Martí-Arbona Ricardo, Mu Fangping, Nowak-Lovato Kristy L, Wren Melinda S, Unkefer Clifford J, Unkefer Pat J
Bioscience Division, Los Alamos National Laboratory, PO Box 1663, Los Alamos, NM 87545, USA.
BMC Genomics. 2014 Dec 18;15(1):1142. doi: 10.1186/1471-2164-15-1142.
The clustering of genes in a pathway and the co-location of functionally related genes is widely recognized in prokaryotes. We used these characteristics to predict the metabolic involvement for a Transcriptional Regulator (TR) of unknown function, identified and confirmed its biological activity.
A software tool that identifies the genes encoded within a defined genomic neighborhood for the subject TR and its homologs was developed. The output lists of genes in the genetic neighborhoods, their annotated functions, the reactants/products, and identifies the metabolic pathway in which the encoded-proteins function. When a set of TRs of known function was analyzed, we observed that their homologs frequently had conserved genomic neighborhoods that co-located the metabolically related genes regulated by the subject TR. We postulate that TR effectors are metabolites in the identified pathways; indeed the known effectors were present. We analyzed Bxe_B3018 from Burkholderia xenovorans, a TR of unknown function and predicted that this TR was related to the glycine, threonine and serine degradation. We tested the binding of metabolites in these pathways and for those that bound, their ability to modulate TR binding to its specific DNA operator sequence. Using rtPCR, we confirmed that methylglyoxal was an effector of Bxe_3018.
These studies provide the proof of concept and validation of a systematic approach to the discovery of the biological activity for proteins of unknown function, in this case a TR. Bxe_B3018 is a methylglyoxal responsive TR that controls the expression of an operon composed of a putative efflux system.
基因在代谢途径中的聚类以及功能相关基因的共定位在原核生物中已得到广泛认可。我们利用这些特征来预测一个功能未知的转录调节因子（TR）的代谢参与情况，鉴定并证实了其生物学活性。
开发了一种软件工具，用于识别目标TR及其同源物在特定基因组邻域内编码的基因。该工具输出的列表包括基因邻域中的基因、它们的注释功能、反应物/产物，并确定编码蛋白发挥作用的代谢途径。在分析一组已知功能的TR时，我们观察到它们的同源物通常具有保守的基因组邻域，其中代谢相关基因与目标TR调控的基因共定位。我们推测TR效应物是已鉴定途径中的代谢物；实际上已知的效应物确实存在。我们分析了来自嗜麦芽窄食单胞菌的Bxe_B3018，这是一个功能未知的TR，并预测该TR与甘氨酸、苏氨酸和丝氨酸降解有关。我们测试了这些途径中代谢物的结合情况，以及那些能够结合的代谢物调节TR与其特定DNA操纵序列结合的能力。使用逆转录聚合酶链反应（rtPCR），我们证实甲基乙二醛是Bxe_3018的一种效应物。
这些研究为发现功能未知蛋白质（在本案例中为TR）的生物学活性提供了概念验证和系统方法的验证。Bxe_B3018是一种甲基乙二醛响应性TR，它控制着一个由假定的外排系统组成的操纵子的表达。
