Laboratoire Adaptation et Pathogénie des Microorganismes, Université Joseph Fourier, CNRS UMR5163, 38700 La Tronche, France and INRIA Grenoble-Rhône-Alpes, 38334 Saint Ismier Cedex, France.
Nucleic Acids Res. 2013 Sep;41(17):e164. doi: 10.1093/nar/gkt655. Epub 2013 Jul 26.
We have developed a new screening methodology for identifying all genes that control the expression of a target gene through genetic or metabolic interactions. The screen combines mutant libraries with luciferase reporter constructs, whose expression can be monitored in vivo and over time in different environmental conditions. We apply the method to identify the genes that control the expression of the gene acs, encoding the acetyl coenzyme A synthetase, in Escherichia coli. We confirm most of the known genetic regulators, including CRP-cAMP, IHF and components of the phosphotransferase system. In addition, we identify new regulatory interactions, many of which involve metabolic intermediates or metabolic sensing, such as the genes pgi, pfkA, sucB and lpdA, encoding enzymes in glycolysis and the TCA cycle. Some of these novel interactions were validated by quantitative reverse transcriptase-polymerase chain reaction. More generally, we observe that a large number of mutants directly or indirectly influence acs expression, an effect confirmed for a second promoter, sdhC. The method is applicable to any promoter fused to a luminescent reporter gene in combination with a deletion mutant library.
我们开发了一种新的筛选方法，用于通过遗传或代谢相互作用识别控制靶基因表达的所有基因。该筛选将突变文库与荧光素酶报告基因构建体相结合，其表达可以在不同的环境条件下进行体内和随时间的监测。我们应用该方法来鉴定控制基因 acs（编码乙酰辅酶 A 合成酶）在大肠杆菌中表达的基因。我们确认了大多数已知的遗传调节剂，包括 CRP-cAMP、IHF 和磷酸转移酶系统的组成部分。此外，我们还发现了新的调节相互作用，其中许多涉及代谢中间产物或代谢感应，例如编码糖酵解和 TCA 循环中酶的 pgi、pfkA、sucB 和 lpdA 基因。其中一些新的相互作用通过定量逆转录-聚合酶链反应得到了验证。更一般地说，我们观察到大量突变体直接或间接影响 acs 表达，这一效应在第二个启动子 sdhC 中得到了证实。该方法适用于任何与发光报告基因融合的启动子，与缺失突变体文库相结合。
