Directed evolution of Vibtio fischeri LuxR for improved response to butanoyl-homoserine lactone
Directed evolution of Vibtio fischeri LuxR for improved response to butanoyl-homoserine lactone
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DOI：
10.1128/aem.00060-07
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发表时间：
2007-09-01
影响因子：
4.4
通讯作者：
Leadbetter, Jared R.
中科院分区：
文献类型：
--
作者：
Hawkins, Andrew C.;Arnold, Frances H.;Leadbetter, Jared R.
LuxR是费氏弧菌典型的酰基高丝氨酸内酯(AHL)群体感应系统中依赖于3-氧基己酰高丝氨酸内酯(3OC6HSL)的转录激活因子。在定量生物测定中，野生型LuxR对丁酰-HSL(C4HSL)没有反应；先前描述的LuxR变异体(LuxR-G2E)对包括戊酰-HSL(C5HSL)在内的各种AHL表现出广泛的反应，但不对C4HSL。在这里，LuxR-G2E的两轮定向进化产生了LuxR的变体，这些变体在低至10 nM的浓度下对C4HSL产生反应。一个突变体LuxR-G4E相对于亲本LuxR-G2E只有一个变化，145F，它本身在三个残基上与野生型不同。对LuxR-G4E内四个突变的剖析表明，至少其中三个突变同时需要实现任何可测量的C4HSL反应。相对于这些残基的其他14种可能组合中的任何一种，这四种变化都提高了对C4HSL的敏感性和特异性。这些数据证实了LuxR在进化上是柔韧的，并表明LuxR在对不同酰基侧链长度的群体感应信号的响应方面并不固有地不对称。
LuxR is the 3-oxohexanoyl-homoserine lactone (3OC6HSL)-dependent transcriptional activator of the prototypical acyl-homoserine lactone (AHL) quorum-sensing system of Vibrio fischeri. Wild-type LuxR exhibits no response to butanoyl-HSL (C4HSL) in quantitative bioassays at concentrations of up to I mu M; a previously described LuxR variant (LuxR-G2E) exhibits a broadened response to diverse AHLs, including pentanoyl-HSL (C5HSL), but not to C4HSL. Here, two rounds of directed evolution of LuxR-G2E generated variants of LuxR that responded to C4HSL at concentrations as low as 10 nM. One variant, LuxR-G4E, had only one change, 145F, relative to the parent LuxR-G2E, which itself differs from the wild type at three residues. Dissection of the four mutations within LuxR-G4E demonstrated that at least three of these changes were simultaneously required to achieve any measurable C4HSL response. The four changes improved both sensitivity and specificity towards C4HSL relative to any of the other 14 possible combinations of those residues. These data confirm that LuxR is evolutionarily pliable and suggest that LuxR is not intrinsically asymmetric in its response to quorum-sensing signals with different acyl-side-chain lengths.