨基酸序列与真细菌Parteurianum的氨基酸序列相似，这表明基因在两个遥远的亲缘生物之间转移。为了更好地了解这些酶复合体的进化和生物多样性，我们将用来自各种细胞的固氮酶进一步研究这些差异。*；哦+‘0$H L S u m a r y in o r m a t i o n(D h R：\WWUSER\TEMPLATE\NORMAL.DOT Zinder，Stephen H.Robert Uffen Robert Uffen@E J@@？邮箱：J@L Microsoft Word 6.0 2；e=e L j 1%

; 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:\WWUSER\TEMPLATE\NORMAL.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 %