Genetic analysis of the modABCD (molybdate transport) operon of Escherichia coli

Abstract

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 ...

Promoters controlling expression of the alternative nitrogenase and the molybdenum uptake system in Rhodobacter capsulatus are activated by NtrC, independent of sigma54, and repressed by molybdenum

“…Transcriptional regulation of the mopA-modABCD operon. The high-affinity molybdate uptake system of E. coli (26,45), which corresponds to ModA, ModB, and ModC of R. capsulatus, was shown to be negatively regulated by high concentrations of molybdate (28,35,36). To analyze the expression of the corresponding R. capsulatus genes, a translational lacZ fusion to modA, which encodes the periplasmic molybdatebinding protein, was constructed.…”

Incorporation of Molybdenum into the Iron-Molybdenum Cofactor of Nitrogenase

“…2 and elsewhere is proposed to be the same protein based on its accumulation of Mo, its position of migration on nondenaturing gels, and its expression in NH 4 ϩ -grown cells. Based on its size and on results presented here, Mo-Sto would not appear to be the periplasmic molybdate-binding protein, ModA, or the molybdate-binding regulatory protein, ModE (24,25). Mo-Sto is soluble, and thus it is not likely to be the product of the modB, modC, or modD genes, which encode the membrane-bound molybdate transport system that has been described in A. vinelandii and Escherichia coli.…”

The yvsA-yvqA (293°-289°) region of the Bacillus subtilis chromosome containing genes involved in metal ion uptake and a putative sigma factor

“…(Kaneko et al, 1995) and with ModB, a membrane protein of E . coli involved in the uptake of molybdenum by the products of the modABCD operon (Maupin-Furlow et al, 1995).…”