Mazón G, Lucena J M, Campoy S, Fernández de Henestrosa A R, Candau P, Barbé J
Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.
Mol Genet Genomics. 2004 Feb;271(1):40-9. doi: 10.1007/s00438-003-0952-x. Epub 2003 Dec 2.
The lexA gene of the cyanobacterium Anabaena sp. strain PCC7120 has been cloned by PCR amplification with primers designed after TBLASTN analysis of its genome sequence using the Escherichia coli LexA sequence as a probe. After over-expression in E. coli and subsequent purification, footprinting experiments demonstrated that the Anabaena LexA protein binds to the sequence TAGTACTAATGTTCTA, which is found upstream of its own coding gene. Directed mutagenesis and sequence comparison of promoters of other Anabaena genes, as well as those of several cyanobacteria, allowed us to define the motif RGTACNNNDGTWCB as the LexA box in this bacterial phylum. Substitution of a single nucleotide in this motif present in the Anabena lexA promoter is sufficient to enable it to bind the Bacillus subtilis LexA protein. These data indicate that Cyanobacteria and Gram-positive bacteria are phylogenetically closely related.
通过使用大肠杆菌LexA序列作为探针,对蓝藻鱼腥藻Anabaena sp.菌株PCC7120的基因组序列进行TBLASTN分析后设计引物,通过PCR扩增克隆了该菌株的lexA基因。在大肠杆菌中过量表达并随后纯化后,足迹实验表明鱼腥藻LexA蛋白与序列TAGTACTAATGTTCTA结合,该序列位于其自身编码基因的上游。对其他鱼腥藻基因以及几种蓝藻的启动子进行定向诱变和序列比较,使我们能够将基序RGTACNNNDGTWCB定义为该细菌门中的LexA框。鱼腥藻lexA启动子中存在的该基序中的单个核苷酸替换足以使其结合枯草芽孢杆菌LexA蛋白。这些数据表明蓝藻和革兰氏阳性菌在系统发育上密切相关。