Molybdate and regulation of mod (molybdate transport), fdhF, and hyc (formate hydrogenlyase) operons in Escherichia coli

Abstract

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. ⌽(hyclacZ ؉ ) mod double mutants produced ␤-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 ␤-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 ⌽(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 systems 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. Synthesis of active molybdoenzymes byEscherichia coli is dependent on the various gene products required for the transport of molybdate into cells (5,9,12,25,29,39,40,43), metabolic conversion of molybdate to an appropriate form, and incorporation into the pterin component of molybdopterin guanine dinucleotide (MGD) during maturation of the apoprotein to active enzyme (6,8,34,46; see references 13, 38, and 46 for reviews). Several mutant strains of E. coli which are defective in one or more of these steps have been isolated and described. Mutants defective in the first step of this process, molybdate transport, were isolated as chlorate-resistant derivatives (chlD [renamed as mod]) (41) whose phenotype can be suppressed by supplementation of the growth medium with molybdate (5,9,12,13,29,40,43,45). The rates of molybdate transport and internal molybdate concentrations of some of these mutants were determined (5, 12, 40). Recent studies have established that the mod operon contains four genes, of which three r...

Transcriptional regulation of molybdoenzyme synthesis in Escherichia coli in response to molybdenum: ModE-molybdate, a repressor of the modABCD (molybdate transport) operon is a secondary transcriptional activator for the hyc and nar operons

“…Lambda phages, isolated from various strains, were transduced into strain BW545 and single lysogens were selected as described by Rosentel et al (1995) and further verified by comparing the levels of P-galactosidase activity produced by the cultures in both the presence and absence of nitrate to that of the original lysogens. All other mutations were transduced MRi93 RK4353 RK5278 V JS720 V JS2009 V JS4809 SE1910 SE1978 SE1980 SE2163 SE2176 SE2200 SE2202 SE2213 AH78 AH79 AH106 AH107 AH121 AH122 AH179 WS160 WS161 WS162 WS168 WS169 WS178 WS192 Phages P1 LRZS LWSl Plasmids P V J S~ pAGl pAH69 pFGH 100 pHYCPl pHYCl pHYC3 pws40 pws31 pws33 pws34 pws35 pZ1918 flb-5301 deoCl ptsF25 A@(narG'-'lacZ) pcnB80 zad2084 : : TnlO MC4100 gyrA219 non-9 RK4353 narL22 5 : : TnlO modB247: Plasmid constructions.…”

“…Media used for bacterial growth have been described previously (Lee et af., 1985(Lee et af., ,1990Rosentel et al, 1995). L-broth (LB), which served as rich medium, was supplemented with glucose (0.3 ' / o ; LBG), sodium formate (15 mM; LBF), sodium molybdate (1 mM; LB + Mo) or sodium nitrate (30 mM), as needed.…”

“…The modABCD operon, encoding rnolybdate-transport components, is also regulated by the availability of molybdate (Miller et al, 1987;Rech et al, 1995;Rosentel et al, 1995). The molybdate-dependent repression of the modABCD operon is mediated through ModE (Grunden, 1996;Grunden et al, 1996;McNicholas et al, 1996;Walkenhorst et al, 1995).…”

“…Transcription of the genes encoding molybdoenzymes also requires the presence of molybdate in the cytoplasm (Cotter & Gunsalus, 1989;Iuchi & Lin, 1987;Pascal et al, 1982;Rosentel et at., 1995;Schlensog et al, 1989;Stewart, 1988). The mechanism by which molybdate availability is transduced to transcription of genes encoding the molybdoenzymes is not known.…”

Functional characterization of the Bradyrhizobium japonicum modA and modB genes involved in molybdenum transport

“…Because of the mutation in the modA gene, traces of molybdate in the medium were not taken up by the mutant cells, and the expression of the mod genes was not repressed. In E. coli, expression of the mod operon also requires molybdate starvation (Rech et al, 1995;Rosentel et al, 1995). The repressor protein ModE binds molybdate to form a complex which, in turn, binds to the promoter region to inhibit transcription of mod genes (Grunden et al, 1996(Grunden et al, , 1999Anderson et al, 1997;McNicholas et al, 1998).…”