Molybdenum-dependent repression of transcription of the Escherichia coli modABCD operon, which encodes the high-affinity molybdate transporter, is mediated by the ModE protein. This regulatory protein was purified as an N-terminal His,-tagged derivative and characterised both with and without the Nterminal oligohistidine extension. Equilibrium centrifugation showed that ModE is at least a 57-kDa homodimer. Circular dichroism spectroscopy indicated that when molybdate or tungstate bind to ModE there is little change in its a-helical content, but a major change in the environment of tryptophan and tyrosine residues occurs. Addition of molybdate or tungstate to the protein results in almost 50% quenching of the fluorescence attributed to tryptophan. Titration of fluorescence quenching showed that two molecules of molybdenum bind to each dimer of ModE with a Kd of 0.8 pM. DNA mobility-shift assays showed that ModE requires molybdenum, or tungstate, to bind with high affinity (approximate K,, of 30 nM ModE) to the modABCD promoter region. In accord with ModE's role as a molybdenum-dependent transcriptional repressor, DNase 1 footprinting experiments showed that the ModE-molybdenum complex binds to a single 31-bp region around the transcription start of the modABCD promoter. This region contains a 6-base palindromic sequence CGTTAT-N,,-ATAACG.

The nucleotide sequence (6,559 base pairs) of the genomic region containing the structural genes for nitrogenase 2 (V nitrogenase) from Azotobacter vinelandii was determined. The open reading frames present in this region are organized into two transcriptional units. One contains vnJH (encoding dinitrogenase reductase 2) and a ferredoxinlike open reading frame (Fd). The second one includes vnJf) (encoding the a subunit of dinitrogenase 2), vnfG (encoding a product similar to the 8 subunit of dinitrogenase 2 from A. chroococcum), and vnfK (encoding the 3 subunit of dinitrogenase 2). The 5'-flanking regions of vnfH and vnJf) contain sequences similar to ntrA-dependent promoters. This gene arrangement allows independent expression of vn.f-Fd and vnfDGK. Mutant strains (CA80 and CA11.80) carrying an insertion in vnjf are still able to synthesize the a and 1 subunits of dinitrogenase 2 when grown in N-free, Mo-deficient, V-containing medium.A strain (RP1.1l) carrying a deletion-plus-insertion mutation in the vnfDGK region produced only dinitrogenase reductase 2.

The expression of the moa locus, which encodes enzymes required for molybdopterin biosynthesis, is enhanced under anaerobiosis but repressed when the bacterium is able to synthesize active molybdenum cofactor. In addition, moa expression exhibits a strong requirement for molybdate. The molybdate enhancement of moa transcription is fully dependent upon the molybdate-binding protein, ModE, which also mediates molybdate repression of the mod operon encoding the high-affinity molybdate uptake system. Due to the repression of moa in molybdenum cofactor-sufficient strains, the positive molybdate regulation of moa is revealed only in strains unable to make the active cofactor. Transcription of moa is controlled at two sigma-70-type promoters immediately upstream of the moaA gene. Deletion mutations covering the region upstream of moaA have allowed each of the promoters to be studied in isolation. The distal promoter is the site of the anaerobic enhancement which is Fnr-dependent. The molybdate induction of moa is exerted at the proximal promoter. Molybdate-ModE binds adjacent to the −35 region of this promoter, acting as a direct positive regulator of moa. The molybdenum cofactor repression also appears to act at the proximal transcriptional start site, but the mechanism remains to be established. Tungstate in the growth medium affects moaexpression in two ways. Firstly, it can act as a functional molybdate analogue for the ModE-mediated regulation. Secondly, tungstate brings about the loss of the molybdenum cofactor repression ofmoa. It is proposed that the tungsten derivative of the molybdenum cofactor, which is known to be formed under such conditions, is ineffective in bringing about repression of moa. The complex control of moa is discussed in relation to the synthesis of molybdoenzymes in the bacterium.

99Mo also exchanges with tungstate but not with vanadate or sulfate. modA, modB, and modC mutants exhibit nitrate reductase activity and 99 Mo accumulation only when grown in more than 10 M Mo, indicating that A. vinelandii also has a low-affinity Mo uptake system. The low-affinity system is not expressed in a modE mutant that synthesizes the high-affinity Mo transporter constitutively or in a spontaneous tungstate-tolerant mutant. Like the wild type, modG mutants only show nitrate reductase activity when grown in >10 nM Mo. However, a modE modG double mutant exhibits maximal nitrate reductase activity at a 100-fold lower Mo concentration. This indicates that the products of both genes affect the supply of Mo but are not essential for nitrate reductase cofactor synthesis. However, nitrogenase-dependent growth in the presence or absence of Mo is severely impaired in the double mutant, indicating that the products of modE and modG may be involved in the early steps of nitrogenase cofactor biosynthesis in A. vinelandii.

Background: Periplasmic receptors constitute a diverse class of binding proteins that differ widely in size, sequence and ligand specificity. Nevertheless, almost all of them display a common β/α folding motif and have similar tertiary structures consisting of two globular domains. The ligand is bound at the bottom of a deep cleft, which lies at the interface between these two domains. The oxyanion-binding proteins are notable in that they can discriminate between very similar ligands.Results: Azotobacter vinelandii is unusual in that it possesses two periplasmic molybdate-binding proteins. The crystal structure of one of these with bound ligand has been determined at 1.2 Å resolution. It superficially resembles the structure of sulphate-binding protein (SBP) from Salmonella typhimurium and uses a similar constellation of hydrogen-bonding interactions to bind its ligand. However, the detailed interactions are distinct from those of SBP and the more closely related molybdate-binding protein of Escherichia coli.Conclusions: Despite differences in the residues involved in binding, the volumes of the binding pockets in the A. vinelandii and E. coli molybdatebinding proteins are similar and are significantly larger than that of SBP. We conclude that the discrimination between molybdate and sulphate shown by these binding proteins is largely dependent upon small differences in the sizes of these two oxyanions.