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 The presence of molybdenum is required for the activity of several enzymes found in animals, plants, and bacteria, such as sulfite oxidase, xanthine dehydrogenase, nitrate reductase, formate dehydrogenase, and nitrogenase (46). In these organisms, the molybdenum is present in the molybdoenzymes in the form of a pterin-containing molybdenum cofactor (MoCo), with the exception of that found in nitrogenase, which has an ironmolybdenum cofactor (FeMoCo) (1,16,34).In Escherichia coli, the successful production of molybdoenzymes relies upon the efficient uptake of molybdate via the molybdate-specific transporter encoded by the modABCD operon (formerly known as the chlD locus) (18,26,35,42). The E. coli molybdate transport machinery, in which modA encodes a molybdate-specific periplasmic binding protein, modB encodes an integral membrane channel-forming protein, and modC encodes an ATP-binding energizer, closely resembles the established ATP-binding cassette (ABC) transporter motif (15, 26). Homologous molybdate transport systems have also been described for Azotobacter vinelandii, Rhodobacter capsulatus, and Haemophilus influenzae (9,23,45).Initial studies of the characterization of mod mutants revealed that strains harboring these mutations were incapable of producing nitrate reductase or formate dehydrogenase activity without molybdate supplementation (11,14,27,36,39,41,43). There are data to suggest that when mod mutants are grown in molybdate-supplemented media, molybdate enters the cells by means of the sulfate transport system as well as other nonspecific anion transporters (22,36). Some of the mod mutants were also analyzed for their molybdate uptake kinetics and were shown to transport molybdate at a much lower rate than mod ϩ strains did (5, 14). Further investigations of mod mutants have demonstrated that the modABCD operon is regulated by the intracellular concentration of molybdate. Specifically, high levels of molybdate reduced the level of transcription of the transport genes (27,35,36). This reduction in the level of transcription of the modABCD operon is most likely mediated by a molybdate-activated repressor protein.An analysis of the modABCD DNA sequence revealed a region in between the transcription and translation start sites of the modA gene which contains an 8-base inverted repeat (TAAC ⅐ GTTA) flanked by two CAT (CA) boxes (26,35,36). There are indications that either the CAT boxes or the inverted repeat or both are involved in the binding of the mod operon repressor (35). The inverted repeat found in this region is analogous to the inverted-repeat sequences implicated as the target binding sites for both the Met and Trp repressor (MetJ and TrpR) proteins (21,32,33).In our laboratory, a derivative of strain SE2069 ...

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...

Although they are few in number, molybdoenzymes play an essential role in microbial metabolism. These enzymes (except dinitrogenase) contain a unique form of molybdopterin-nucleotide as the cofactor (33). In Escherichia coli, the main cofactor found in molybdoproteins (formate dehydrogenase, nitrate reductase, etc.) is molybdopterin guanine dinucleotide (33). The biosynthesis of molybdopterin guanine dinucleotide and thus active molybdoenzymes starts with the transport of molybdate into cells. Mutant strains which are defective in one molybdoenzyme, nitrate reductase activity, have been isolated from several microorganisms as chlorate-resistant strains (11,19,42). Pleiotropic molybdoenzyme-defective mutants whose phenotype can be suppressed by increasing the molybdate concentration in the growth medium were defined as transport-negative (mod; previously termed chlD [39]) mutants (11). By using this rationale, a fraction of chlorate-resistant mutants were identified as mod mutants (19,42). Biochemical analysis of some of these mutants confirmed that the mod mutation decreased the rate of molybdate transport and thus its accumulation by cells (9,16,38). By complementing these mutants, the wild-type genes coding for various components of the molybdate transport system have been isolated from E. coli (16,21,35). Johann and Hinton (21) determined the DNA sequence of an internal segment of the mod operon (modC gene) from E. coli. However, the complete DNA sequence of the E. coli mod operon is not available, although a large number of presumptive mod mutants of E. coli have been described. Besides E. coli, mod ϩ DNA was also isolated from Azotobacter vinelandii and Rhodobacter capsulatus and sequenced (27,43). Analysis of the mod DNA sequences from these organisms suggests that the transport of molybdate into cells is achieved by a typical periplasmic binding protein and an ATP-dependent transport system similar to the ones reported for other solutes, like sulfate, histidine, maltose, etc. (2,17,40,41).In this communication, the complete DNA sequence of the mod operon from E. coli is presented. By using cloned mod ϩ DNA, the mutations in a number of mod mutants were mapped within the mod operon. However, several chlorateresistant mutants initially identified as Mod Ϫ had unique phenotypic characteristics, and on the basis of complementation analysis, the mutation in these mutants was found to be outside the mod operon. MATERIALS AND METHODSBacterial strains. The bacterial strains used in this study are presented in Table 1 and are derivatives of E. coli K-12.Media and growth conditions. L broth which served as the rich medium was supplemented with glucose (0.3%; LBG) (25), formate, or molybdate as needed at the concentrations indicated for each experiment. Glucose-minimal medium and low-sulfur medium (LSM) have been described previously (26). For molybdate-free glucose-minimal medium, sodium molybdate (normally present at a final concentration of 40 M) was omitted. No attempt was made to remove contaminating molybdate ...