The regulatory network of nitrogen assimilation and fixation in the methanogenic Archaeon Methanosarcina mazei strain Gö1

产甲烷古生菌Gö1菌株在氮素限制条件下生长时，能够固定分子氮。通过对基因组序列的分析，我们鉴定并鉴定了含钼固氮酶的编码基因。通过这个项目，我们试图使用经典的遗传方法和现代分子工具来分析Mazei女士的氮代谢调控网络，这些方法来自于对完整基因组序列的了解。第一个主要目标是利用二维凝胶电泳法和Northern印迹分析来识别和表征受铵调控的基因。第二个目标是使用两种策略来分析氮调控基因的调控元件：(I)对已鉴定的氮调控基因进行启动子比较分析；(Ii)利用已鉴定的氮调控启动子区域，通过亲和层析和凝胶滞留分析从不同氮素利用率下生长的细胞中鉴定启动子结合蛋白。为了获得更多关于氮调控子的信息以及氮和碳调控子之间的潜在交互作用，将利用DNA微阵列来分析整个基因组在不同氮素可用性和不同碳源存在下的表达模式。

The mesophilic methanogenic Archaeon Methanosarcina mazei strain Gö1 is able to fix molecular nitrogen when growing under nitrogen-limited conditions. Analyzing the sequence of the genome we have identified and characterized the genes encoding the molybdenum-containing nitrogenase. With this project we seek to analyze the regulatory network of nitrogen metabolism in Ms. mazei using classical genetic approaches and modern molecular tools arising from the knowledge of the complete genome sequence. The first major goal is to identify and to characterize genes, which are regulated by ammonium, using 2-dimensional gel electrophoresis and northern blot analyses. The second goal is to analyze regulatory elements of the nitrogen regulon using two strategies: (i) comparative promoter analyses of the identified nitrogen regulated genes and (ii) identification of promoter binding proteins from cells grown at different nitrogen availabilities by affinity chromatography and gel retardation assays using the identified nitrogen regulated promoter regions. To obtain additional information about the nitrogen regulon and potential cross talks between the nitrogen and the carbon regulon, expression patterns of the whole genome at different nitrogen availabilities and in the presence of different carbon sources will be analyzed by employing DNA-microarrays.