Collins Cynthia H, Leadbetter Jared R, Arnold Frances H
Biochemistry & Molecular Biophysics, California Institute of Technology, Pasadena, California 91125, USA.
Nat Biotechnol. 2006 Jun;24(6):708-12. doi: 10.1038/nbt1209. Epub 2006 May 21.
The transcription factor LuxR activates gene expression in response to binding the signaling molecule 3-oxo-hexanoyl-homoserine lactone (3OC6HSL), an acyl-HSL with a carbonyl substituent at the third carbon of the acyl chain. We previously described a LuxR variant, LuxR-G2E, that activates gene expression by binding a broader range of acyl-HSLs, including straight-chain acyl-HSLs to which LuxR does not respond. Here, we use a dual positive-negative selection system to identify a variant of LuxR-G2E that retains the response to straight-chain acyl-HSLs, but no longer responds to 3OC6HSL. A single mutation, R67M, reduces LuxR-G2E's response to acyl-HSLs having a carbonyl substituent at the third carbon of the acyl chain. This specificity-enhancing mutation would not have been identified by positive selection alone. The dual selection system provides a rapid and reliable method for identifying LuxR variants that have or lack the desired response to a given set of acyl-HSL signals. LuxR variants with altered signaling specificities might become useful components for constructing artificial cell-cell communication systems that program population level behaviors.
转录因子LuxR在结合信号分子3-氧代己酰高丝氨酸内酯（3OC6HSL，一种在酰基链的第三个碳原子上带有羰基取代基的酰基高丝氨酸内酯）时会激活基因表达。我们之前描述过一种LuxR变体，即LuxR-G2E，它通过结合更广泛的酰基高丝氨酸内酯来激活基因表达，包括LuxR不响应的直链酰基高丝氨酸内酯。在这里，我们使用一种双阳性-阴性选择系统来鉴定LuxR-G2E的一个变体，该变体保留了对直链酰基高丝氨酸内酯的响应，但不再对3OC6HSL作出反应。单个突变R67M降低了LuxR-G2E对在酰基链的第三个碳原子上带有羰基取代基的酰基高丝氨酸内酯的响应。这种增强特异性的突变仅通过阳性选择是无法鉴定出来的。这种双选择系统为鉴定对给定的一组酰基高丝氨酸内酯信号具有或缺乏所需响应的LuxR变体提供了一种快速且可靠的方法。具有改变的信号特异性的LuxR变体可能会成为构建能够编程群体水平行为的人工细胞间通信系统的有用组件。
