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

中温产甲烷古细菌Methanosarcina mazei菌株Gö1在氮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，我们鉴定并鉴定了编码含钼氮酶的基因。在这个项目中，我们试图利用经典的遗传方法和现代分子工具来分析Ms. mazei的氮代谢调控网络。第一个主要目标是利用二维凝胶电泳和northern blot分析鉴定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的调控元件：(i)对已鉴定的氮调控基因进行比较启动子分析；（ii）利用已鉴定的氮调控启动子区域，通过亲和层析和凝胶阻滞试验，从不同氮可用性下生长的细胞中鉴定启动子结合蛋白。为了获得氮调控子的更多信息以及氮和碳调控子之间潜在的交叉对话，将利用dna微阵列分析不同氮可用性和不同碳源存在下全基因组的表达模式。

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.