Overton Tim, Reid Eleanor G F, Foxall Robin, Smith Harry, Busby Stephen J W, Cole Jeffrey A
School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom.
J Bacteriol. 2003 Aug;185(16):4734-47. doi: 10.1128/JB.185.16.4734-4747.2003.
The Neisseria gonorrhoeae genome encodes a homologue of the Escherichia coli FNR protein (the fumarate and nitrate reductase regulator). Despite its similarity to E. coli FNR, the gonococcal FNR only partially complemented an E. coli fnr mutation. After error-prone PCR mutagenesis of the gonococcal fnr gene, we identified four mutant fnr derivatives carrying the same S18F substitution, and we showed that the mutant FNR could activate transcription from a range of class I and class II FNR-dependent promoters in E. coli. Prompted by the similarities between gonococcal and E. coli FNR, we made changes in gonococcal fnr that created substitutions that are equivalent to previously characterized substitutions in E. coli FNR. First, our experiments showed that cysteine, C116, in the gonococcal FNR, equivalent to C122 in E. coli FNR, is essential, presumably because, as in E. coli FNR, it binds to an iron-sulfur center. Second, the L22H and D148A substitutions in gonococcal FNR were made. These changes are equivalent to the L28H and D154A changes in E. coli FNR, which had been shown to increase FNR activity in the presence of oxygen. We show that the effects of these substitutions in gonococcal FNR are distinct from those of the S18F substitution. Similarly, substitutions in the putative activating regions of gonococcal FNR were made. We show that the activity of gonococcal FNR in E. coli can be increased by transplanting certain activating regions from E. coli FNR. The effects of these substitutions are additive to those due to S18F. From these data, we conclude that the effects of the S18F substitution in gonococcal FNR are distinct from the effects of the other substitutions. S18 is immediately adjacent to one of three N-terminal cysteine residues that coordinate the iron-sulfur center, and thus the S18F substitution is most likely to stabilize this center. Support for this came from complementary experiments in which we created the S24F substitution in E. coli FNR, which is equivalent to the S18F substitution in gonococcal FNR. Our results show that the S24F substitution changes the activity of E. coli FNR and that the changes are distinct from those due to previously characterized substitutions.
淋病奈瑟菌基因组编码一种与大肠杆菌FNR蛋白（延胡索酸和硝酸盐还原酶调节因子）同源的蛋白。尽管淋病奈瑟菌FNR与大肠杆菌FNR相似，但它只能部分互补大肠杆菌的fnr突变。在对淋病奈瑟菌fnr基因进行易错PCR诱变后，我们鉴定出四个携带相同S18F替换的突变fnr衍生物，并表明突变型FNR能够激活大肠杆菌中一系列I类和II类FNR依赖性启动子的转录。受淋病奈瑟菌和大肠杆菌FNR之间相似性的启发，我们对淋病奈瑟菌fnr进行了改变，产生了与大肠杆菌FNR中先前表征的替换等效的替换。首先，我们的实验表明，淋病奈瑟菌FNR中的半胱氨酸C116（相当于大肠杆菌FNR中的C122）是必需的，推测原因是，与大肠杆菌FNR一样，它与一个铁硫中心结合。其次，在淋病奈瑟菌FNR中进行了L22H和D148A替换。这些变化等同于大肠杆菌FNR中的L28H和D154A变化，已证明这些变化在有氧条件下会增加FNR活性。我们表明，淋病奈瑟菌FNR中这些替换的效果与S18F替换的效果不同。同样，在淋病奈瑟菌FNR的假定激活区域进行了替换。我们表明，通过移植大肠杆菌FNR的某些激活区域，可以提高淋病奈瑟菌FNR在大肠杆菌中的活性。这些替换的效果与S18F引起的效果是相加的。从这些数据中，我们得出结论，淋病奈瑟菌FNR中S18F替换的效果与其他替换的效果不同。S18紧邻协调铁硫中心的三个N端半胱氨酸残基之一，因此S18F替换最有可能稳定这个中心。这一点得到了互补实验的支持，在这些实验中，我们在大肠杆菌FNR中创建了S24F替换，它等同于淋病奈瑟菌FNR中的S18F替换。我们的结果表明，S24F替换改变了大肠杆菌FNR的活性，并且这些变化与先前表征的替换引起的变化不同。
