Grunden A M, Ray R M, Rosentel J K, Healy F G, Shanmugam K T
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611, USA.
J Bacteriol. 1996 Feb;178(3):735-44. doi: 10.1128/jb.178.3.735-744.1996.
The modABC gene products constitute the molybdate-specific transport system in Escherichia coli. Another operon coding for two proteins which diverges from the modABCD operon has been identified. The first gene of this operon codes for a 262-amino-acid protein, designated ModE (28 kDa), and the second genes codes for a 490-amino-acid protein. ModF (54 kDa). The role of ModF has not yet been determined; however, mutations in modE depressed modABCD transcription even in the presence of molybdate, suggesting that ModE is a repressor. ModE, in the presence of 1 mM molybdate, repressed the production of plasmid-encoded ModA and ModB' proteins in an in vitro transcription-translation system. DNA mobility shift experiments confirmed that ModE binds to an oligonucleotide derived from the operator region of the modABCD operon. Further experimentation indicated that ModE binding to target DNA minimally requires an 8-bp inverted-repeat sequence, TAAC GITA. A highly conserved amino acid sequence, TSARNOXXG (amino acids 125 to 133), was identified in ModE and homologs from Azotobacter vinelandii, Haemophilus influenzae, Rhodobacter capsulatus, and Clostridium pasterianum. Mutants with mutations in either T or G of this amino acid sequence were isolated as "superrepressor" mutants. These mutant proteins repressed modABCD transcription even in the absence of molybdate, which implies that this stretch of amino acids is essential for the binding of molybdate by the ModE protein. These results show that molybdate transport in E. coli is regulated by ModE, which acts as a repressor when bound to molybdate.
ModABC基因产物构成了大肠杆菌中钼酸盐特异性转运系统。已鉴定出另一个与modABCD操纵子不同的编码两种蛋白质的操纵子。该操纵子的第一个基因编码一种262个氨基酸的蛋白质，命名为ModE（28 kDa），第二个基因编码一种490个氨基酸的蛋白质，即ModF（54 kDa）。ModF的作用尚未确定；然而，即使在存在钼酸盐的情况下，modE中的突变也会抑制modABCD的转录，这表明ModE是一种阻遏物。在1 mM钼酸盐存在的情况下，ModE在体外转录-翻译系统中抑制了质粒编码的ModA和ModB'蛋白的产生。DNA迁移率变动实验证实ModE与源自modABCD操纵子操纵区的寡核苷酸结合。进一步的实验表明，ModE与靶DNA的结合至少需要一个8 bp的反向重复序列TAAC GITA。在ModE以及来自棕色固氮菌、流感嗜血杆菌、荚膜红细菌和巴氏梭菌的同源物中鉴定出了一个高度保守的氨基酸序列TSARNOXXG（第125至133位氨基酸）。在该氨基酸序列的T或G处发生突变的突变体被分离为“超级阻遏物”突变体。这些突变蛋白即使在没有钼酸盐的情况下也能抑制modABCD的转录，这意味着这段氨基酸序列对于ModE蛋白结合钼酸盐至关重要。这些结果表明，大肠杆菌中的钼酸盐转运受ModE调控，ModE在与钼酸盐结合时作为阻遏物发挥作用。
