Baldwin T O, Devine J H, Heckel R C, Lin J W, Shadel G S
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843.
J Biolumin Chemilumin. 1989 Jul;4(1):326-41. doi: 10.1002/bio.1170040145.
We have determined the complete nucleotide sequence of a 7622 base pair fragment of DNA from Vibrio fischeri strain ATCC7744 that contains all the information required to confer plasmid-borne, regulated bioluminescence upon strains of Escherichia coli. The lux regulon from V. fischeri consists of two divergently transcribed operons, L (left) and R (right), and at least seven genes, luxR (L operon) and luxICDABE (R operon) and the intervening control region. The luxA and luxB genes encode respectively the alpha and beta subunits of luciferase. The gene order luxCDABE seen in V. fischeri is the same as for V. harveyi. We have determined the sequence of the luxAB and flanking regions from Photobacterium leiognathi and have found upstream sequences homologous with luxC from the Vibrio species, but between luxB and luxE, there is an open reading frame encoding a protein of 227 amino acids (26,229 molecular weight) that is not found in this location in the Vibrio species. The amino terminal amino acid sequence of the encoded protein is nearly identical to that determined by O'Kane and Lee (University of Georgia) for the non-fluorescent flavoprotein from a closely related Photobacterium species (Dr Dennis O'Kane, personal communication). We have therefore designated this gene luxN. There is a 20-base inverted repeat ACCTGTAGGAxTCGTACAGGT, centred between bases 927 and 928 in the region between the two operons of V. fischeri. This region appears to fulfil two functions: it is critical for the LuxR protein to exert its effect and it is a consensus binding site for the E. coli LexA protein, a negative regulatory protein involved with the SOS response. There are sequences within the luxR coding region that appear to function in a cis-acting fashion to repress transcription from both the leftward and rightward promoters in the absence of the respective transcriptional activator proteins, thereby resulting in low basal levels of transcription. It now appears clear that there are multiple levels of control on the lux system allowing for a modulation of the intensity of bioluminescence of over four orders of magnitude.
我们已经确定了费氏弧菌菌株ATCC7744中一段7622个碱基对的DNA片段的完整核苷酸序列，该片段包含赋予大肠杆菌菌株质粒携带的、可调控生物发光所需的所有信息。费氏弧菌的lux操纵子由两个反向转录的操纵子L（左）和R（右）以及至少七个基因组成，即luxR（L操纵子）和luxICDABE（R操纵子）以及中间的控制区域。luxA和luxB基因分别编码荧光素酶的α和β亚基。在费氏弧菌中看到的luxCDABE基因顺序与哈氏弧菌相同。我们已经确定了发光杆菌的luxAB及其侧翼区域的序列，并且发现了与弧菌属的luxC同源的上游序列，但在luxB和luxE之间，有一个开放阅读框，编码一个227个氨基酸（分子量26,229）的蛋白质，该蛋白质在弧菌属的这个位置上不存在。所编码蛋白质的氨基末端氨基酸序列与奥凯恩和李（佐治亚大学）确定的来自密切相关的发光杆菌属物种的非荧光黄素蛋白的序列几乎相同（丹尼斯·奥凯恩博士，个人交流）。因此，我们将这个基因命名为luxN。在费氏弧菌两个操纵子之间的区域中，碱基927和928之间的中心位置有一个20个碱基的反向重复序列ACCTGTAGGAxTCGTACAGGT。该区域似乎履行两种功能：它对于LuxR蛋白发挥其作用至关重要，并且它是大肠杆菌LexA蛋白的共有结合位点，LexA蛋白是一种参与SOS反应的负调控蛋白。在luxR编码区域内存在一些序列，在没有各自的转录激活蛋白的情况下，这些序列似乎以顺式作用方式抑制来自向左和向右启动子的转录，从而导致低水平的基础转录。现在看来很清楚，lux系统存在多个控制水平，允许对生物发光强度进行超过四个数量级的调节。
