Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China.
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, PR China.
Microbiology (Reading). 2013 Dec;159(Pt 12):2571-2583. doi: 10.1099/mic.0.071449-0. Epub 2013 Sep 25.
In actinomycetes, two main regulators, the OmpR-like GlnR and the TetR-type AmtR, have been identified as the central regulators for nitrogen metabolism. GlnR-mediated regulation was previously identified in different actinomycetes except for members of the genus Corynebacterium, in which AmtR plays a predominant role in nitrogen metabolism. Interestingly, some actinomycetes (e.g. Streptomyces avermitilis) harbour both glnR- and amtR-homologous genes in the chromosome. Thus, it will be interesting to determine how these two different types of regulators function together in nitrogen regulation of these strains. In this study, AmtRsav (sav_6701) in S. avermitilis, the homologue of AmtR from Corynebacterium glutamicum, was functionally characterized. We showed, by real-time reverse transcription (RT)-PCR (qPCR) in combination with electrophoretic mobility shift assays (EMSAs), that gene cluster sav_6697-6700 encoding a putative amidase, a urea carboxylase and two hypothetical proteins, respectively, and sav_6709 encoding a probable amino acid permease are under the direct control of AmtRsav. Using approaches of comparative analysis combined with site-directed DNA mutagenesis, the AmtRsav binding sites in the respective intergenic regions of sav_6700/6701 and sav_6709/6710 were defined. By genome screening coupled with EMSAs, two novel AmtRsav binding sites were identified. Taken together, AmtRsav seems to play a marginal role in regulation of nitrogen metabolism of S. avermitilis.
在放线菌中，已经确定了两种主要的调节剂，OmpR 样的 GlnR 和 TetR 型 AmtR，它们是氮代谢的中心调节剂。GlnR 介导的调节以前在不同的放线菌中被鉴定出来，除了棒状杆菌属的成员，在这些成员中 AmtR 在氮代谢中起主要作用。有趣的是，一些放线菌（例如阿维链霉菌）在染色体中同时含有 glnR-和 amtR-同源基因。因此，确定这两种不同类型的调节剂如何在这些菌株的氮调节中协同作用将是有趣的。在这项研究中，阿维链霉菌中的 AmtRsav（sav_6701），即谷氨酸棒状杆菌 AmtR 的同源物，其功能得到了表征。我们通过实时 RT-PCR（qPCR）与电泳迁移率变动分析（EMSA）相结合表明，基因簇 sav_6697-6700 分别编码一个假定的酰胺酶、一个脲酶和两个假定的蛋白质，而 sav_6709 编码一个可能的氨基酸渗透酶，直接受 AmtRsav 的控制。通过比较分析与定点 DNA 诱变相结合的方法，确定了 sav_6700/6701 和 sav_6709/6710 中相应基因间区的 AmtRsav 结合位点。通过基因组筛选与 EMSA 相结合，鉴定了两个新的 AmtRsav 结合位点。总之，AmtRsav 似乎在阿维链霉菌氮代谢调节中发挥着次要作用。
