Hawkins Andrew C, Arnold Frances H, Stuermer Rainer, Hauer Bernhard, Leadbetter Jared R
Environmental Science & Engineering, California Institute of Technology, 1200 E. California Blvd., Mail Code 138-78, Pasadena, CA 91125-7800, USA.
Appl Environ Microbiol. 2007 Sep;73(18):5775-81. doi: 10.1128/AEM.00060-07. Epub 2007 Aug 3.
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 1 microM; 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, I45F, 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.
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3-氧代己酰高丝氨酸内酯（3OC6HSL）的转录激活因子。在浓度高达1微摩尔的定量生物测定中，野生型LuxR对丁酰高丝氨酸内酯（C4HSL）无反应；先前描述的LuxR变体（LuxR-G2E）对包括戊酰高丝氨酸内酯（C5HSL）在内的多种AHL表现出更广泛的反应，但对C4HSL无反应。在这里，对LuxR-G2E进行两轮定向进化产生了对低至10纳摩尔浓度的C4HSL有反应的LuxR变体。一个变体LuxR-G4E相对于亲本LuxR-G2E只有一个变化，即I45F，而LuxR-G2E本身在三个残基上与野生型不同。对LuxR-G4E中的四个突变进行剖析表明，这些变化中至少有三个是实现任何可测量的C4HSL反应所必需的。相对于这些残基的其他14种可能组合中的任何一种，这四个变化提高了对C4HSL的敏感性和特异性。这些数据证实LuxR在进化上具有可塑性，并表明LuxR在对具有不同酰基侧链长度的群体感应信号的反应中并非本质上不对称。
