Molybdate Uptake Research Articles

Complete Genome Sequences of Azotobacter vinelandii Wild-Type Strain CA and Tungsten-Tolerant Mutant Strain CA6

We report the complete genome sequences of Azotobacter vinelandii mutant strain CA6 and its parent wild-type strain, CA. When fixing nitrogen, strain CA6 displays slow growth and impaired molybdate uptake, tolerance to tungstates, and production of hydrogen gas, compared to results for strain CA. Comparing these genome sequences may provide a genetic basis for these mutant phenotypes.

ZnAl Layered Double Hydroxides As Potential Molybdate Sorbents and Valorize the Exchanged Sorbent for Catalytic Wet Peroxide Oxidation of Phenol

Molybdate uptake was studied using ZnAl layered double hydroxides (LDHs) having different interlayer anions namely chloride, carbonate, or nitrate with various Zn/Al atomic ratios. A maximum molybdate uptake of 114 mg Mo/g was observed for ZnAl3-Cl-LDH. The uptake is due to ion-exchange for chloride and nitrate containing samples and surface adsorption for carbonate bearing samples. Temperature of the medium had little influence on the uptake while nearly 100% uptake was observed for a wide pH range (2.3 to 10.0). The material after molybdate uptake was valorized for catalytic wet peroxide oxidation of aqueous phenol (100 mg/L). A maximum phenol degradation of 43% was achieved at catalyst loading of 0.5 g/L. The potential for dual application of LDHs for the removal of both inorganic and organic pollutants in the perspective of wastewater remediation by acting as sorbents for molybdate and valorize the used sorbent as catalyst for the removal of phenol is demonstrated.

Comparison of arsenate, chromate and molybdate binding on schwertmannite: Surface adsorption vs anion-exchange

The presence of iron oxides may play an important role in controlling the mobility and availability of contaminants in soils and waters affected by acid mine drainage. The present study describes the uptake of arsenate, chromate and molybdate from solution by synthetic schwertmannite. Batch experiments were performed at different pH values in order to obtain the adsorption isotherms for the three oxyanions. In addition to the formation of surface complexes between the oxyanions and the iron surface reactive groups, it is also expected that anion exchange will occur between sulphate anions from the schwertmannite structure and the oxyanions present in the solid/solution interface. Comparison of the experimental adsorption results for the different oxyanions showed large differences, not only the amount adsorbed, which was much higher for arsenate, but also in the sulphate exchange with the anions in solution. In case of chromate, the main mechanism of adsorption process is the exchange reaction with the sulphate groups present in the schwertmannite. The observed results suggest a different adsorption mechanisms for each of the three oxyanions, with important implications for the mobility of these anions in acid mine drainage systems.

Mapping of ionomic traits in Mimulus guttatus reveals Mo and Cd QTLs that colocalize with MOT1 homologues.

Natural variation in the regulation of the accumulation of mineral nutrients and trace elements in plant tissues is crucial to plant metabolism, development, and survival across different habitats. Studies of the genetic basis of natural variation in nutrient metabolism have been facilitated by the development of ionomics. Ionomics is a functional genomic approach for the identification of the genes and gene networks that regulate the elemental composition, or ionome, of an organism. In this study, we evaluated the genetic basis of divergence in elemental composition between an inland annual and a coastal perennial accession of Mimulus guttatus using a recombinant inbred line (RIL) mapping population. Out of 20 elements evaluated, Mo and Cd were the most divergent in accumulation between the two accessions and were highly genetically correlated in the RILs across two replicated experiments. We discovered two major quantitative trait loci (QTL) for Mo accumulation, the largest of which consistently colocalized with a QTL for Cd accumulation. Interestingly, both Mo QTLs also colocalized with the two M. guttatus homologues of MOT1, the only known plant transporter to be involved in natural variation in molybdate uptake.

Algae and humans share a molybdate transporter

Almost all living organisms need to obtain molybdenum from the external medium to achieve essential processes for life. Activity of important enzymes such as sulfite oxidase, aldehyde oxidase, xanthine dehydrogenase, and nitrate reductase is strictly dependent on the presence of Mo in its active site. Cells take up Mo in the form of the oxianion molybdate, but the molecular nature of the transporters is still not well known in eukaryotes. MOT1 is the first molybdate transporter identified in plant-type eukaryotic organisms, but it is absent in animal genomes. Here we report a molybdate transporter different from the MOT1 family, encoded by the Chlamydomonas reinhardtii gene MoT2, that is also present in animals including humans. The knockdown of CrMoT2 transcription leads to the deficiency of molybdate uptake activity in Chlamydomonas. In addition, heterologous expression in Saccharomyces cerevisiae of MoT2 genes from Chlamydomonas and humans support the functionality of both proteins as molybdate transporters. Characterization of CrMOT2 and HsMOT2 activities showed an apparent Km of about 550 nM that, though higher than the Km reported for MOT1, still corresponds to high affinity systems. CrMoT2 transcription is activated when extracellular molybdate concentration is low but in contrast to MoT1 is not activated by nitrate. Analysis of protein databases revealed the presence of four motifs present in all the proteins with high similarity to MOT2, that label a previously undescribed family of proteins probably related to molybdate transport. Our results open the way toward the understanding of molybdate transport as part of molybdenum homeostasis and Moco biosynthesis in animals.

Molybdenum—nitrogen co‐limitation in freshwater and coastal heterocystous cyanobacteria

Molybdenum (Mo) is essential for the biological assimilation of inorganic nitrogen (N). We compared Mo requirements for N2‐fixation in two species of filamentous heterocystous cyanobacteria (HC) to test the hypothesis that coastal HC require higher Mo concentrations than freshwater HC. This expectation follows from the fact that Mo is more concentrated in seawater (~ 105 nmol L−1) than in most freshwaters (< 20 nmol L−1). Contrary to this hypothesis, we found that both strains maintained N2‐fixation for 30 d at 10 nmol L−1. Mo concentrations < 1 nmol L−1 induced N‐limitation in both species, as indicated by increased C:N ratios and decreased nitrogenase expression and activity. This response took time to induce, likely due to high‐affinity molybdate uptake by both species. Measurable N2‐fixation persisted in the coastal strain (Nostoc sp. CCMP 2511) for at most 12 d; 3 d were required for chlorophyll a concentrations to fall below those of Mo‐replete cultures. An additional 7 d and 11 d, respectively, were required for N2‐fixation rates and chlorophyll levels to decline in Mo‐limited freshwater cultures (Nostoc sp. PCC 7120). When Mo was high (> 1 µmol L−1), the freshwater strain exhibited considerable Mo storage (> 100 µmol mol−1 Mo: C) whereas cellular Mo remained < 10 µmol mol−1 Mo:C in the coastal strain. The high Mo content and extended time required for N2‐fixation to decrease in the freshwater strain could be due to expression of the gene mop, which encodes a putative molybdate‐storage protein. This study suggests the importance of Mo storage in freshwater HC.

A role for tungsten in the biology of Campylobacter jejuni: tungstate stimulates formate dehydrogenase activity and is transported via an ultra‐high affinity ABC system distinct from the molybdate transporter

The food-borne pathogen Campylobacter jejuni possesses no known tungstoenzymes, yet encodes two ABC transporters (Cj0300-0303 and Cj1538-1540) homologous to bacterial molybdate (ModABC) uptake systems and the tungstate transporter (TupABC) of Eubacterium acidaminophilum respectively. The actual substrates and physiological role of these transporters were investigated. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry of the purified periplasmic binding proteins of each system revealed that while Cj0303 is unable to discriminate between molybdate and tungstate (K(D) values for both ligands of 4-8 nM), Cj1540 binds tungstate with a K(D) of 1.0 +/- 0.2 pM; 50 000-fold more tightly than molybdate. Induction-coupled plasma mass spectroscopy of single and double mutants showed that this large difference in affinity is reflected in a lower cellular tungsten content in a cj1540 (tupA) mutant compared with a cj0303c (modA) mutant. Surprisingly, formate dehydrogenase (FDH) activity was decreased approximately 50% in the tupA strain, and supplementation of the growth medium with tungstate significantly increased FDH activity in the wild type, while inhibiting known molybdoenzymes. Our data suggest that C. jejuni possesses a specific, ultra-high affinity tungstate transporter that supplies tungsten for incorporation into FDH. Furthermore, possession of two MoeA paralogues may explain the formation of both molybdopterin and tungstopterin in this bacterium.

Molybdate transport through the plant sulfate transporter SHST1

Molybdenum is an essential micronutrient required by plants. The mechanism of molybdenum uptake in plants is poorly understood, however, evidence has suggested that sulfate transporters may be involved. The sulfate transporter from Stylosanthes hamata, SHST1, restored growth of the sulfate transport yeast mutant, YSD1, on media containing low amounts of molybdate. Kinetic analysis using 99 MoO 4 2 - demonstrated that SHST1 enhanced the uptake of molybdate into yeast cells at nM concentrations. Uptake was not inhibited by sulfate, but sulfate transport via SHST1 was reduced with molybdate. These results are the first measurement of molybdate transport by a characterised plant sulfate transport protein.

A high-affinity molybdate transporter in eukaryotes

Molybdenum is an essential element for almost all living beings, which, in the form of a molybdopterin-cofactor, participates in the active site of enzymes involved in key reactions of carbon, nitrogen, and sulfur metabolism. This metal is taken up by cells in form of the oxyanion molybdate. Bacteria acquire molybdate by an ATP-binding-cassette (ABC) transport system in a widely studied process, but how eukaryotic cells take up molybdenum is unknown because molybdate transporters have not been identified so far. Here, we report a eukaryotic high-affinity molybdate transporter, encoded by the green alga Chlamydomonas reinhardtii gene MoT1. An antisense RNA strategy over the MoT1 gene showed that interference of the expression of this gene leads to the inhibition of molybdate transport activity and, in turn, of the Mo-containing enzyme nitrate reductase, indicating a function of MoT1 in molybdate transport. MOT1 functionality was also shown by heterologous expression in Saccharomyces cerevisiae. Molybdate uptake mediated by MOT1 showed a K(m) of approximately 6 nM, which is the range of the lowest K(m) values reported and was activated in the presence of nitrate. Analysis of deduced sequence from the putative protein coded by MoT1 showed motifs specifically conserved in similar proteins present in the databases, and defines a family of membrane proteins in both eukaryotes and prokaryotes probably involved in molybdate transport and distantly related to plant sulfate transporters SULTR. These findings represent an important step in the understanding of molybdate transport, a crucial process in eukaryotic cells.

An Arabidopsis thaliana high-affinity molybdate transporter required for efficient uptake of molybdate from soil

Molybdenum (Mo) is a trace element essential for living organisms, however no molybdate transporter has been identified in eukaryotes. Here, we report the identification of a molybdate transporter, MOT1, from Arabidopsis thaliana. MOT1 is expressed in both roots and shoots, and the MOT1 protein is localized, in part, to plasma membranes and to vesicles. MOT1 is required for efficient uptake and translocation of molybdate and for normal growth under conditions of limited molybdate supply. Kinetics studies in yeast revealed that the K(m) value of MOT1 for molybdate is approximately 20 nM. Furthermore, Mo uptake by MOT1 in yeast was not affected by coexistent sulfate, and MOT1 did not complement a sulfate transporter-deficient yeast mutant strain. These data confirmed that MOT1 is specific for molybdate and that the high affinity of MOT1 allows plants to obtain scarce Mo from soil.

Uptake of Molybdate by Adsorption onto Industrial Solid Waste Fe(III)/Cr(III) Hydroxide: Kinetic and Equilibrium studies

Removal of molybdate on to industrial solid waste Fe(III)/Cr(III) hydroxide as adsorbent has been investigated. Pretreated adsorbent was found to be more efficient in the uptake of molybdate compared to untreated adsorbent. Effect of pH on the adsorption was studied in the pH range 4.0 to 10.0. Optimum pH for maximum removal was found to be 4.0. Langmuir and Freundlich isotherm models were used to study the adsorption capacity of the adsorbent. The adsorption follows second order kinetics. No significant effect of temperature has been observed. Desorption studies showed that ion exchange mechanism is predominant.

Effects of Gas Flaring on Soil Fertility

This article, written by Technology Editor Dennis Denney, contains highlights of paper SPE 93666, "Impact of Gas Flaring on Soil Fertility," by R.E. Akpojivi, SPE, and P.E. Akumagba, SPE, Petroleum Training Inst., Effurun, prepared for the 2005 SPE Middle East Oil & Gas Show and Conference, Bahrain, 12-15 March. Gas flaring is used in Nigeria to dispose of associated gas. Low flare stacks bring the flare close to nearby vegetation and soil. This study investigated the effect of oil-sector gas-flaring activity on soil-fertility parameters. Introduction Gas flaring is the burning of natural gas and other petroleum hydrocarbons at flare stacks in oil fields during operation. The casing-head gas produced as associated natural gas during oil production varies in composition from location to location; however, the basic components include methane, ethane, propane, iso-butane, n-butane, iso-pentane, n-pentane, n-hexane, CO2, H2S, He, and N2. Modern technology and commercial opportunities have permitted industrialized nations, which often flared natural gas during the early history of oil production, to now process it for commercial sale or reinjection into the reservoirs. However, several sub-Saharan African nations including Nigeria still use gas flaring as a method to dispose of associated gas during oil-sector operations, ostensibly because gas infrastructure is extremely low. For example, despite efforts to expand the gas-to-liquid industry in Nigeria, companies flared [667 Bscf of natural gas at 176 locations in 2002, according to the U.S. Energy Information Agency, Dept. of Energy]. This gas flaring represents [49%] of the total gas produced in Nigeria in 2002. Flaring of associated gas releases emissions rich in carbon, nitrogen, and sulfur oxides and soot. These acid gases are carried downward as acid deposition (wet and dry depositions) onto vegetation, soil, and water bodies in communities close to the flare sites. Also, intense heat and continuous illumination are associated with gas flaring. The low height of flare stacks ensures local pollution at ground level and nearby dry deposition, as well as close heating of surrounding vegetation and soil. Likely acid deposition and intense heat from gas flares are likely to harm the fertility of the surrounding soils. The obvious signs are the poor vegetation growth and scorched soils around gas-flare locations. Acid deposition decreases the soil pH. However, the effect of acid deposition on a particular soil ecosystem is influenced by such factors as acid sensitivity, neutralization capability, concentration and composition of acid reaction products, and the amount of acid added to the system. The basic cations (Ca2+, Mg2+, and K+) are replaced by hydrogen ions or soluble metals and are lost through leaching. Increased acidity reduces activity of soil microorganisms sensitive to low pH, thus decreasing decomposition of plant residues and the recycling of essential plant nutrients. The concentration of trace-metal ions in soil solution increases (including aluminium, copper, iron, zinc, boron, manganese, chromium, and nickel) to levels that may be phytotoxic. Phosphorus compounds in the soil become mostly aluminum and iron phosphates, resulting in a reduced availability of plant phosphorus. Plant uptake of molybdate is reduced. Nitrification by the main autotrophic genera involved (Nitrosononas and Nitrobacter) is inhibited; thus, NH4+ is the main form of nitrogen taken up by plants instead of NO3−. There is reduced symbiotic nitrogen fixation by legumes, except the Rhizobium strain, which is acid tolerant.

Global gene expression analysis revealed an unsuspected deo operon under the control of molybdate sensor, ModE protein, in Escherichia coli

ModE protein, a molybdate sensor/regulator, controls the transcription of genes coding for molybdate uptake (mod), molybdopterin synthesis (moa), molybdoenzymes nitrate reductase (nap) and dimethylsulfoxide reductase (dms), as well as fermentative dihydrogen production (fdhF and hyc) and respiratory nitrate reductase (narXL) in Escherichia coli. The catalytic product of a second protein, MoeA, is also required for molybdate-dependent positive regulation of hyc and nar operons. To explore the potential role of ModE and MoeA in the regulation of other E. coli genes, the global gene expression profile of a wild type and a modE, moeA double mutant grown in glucose-minimal medium under anaerobic conditions were compared. Expression of 67 genes was affected by the modE and moeA mutations (P value <0.01). Of these, 17 differed by at least 2-fold or higher. Fourteen genes were expressed at a higher level in the mutant (2.4- to 23.9-fold) (notably, mod-molybdate transport, deo-nucleoside catabolism and opp-oligopeptide transport operons) and dmsA and yli operon were expressed at a higher level in the wild type parent (2.6- to 5.7-fold). One of the unexpected findings was repression of the deo operon by ModE. This was confirmed by quantitative RT-PCR and by the analysis of a deoC-lacZ fusion. The deo promoter/operator region contains a putative ModE-consensus sequence centered at -35 in which the adenines are replaced by guanines (TGTGT-N7-TGTGT). The ModE protein did bind to the deo upstream DNA and shifted its electrophoretic mobility. Bioinformatics analysis of the E. coli genome for ModE-consensus motif (TATAT-N7-TAYAT) identified 21 additional genes/operons including the moa as potential targets for Mo-control. The physiological role of many of the genes identified solely by bioinformatics (19/21) is unknown. Expression levels of these genes were similar in the parent and the isogenic modE, moeA mutant when cultured anaerobically in glucose-minimal medium. This study identified additional targets, such as deo and opp, for the Mo-dependent control in E. coli.

ModE-dependent molybdate regulation of the molybdenum cofactor operon moa in Escherichia coli.

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 moa expression 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 of moa. 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.

Molybdenum cofactor amounts in Chlamydomonas reinhardtii depend on the Nit5 gene function related to molybdate transport

ABSTRACTStrain 21gr from Chlamydomonas reinhardtii is a cryptic mutant defective in the Nit5 gene related to the biosynthesis of molybdenum cofactor (MoCo). In spite of this mutation, this strain has active MoCo and can grow on nitrate media. In genetic crosses, the Nit5 mutation cosegregated with a phenotype of resistance to high concentrations of molybdate and tungstate. Molybdate/tungstate toxicity was much higher in nitrate than in ammonium media. Strain 21gr showed lower amounts of MoCo activity than the wild type both when grown in nitrate and after growth in ammonium and nitrate induction. However, nitrate reductase (NR) specific activity was similar in wild type and 21gr cells. Tungstate, either at nanomolar concentrations in nitrate media or at micromolar concentrations during growth in ammonium and nitrate induction, strongly decreased MoCo and NR amounts in wild‐type cells but had a slight effect in 21gr cells. Molybdate uptake activity of ammonium‐grown cells from both the wild‐type and 21gr strains was small and blocked by sulphate 0·3 mM. However, cells from nitrate medium showed a molybdate uptake activity insensitive to sulphate. This uptake activity was much higher and more sensitive to inhibition by tungstate in the wild type than in strain 21gr. These results suggest that strain 21gr has a high affinity and low capacity molybdate transport system able to discriminate efficiently tungstate, and lacks a high capacity molybdate/tungstate transport system, which operates in wild‐type cells upon nitrate induction. This high capacity molybdate transport system would account for both the stimulating effect of molybdate on MoCo amounts and the toxic effects of tungstate and molybdate when present at high concentrations.

Changes in the growth rates of saline-lake diatoms in response to variation in salinity, brine type and nitrogen form

The growth rates of four saline-lake diatom taxa were measured under varying conditions of salinity (5, 8 and 11‰), brine type (sulfate- versus bicarbonate-dominated) and nitrogen form (NH4 + versus NO3 - ), using a full factorial design. With NO3 - as the nitrogen source, Cyclotella quil- lensis, Cymbella pusilla and Anomoeoneis costata exhibited lower growth rates in the sulfate versus bicarbonate media. The strain of Chaetoceros elmorei used in these experiments, isolated from a sulfate-dominated lake, was unable to grow on NO3 - alone. In the NH4 + treatments, neither salinity nor brine type affected the growth rates of C.quillensis or C.elmorei. When supplied with NH4 + , C.pusilla and A.costata had higher growth rates in the bicarbonate versus sulfate media, although for C.pusilla the difference on NH4 + was not as great as on NO3 -. The impact of brine type on NO3 - use is consistent with the theory that sulfate inhibits molybdate uptake, as molybdenum is required for NO3 - use but not NH4 +. Cymbella pusilla was the only taxon affected by changes in salinity. The four taxa used in these experiments are frequently found in saline lakes and saline-lake sediments, hence they are used in paleoclimate reconstructions; the results presented here provide additional infor- mation that may enhance these diatom-based reconstructions.

Molybdenum Metabolism in Plants

Abstract: Among the micronutrients essential for plant growth and for microsymbionts, Mo is required in minute amounts. However, since Mo is often sequestered by Fe‐ or Al‐oxihydrox‐ides, especially in acidic soils, the concentration of the water‐soluble molybdate anion available for uptake by plants may be limiting for the plant, even when the total Mo content of the soil is sufficient. In contrast to bacteria, no specific molybdenum uptake system is known for plants, but since molybdate and sulfate behave similarly and have similar structure, uptake of molybdate could be mediated unspecifically by one of the sulfate transporters. Transport into the different plant organs proceeds via xylem and phloem. A pterin‐bound molybdenum is the cofactor of important plant enzymes involved in redox processes: nitrate reductase, xanthine dehydrogenase, aIdehyde oxidase, and probably sulfite oxidase. Biosynthesis of the molybdenum cofactor (Moco) starts with a guanosine‐X‐phos‐phate. Subsequently, a sulfur‐free pterin is synthesized, sulfur is added, and finally molybdenum is incorporated. In addition to the molybdopterin enzymes, small molybdopterin binding proteins without catalytic function are known and are probably involved in the storage of Moco. In symbiotic systems the nitrogen supply of the host plant is strongly influenced by the availability of Mo in soil, since both bacterial nitrogenase and NADPH‐dependent nitrate reductase of mycorrhizal fungi are Mo enzymes.

Two crystal forms of ModE, the molybdate-dependent transcriptional regulator from Escherichia coli.

The molybdenum-responsive ModE regulatory protein from Escherichia coli has been purified and used in crystallization trials. Two crystal forms have been observed. Form I is tetragonal, P41212 (or enantiomorph), with a = b = 72.3, c = 246.2 A and diffracts to medium resolution. Form II is orthorhombic, P21212, with a = 82.8, b = 127.9, c = 64.0 A and diffraction has been observed beyond 2.8 A resolution. Structural analysis, in combination with ongoing biochemical characterization, will assist the elucidation of the structure-activity relationship in regulating the uptake of molybdate in bacteria.

Anaerobic regulation of the Escherichia coli dmsABC operon requires the molybdate-responsive regulator ModE.

Expression of the Escherichia coli dmsABC operon that encodes a molybdenum-containing DMSO/TMAO reductase is increased in response to anaerobiosis and repressed by nitrate. These changes are mediated by the transcription factors Fnr and NarL respectively. Interestingly, modC strains that are defective in molybdate uptake exhibit impaired anaerobic induction and no nitrate-dependent repression of the dmsABC operon. To determine if the molybdate-responsive transcription factor ModE is involved in this process, a set of dmsA-lacZ operon fusions were constructed and analysed. The pattern of dmsA-lacZ expression in response to anaerobiosis and nitrate addition was identical in both modC and modE strains, thus suggesting a regulatory role for ModE. In vitro studies confirmed that ModE bound the dmsA promoter at a high-affinity site typical of other E. coli ModE operator sites. Mutations in this site abolished ModE binding in vitro and displayed the same phenotype as a modE mutation. In contrast to previously characterized ModE operator sites, which either overlap or are located immediately upstream of the ModE-regulated promoter, the ModE site is centred 52.5 bp downstream of the major dmsA transcript start site. We identified a putative integration host factor (IHF) binding site in the intervening sequence, and in vitro studies confirmed that IHF bound this site with high affinity. Using himA mutants, we confirmed that IHF plays a role in the molybdate-dependent regulation of dmsA-lacZ expression in vivo. This study provides the first example in which ModE affects gene regulation in concert with another transcription factor.

The Escherichia coli modE gene: effect of modE mutations on molybdate dependent modA expression

The Escherichia coli modABCD operon, which encodes a high-affinity molybdate uptake system, is transcriptionally regulated in response to molybdate availability by ModE. Here we describe a highly effective enrichment protocol, applicable to any gene with a repressor role, and establish its application in the isolation of transposon mutations in modE. In addition we show that disruption of the ModE C-terminus abolishes derepression in the absence of molybdate, implying this region of ModE controls the repressor activity. Finally, a mutational analysis of a proposed molybdate binding motif indicates that this motif does not function in regulating the repressor activity of ModE.