State Key Laboratory of Microbial Technology, College of Life Science, Shandong University, Jinan, 250100, People's Republic of China.
Curr Microbiol. 2011 Jul;63(1):32-8. doi: 10.1007/s00284-011-9936-y. Epub 2011 Apr 17.
NtcA, highly conserved in cyanobacteria, regulates the expression of a large number of genes involved in nitrogen and carbon metabolisms, photosynthesis, and stress responses. In the filamentous diazotrophic cyanobacterium Anabaena PCC 7120, NtcA is also required for the initiation of heterocyst differentiation, triggered by the accumulation of 2-oxoglutarate (2-OG) following nitrogen starvation. Recent structural studies reveal the binding pocket of 2-OG on each of the two subunits of the NtcA homodimer, and indicate a route of signal transmission upon 2-OG binding. In this study, we studied the effect of mutations of two critical residues in the effector-binding domain of NtcA on heterocyst differentiation. Mutations of these residues could change strongly the ability of NtcA to sense the nitrogen-starvation signal in vivo. As a result of these mutations, the corresponding strains were unable to form any heterocysts, or form a few heterocysts at a very low frequency. Consistent with these phenotypes, these mutations were defective in initiating transcription by the RNA polymerase in the presence of 2-OG as determined by a reconstituted in vitro transcriptional assay. The different effects of the two mutations were consistent with the roles of the two corresponding residues in 2-OG binding highlighted by recent structural analysis of the NtcA-2-OG complex. These studies provided genetic evidence for the importance of the effector-binding domain in the regulatory function of NtcA.
NtcA 在蓝藻中高度保守，调节涉及氮碳代谢、光合作用和应激反应的大量基因的表达。在丝状固氮蓝藻 Anabaena PCC 7120 中，NtcA 也需要 2-氧戊二酸 (2-OG) 的积累来触发异形胞分化，氮饥饿会导致 2-OG 的积累。最近的结构研究揭示了 NtcA 同源二聚体的每个亚基上 2-OG 的结合口袋，并指出了 2-OG 结合后的信号传递途径。在这项研究中，我们研究了 NtcA 效应物结合域中两个关键残基突变对异形胞分化的影响。这些残基的突变可以强烈改变 NtcA 体内感知氮饥饿信号的能力。由于这些突变，相应的菌株无法形成任何异形胞，或者以非常低的频率形成少数异形胞。与这些表型一致，这些突变在存在 2-OG 的情况下，通过体外转录实验确定的 RNA 聚合酶起始转录的能力有缺陷。这两种突变的不同影响与最近 NtcA-2-OG 复合物结构分析突出的两个相应残基在 2-OG 结合中的作用一致。这些研究为效应物结合域在 NtcA 调节功能中的重要性提供了遗传证据。
