An analysis of the binding of repressor protein ModE to modABCD (molybdate transport) operator/promoter DNA of Escherichia coli.

Expression of the modABCD operon in Escherichia coli, which codes for a molybdate-specific transporter, is repressed by ModE in vivo in a molybdate-dependent fashion. In vitro DNase I-footprinting experiments identified three distinct regions of protection by ModE-molybdate on the modA operator/promoter DNA, GTTATATT (-15 to -8; region 1), GCCTACAT (-4 to +4; region 2), and GTTACAT (+8 to +14; region 3). Within the three regions of the protected DNA, a pentamer sequence, TAYAT (Y = C or T), can be identified. DNA-electrophoretic mobility experiments showed that the protected regions 1 and 2 are essential for binding of ModE-molybdate to DNA, whereas the protected region 3 increases the affinity of the DNA to the repressor. The stoichiometry of this interaction was found to be two ModE-molybdate per modA operator DNA. ModE-molybdate at 5 nM completely protected the modABCD operator/promoter DNA from DNase I-catalyzed hydrolysis, whereas ModE alone failed to protect the DNA even at 100 nM. The apparent K(d) for the interaction between the modA operator DNA and ModE-molybdate was 0.3 nM, and the K(d) increased to 8 nM in the absence of molybdate. Among the various oxyanions tested, only tungstate replaced molybdate in the repression of modA by ModE, but the affinity of ModE-tungstate for modABCD operator DNA was 6 times lower than with ModE-molybdate. A mutant ModE(T125I) protein, which repressed modA-lac even in the absence of molybdate, protected the same region of modA operator DNA in the absence of molybdate. The apparent K(d) for the interaction between modA operator DNA and ModE(T125I) was 3 nM in the presence of molybdate and 4 nM without molybdate. The binding of molybdate to ModE resulted in a decrease in fluorescence emission, indicating a conformational change of the protein upon molybdate binding. The fluorescence emission spectra of mutant ModE proteins, ModE(T125I) and ModE(Q216*), were unaffected by molybdate. The molybdate-independent mutant ModE proteins apparently mimic in its conformation the native ModE-molybdate complex, which binds to a DNA sequence motif of TATAT-7bp-TAYAT.     

Characterization of the molybdate transport system ModABC of Staphylococcus carnosus

Transposon mutagenesis of Staphylococcus carnosus led to the identification of three genes, modABC, which encode an ABC transporter that is involved in molybdate transport. It was shown by [¹⁴C]palmitate labeling that ModA represents a lipoprotein that in gram-positive bacteria is the counterpart of the periplasmic binding proteins of gram-negative organisms. The sequence characteristics identify ModB as the integral-membrane, channel-forming protein and ModC as the ATP-binding energizer for the transport system. Mutants defective in modABC had only 0.4% of the wild-type nitrate reductase activity. Molybdate at a non-physiologically high concentration (100 μM) fully restored nitrate reductase activity, suggesting that at least one other system is able to transport molybdate, but with lower affinity. The expression of modA (and most likely of modBC) was independent of oxygen and nitrate. To date, there are no indications for molybdate-specific regulation of modABC expression since in a modB mutant, modA expression was unchanged in comparison to the wild-type. Received: 5 February 1999 / Accepted: 31 May 1999     

Structure of the molybdate/tungstate binding protein mop from Sporomusa ovata

BACKGROUND: Transport of molybdenum into bacteria involves a high-affinity ABC transporter system whose expression is controlled by a repressor protein called ModE. While molybdate transport is tightly coupled to utilization in some bacteria, other organisms have molybdenum storage proteins. One class of putative molybdate storage proteins is characterized by a sequence consisting of about 70 amino acids (Mop). A tandem repeat of Mop sequences also constitutes the molybdate binding domain of ModE. RESULTS: We have determined the crystal structure of the 7 kDa Mop protein from the methanol-utilizing anaerobic eubacterium Sporomusa ovata grown in the presence of molybdate and tungstate. The protein occurs as highly symmetric hexamers binding eight oxyanions. Each peptide assumes a so-called OB fold, which has previously also been observed in ModE. There are two types of oxyanion binding sites in Mo at the interface between two or three peptides. All oxyanion binding sites were found to be occupied by WO(4) rather than MoO(4). CONCLUSIONS: The biological function of proteins containing only Mop sequences is unknown, but they have been implicated in molybdate homeostasis and molybdopterin cofactor biosynthesis. While there are few indications that the S. ovata Mop binds pterin, the structure suggests that only the type-1 oxyanion binding sites would be sufficiently accessible to bind a cofactor. The observed occupation of the oxyanion binding sites by WO(4) indicates that Mop might also be involved in controlling intracellular tungstate levels.     

Dissection of discrete kinetic events in the binding of antibiotics and substrates to the galactose-H+ symport protein,GalP, ofEscherichia coli

GalP is the membrane protein responsible for H⁺-driven uptake of D-galactose intoEscherichia coli. It is suggested to be the bacterial equivalent of the mammalian glucose transporter, GLUT1, since these proteins share sequence homology, recognise and transport similar substrates and are both inhibited by cytochalasin B and forskolin. The successful over-production of GalP to 35–55% of the total inner membrane protein ofE. coli has allowed direct physical measurements on isolated membrane preparations. The binding of the antibiotics cytochalasin B and forskolin could be monitored from changes in the inherent fluorescence of GalP, enabling derivation of a kinetic mechanism describing the interaction between the ligands and GalP. The binding of sugars to GalP produces little or no change in the inherent fluorescence of the transporter. However, the binding of transported sugars to GalP produces a large increase in the fluorescence of 8-anilino-1-naphthalene sulphonate (ANS) excited via tryptophan residues. This has allowed a binding step, in addition to two putative translocation steps, to be measured. From all these studies a basic kinetic mechanism for the transport cycle under non-energised conditions has been derived. The ease of genetical manipulation of thegalP gene inE. coli has been exploited to mutate individual amino acid residues that are predicted to play a critical role in transport activity and/or the recognition of substrates and antibiotics. Investigation of these mutant proteins using the fluorescence measurements should elucidate the role of individual residues in the transport cycle as well as refine the current model.Abbreviations GalP galactose-H⁺ transporter - AraE arabinose-H⁺ transporter - GLUT1 human erythrocyte glucose transporter requests for offprints: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2UH, UK     

Ferric hydroxamate binding protein FhuD from Escherichia coli: mutants in conserved and non-conserved regions

Uptake of iron complexes into the Gram-negative bacterial cell requires highly specific outer membrane receptors and specific ATP-dependent (ATP-Binding-Cassette (ABC)) transport systems located in the inner membrane. The latter type of import system is characterized by a periplasmic binding protein (BP), integral membrane proteins, and membrane-associated ATP-hydrolyzing proteins. In Gram-positive bacteria lacking the periplasmic space, the binding proteins are lipoproteins tethered to the cytoplasmic membrane. To date, there is little structural information about the components of ABC transport systems involved in iron complex transport. The recently determined structure of the Escherichia coli periplasmic ferric siderophore binding protein FhuD is unique for an ABC transport system (Clarke et al. 2000). Unlike other BP's, FhuD has two domains connected by a long -helix. The ligand binds in a shallow pocket between the two domains. In vivo and in vitro analysis of single amino acid mutants of FhuD identified several residues that are important for proper functioning of the protein. In this study, the mutated residues were mapped to the protein structure to define special areas and specific amino acid residues in E. coli FhuD that are vital for correct protein function. A number of these important residues were localized in conserved regions according to a multiple sequence alignment of E. coli FhuD with other BP's that transport siderophores, heme, and vitamin B₁₂. The alignment and structure prediction of these polypeptides indicate that they form a distinct family of periplasmic binding proteins.     

Isolation of the putP gene of Corynebacterium glutamicum and characterization of a low-affinity uptake system for compatible solutes

Corynebacterium glutamicum accumulates the compatible solutes proline, glycine betaine, and ectoine under conditions of high osmolality. Uptake of proline is mediated by both a high-affinity and a low-affinity secondary transport system. The low-affinity uptake system also accepts glycine betaine and ectoine as substrates. In the present study, the gene encoding the high-affinity proline uptake system PutP was isolated by heterologous complementation of Escherichia coli mutant strain WG389, which lacks the transport systems BetT, PutP, ProP, and ProU and is unable to synthesize proline and glycine betaine. This gene (putP) encodes a protein of 524 amino acids that shares identity with the proline transport systems PutP of E. coli, Staphylococcus aureus, Salmonella typhimurium, Haemophilus influenzae, and Klebsiella pneumoniae. Functional studies of PutP synthesized in E. coli mutant strain MKH13, which also lacks the transport systems for compatible solutes and is unable to synthesize glycine betaine, revealed that this carrier system is not regulated by the external osmolality on the level of activity. K _m values of 7.6 mM for proline and 1.3 mM for sodium as cotransported ion were determined. Deletion of the putP gene allowed the functional characterization of another proline uptake system with low affinity. Received: 27 February 1997 / Accepted: 24 April 1997     

Identification of an Arabidopsis solute carrier critical for intracellular transport and inter-organ allocation of molybdate

Plants represent an important source of molybdenum in the human diet. Recently, MOT1 has been identified as a transport protein responsible for molybdate import in Arabidopsis thaliana L.; however, the function of the homologous protein MOT2 has not been resolved. Interestingly, MOT2‐GFP analysis indicated a vacuolar location of this carrier protein. By site directed mutagenesis at the N‐terminal end of MOT2, we identified a di‐leucine motif that is essential for driving the protein into the vacuolar membrane. Molybdate quantification in isolated vacuoles showed that this organelle serves as an important molybdate store in Arabidopsis cells. When grown on soil, leaves from mot2 T‐DNA mutants contained more molybdate, whereas mot2 seeds contained significantly less molybdate than corresponding wild‐type (Wt) tissues. Remarkably, MOT2 mRNA accumulates in senescing leaves and mot2 leaves from plants that had finished their life cycle had 15‐fold higher molybdate levels than Wt leaves. Reintroduction of the endogenous MOT2 gene led to a Wt molybdate phenotype. Thus, mot2 mutants exhibit impaired inter‐organ molybdate allocation. As total concentrations of the molybdenum cofactor (Moco) and its precursor MPT correlates with leaf molybdate levels, we present novel evidence for an adjustment of Moco biosynthesis in response to cellular MoO₄^2? levels. We conclude that MOT2 is important for vacuolar molybdate export, an N‐terminal di‐leucine motif is critical for correct subcellular localisation of MOT2 and activity of this carrier is required for accumulation of molybdate in Arabidopsis seeds. MOT2 is a novel element in inter‐organ translocation of an essential metal ion.     

Reduction of molybdate by sulfate-reducing bacteria

Molybdate is an essential trace element required by biological systems including the anaerobic sulfate-reducing bacteria (SRB); however, detrimental consequences may occur if molybdate is present in high concentrations in the environment. While molybdate is a structural analog of sulfate and inhibits sulfate respiration of SRB, little information is available concerning the effect of molybdate on pure cultures. We followed the growth of Desulfovibrio gigas ATCC 19364, Desulfovibrio vulgaris Hildenborough, Desulfovibrio desulfuricans DSM 642, and D. desulfuricans DSM 27774 in media containing sub-lethal levels of molybdate and observed a red-brown color in the culture fluid. Spectral analysis of the culture fluid revealed absorption peaks at 467, 395 and 314 nm and this color is proposed to be a molybdate–sulfide complex. Reduction of molybdate with the formation of molybdate disulfide occurs in the periplasm D. gigas and D. desulfuricans DSM 642. From these results we suggest that the occurrence of poorly crystalline Mo-sulfides in black shale may be a result from SRB reduction and selective enrichment of Mo in paleo-seawater.     

The rnf gene products in Rhodobacter capsulatus play an essential role in nitrogen fixation during anaerobic DMSO-dependent growth in the dark

The rnf genes in Rhodobacter capsulatus are essential for nitrogen fixation in the light. Because R. capsulatus grows readily on N₂ in the dark by anaerobic respiration with dimethylsulfoxide, the diazotrophic capacities of various strains in the dark were examined. No rnf mutants tested grew diazotrophically, and a nonpolar fdxN-null mutant showed decreased diazotrophic growth in the dark, suggesting that the Rnf and FdxN proteins form the primary electron donor pathway to nitrogenase in the dark as well as in the light. Nonphotosynthetic mutants lacking the component of cyclic electron transport grew diazotrophically and the levels of Rnf proteins were similar to those of the wild-type. These results indicate that rnf gene products play an essential role in nitrogen fixation without any functional link to the cyclic electron transport system. Received: 19 August 1997 / Accepted: 20 January 1998     

Purification and properties of the Escherichia coli nucleoside transporter NupG,a paradigm for a major facilitator transporter sub-family

NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K_m values of ～20–30 μM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1′-¹³C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly α-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.     

Binding protein dependent transport of C4-dicarboxylates in Rhodobacter capsulatus

The characteristics of malate transport into aerobically grown cells of the purple photosynthetic bacterium Rhodobacter capsulatus were determined. A single transport system was distinguished kinetically which displayed a K_t value of 2.9 ± 1.2 μM and V_max of 43 ± 6 nmol · min^-1 · mg^-1 protein. Competition experiments indicated that the metabolically related C4-dicarboxylates succinate and fumarate are also transported by this system. Malate uptake was sensitive to osmotic shock and evidence from the binding of radiolabelled malate and succinate to periplasmic protein fractions indicated that transport is mediated by a dicarboxylate binding protein. The activity of the transport system was studied as a function of external and internal pH and it was found that a marked activation of uptake occurred at intracellular pH values greater than 7. The use of a high affinity binding protein dependent system to transport a major carbon and energy source suggests that Rhodobacter capsulatus would be capable of obtaining growth sustaining quantities of C4-dicarboxylates even if these were present at very low concentrations in the environment.