Weidenbach Katrin, Ehlers Claudia, Schmitz Ruth A
Institut für Allgemeine Mikrobiologie, Universität Kiel, Germany.
FEBS J. 2014 Aug;281(15):3507-22. doi: 10.1111/febs.12876. Epub 2014 Jul 4.
With the aim of unraveling their potential involvement in the regulation of nitrogen metabolism in Methanosarcina mazei strain Gö1, we characterized five genes that are differentially transcribed in response to changing nitrogen availability and encoding putative transcriptional regulators. Study of the respective mutant strains under nitrogen-limited conditions revealed a growth delay for M. mazei MM0444::pac and MM1708::pac, and strongly reduced diazotrophic growth for MM0872::pac, whereas the absence of MM2441 or MM2525 did not affect growth behaviour. Transcriptome analyses further demonstrated that only MM1708 - encoding a CxxCG zinc finger protein - plays a regulatory role in nitrogen metabolism, most likely by specifically enhancing transcription of the N2 fixation (nif) operon under nitrogen-limited conditions. In agreement with this, a palindromic binding motif was predicted in silico in the nifH promoter region, nine nucleotides upstream of the BRE box, and confirmed to bind purified maltose-binding protein-MM1708 by electromobility shift assays. As MM1708 itself is under the control of the global nitrogen repressor NrpR, this adds a secondary level to the transcriptional regulation of the nif genes, and is most likely crucial for maximal nif induction under nitrogen-limited conditions. This is in accordance with the finding that protein expression of NifH is highly reduced in the absence of MM1708 under nitrogen-limited conditions. On the basis of our findings, we hypothesize that, in M. mazei, nitrogen fixation is controlled by a hierarchical network of two transcriptional regulators, the global nitrogen repressor NrpR, and the newly identified activator NrpA (MM1708), thereby providing tight control of N2 fixation.
为了阐明它们在马氏甲烷八叠球菌菌株Gö1氮代谢调控中的潜在作用，我们对五个基因进行了表征，这些基因在氮供应变化时差异转录，并编码假定的转录调节因子。在氮限制条件下对各个突变菌株的研究表明，马氏甲烷八叠球菌MM0444::pac和MM1708::pac生长延迟，而MM0872::pac的固氮生长显著降低，而缺失MM2441或MM2525不影响生长行为。转录组分析进一步表明，只有编码CxxCG锌指蛋白的MM1708在氮代谢中起调节作用，很可能是通过在氮限制条件下特异性增强固氮（nif）操纵子的转录。与此一致的是，在计算机模拟中预测到nifH启动子区域在BRE框上游九个核苷酸处有一个回文结合基序，并通过电泳迁移率变动分析证实其与纯化的麦芽糖结合蛋白-MM1708结合。由于MM1708本身受全局氮阻遏物NrpR的控制，这为nif基因的转录调控增加了一个二级水平，并且很可能对氮限制条件下nif的最大诱导至关重要。这与在氮限制条件下缺失MM1708时NifH蛋白表达高度降低的发现一致。基于我们的发现，我们假设，在马氏甲烷八叠球菌中，固氮由两个转录调节因子的分级网络控制，即全局氮阻遏物NrpR和新鉴定的激活剂NrpA（MM1708），从而对N2固定提供严格控制。
