MOLYBDATE AND REGULATION OF MOD (MOLYBDATE TRANSPORT), FDHF, AND HYC (FORMATE HYDROGENLYASE) OPERONS IN ESCHERICHIA-COLI

Escherichia coli mutants with defined mutations in specific mod genes that affect molybdate transport were isolated and analyzed for the eff ects of particular mutations on the regulation of the mod operon as we ll as the fdhF and hyc operons which code for the components of the fo rmate hydrogenlyase (FHL) complex. Phi(hyc'-'lacZ(+)) mod double mutan ts produced P-galactosidase activity only when they were cultured in m edium supplemented with molybdate. This requirement was specific for m olybdate and was independent of the moa, mob, and moe gene products ne eded for molybdopterin guanine dinucleotide (MGD) synthesis, as well a s Mog protein. The concentration of molybdate required for FHL product ion by mod mutants wais dependent on medium composition. In low-sulfur medium, the amount of molybdate needed by mod mutants for the product ion 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 incr eased 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 lowere d Phi(modA'-'lacZ(+)) expression. Repression of the mod operon was sen sitive to molybdate but was insensitive to mutations in the MGD synthe tic pathway. These physiological and genetic experiments show that mol ybdate can be transported by one of the following three anion transpor t systems in E. coli: the native system, the sulfate transport system (cysTWA gene products), and an undefined transporter. Upon entering th e cytoplasm, molybdate branches out to mod regulation,fdhF and hyc act ivation, and metabolic conversion, leading to MGD synthesis and active molybdoenzyme synthesis.