Harayama S, Bollinger J, Iino T, Hazelbauer G L
J Bacteriol. 1983 Jan;153(1):408-15. doi: 10.1128/jb.153.1.408-415.1983.
We used transposon insertion mutagenesis, molecular cloning, and a novel procedure for in vitro construction of polar and nonpolar insertion mutations to characterize the genetic organization and gene products of the beta-methylgalactoside (Mgl) transport system, which utilizes the galactose-binding protein. The data indicate that the mgl operon contained three genes, which were transcribed in the order mglB, mglA, and mglC. The first gene coded for the 31,000 Mr galactose-binding protein, which was synthesized as a 3,000-dalton-larger precursor form. The mglA product was a 50,000 Mr protein which was tightly associated with the membrane, and the mglC product was a 38,000 Mr protein which was apparently loosely associated with the membrane and was probably located on the internal face of the cytoplasmic membrane. Identification of gene products was facilitated by in vitro insertion of a fragment of Tn5 containing the gene conferring kanamycin resistance into a restriction site in the operon. The fragment proved to have a polar effect on the expression of promoter-distal genes only when inserted in one of the two possible orientations. The three identified gene products were necessary and apparently sufficient for transport activity, but only the binding protein was required for chemotaxis towards galactose. The transport system appeared to contain the minimum number of components for a binding protein-related system: a periplasmic recognition component, a transmembrane protein, and a peripheral membrane protein that may be involved in energy linkage.
我们利用转座子插入诱变、分子克隆以及一种用于体外构建极性和非极性插入突变的新方法，来表征利用半乳糖结合蛋白的β-甲基半乳糖苷（Mgl）转运系统的遗传组织和基因产物。数据表明，mgl操纵子包含三个基因，它们按mglB、mglA和mglC的顺序转录。第一个基因编码31,000 Mr的半乳糖结合蛋白，它以比其大3,000道尔顿的前体形式合成。mglA产物是一种50,000 Mr的蛋白，与膜紧密结合，mglC产物是一种38,000 Mr的蛋白，显然与膜松散结合，可能位于细胞质膜的内表面。通过将含有赋予卡那霉素抗性基因的Tn5片段体外插入操纵子中的一个限制性位点，有助于鉴定基因产物。仅当以两种可能方向之一插入时，该片段才对启动子远端基因的表达产生极性影响。所鉴定的三种基因产物对于转运活性是必需的且显然是足够的，但只有结合蛋白是对半乳糖趋化性所必需的。该转运系统似乎包含了与结合蛋白相关系统的最少组成成分：一个周质识别成分、一个跨膜蛋白以及一个可能参与能量连接的外周膜蛋白。
