Rosentel J K, Healy F, Maupin-Furlow J A, Lee J H, Shanmugam K T
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611, USA.
J Bacteriol. 1995 Sep;177(17):4857-64. doi: 10.1128/jb.177.17.4857-4864.1995.
Escherichia coli mutants with defined mutations in specific mod genes that affect molybdate transport were isolated and analyzed for the effects of particular mutations on the regulation of the mod operon as well as the fdhF and hyc operons which code for the components of the formate hydrogenlyase (FHL) complex. phi (hyc'-'lacZ+) mod double mutants produced beta-galactosidase activity only when they were cultured in medium supplemented with molybdate. This requirement was specific for molybdate and was independent of the moa, mob, and moe gene products needed for molybdopterin guanine dinucleotide (MGD) synthesis, as well as Mog protein. The concentration of molybdate required for FHL production by mod mutants was dependent on medium composition. In low-sulfur medium, the amount of molybdate needed by mod mutants for the production of half-maximal FHL activity was increased approximately 20 times by the addition of 40 mM of sulfate, mod mutants growing in low-sulfur medium transported molybdate through the sulfate transport system, as seen by the requirement of the cysA gene product for this transport. In wild-type E. coli, the mod operon is expressed at very low levels, and a mod+ merodiploid E. coli carrying a modA-lacZ fusion produced less than 20 units of beta-galactosidase activity. This level was increased by over 175 times by a mutation in the modA, modB, or modC gene. The addition of molybdate to the growth medium of a mod mutant lowered phi (modA'-'lacZ+) expression. Repression of the mod operon was sensitive to molybdate but was insensitive to mutations in the MGD synthetic pathway. These physiological and genetic experiments show that molybdate can be transported by one of the following three anion transport system in E. coli: the native system, the sulfate transport system (cysTWA gene products), and an undefined transporter. Upon entering the cytoplasm, molybdate branches out to mod regulation, fdhF and hyc activation, and metabolic conversion, leading to MGD synthesis and active molybdoenzyme synthesis.
分离出在特定mod基因中具有明确突变且影响钼酸盐转运的大肠杆菌突变体，并分析特定突变对mod操纵子以及编码甲酸氢裂解酶（FHL）复合体组分的fdhF和hyc操纵子调控的影响。phi(hyc'-'lacZ+) mod双突变体仅在添加钼酸盐的培养基中培养时才产生β-半乳糖苷酶活性。这种需求对钼酸盐具有特异性，并且独立于钼蝶呤鸟嘌呤二核苷酸（MGD）合成所需的moa、mob和moe基因产物以及Mog蛋白。mod突变体产生FHL所需的钼酸盐浓度取决于培养基组成。在低硫培养基中，添加40 mM硫酸盐会使mod突变体产生半最大FHL活性所需的钼酸盐量增加约20倍，在低硫培养基中生长的mod突变体通过硫酸盐转运系统转运钼酸盐，这可通过该转运对cysA基因产物的需求看出。在野生型大肠杆菌中，mod操纵子以非常低的水平表达，携带modA-lacZ融合的mod+部分二倍体大肠杆菌产生的β-半乳糖苷酶活性小于20单位。modA、modB或modC基因中的突变使该水平增加了175倍以上。向mod突变体的生长培养基中添加钼酸盐会降低phi(modA'-'lacZ+)的表达。mod操纵子的抑制对钼酸盐敏感，但对MGD合成途径中的突变不敏感。这些生理和遗传实验表明，钼酸盐可通过大肠杆菌中的以下三种阴离子转运系统之一进行转运：天然系统、硫酸盐转运系统（cysTWA基因产物）和一种未定义的转运体。进入细胞质后，钼酸盐分支到mod调控、fdhF和hyc激活以及代谢转化，导致MGD合成和活性钼酶合成。
