Reconstitution of binding protein-dependent ribose transport in spheroplasts of Escherichia coli K-12.

Purified Escherichia coli K-12 ribose binding protein was used to reconstitute the high affinity ribose transport system in spheroplasts derived from ribose-induced cells. It was not possible to reconstitute ribose transport in spheroplasts derived from uninduced cells or from transport-negative mutant strains, suggesting that one or more additional inducible components are required for binding protein-dependent ribose transport. It was possible to reconstitute transport in a ribokinase-deficient mutant which constitutively transports but does not utilize ribose.     

Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.     

Genetically probing the regions of ribose-binding protein involved in permease interaction

The ribose-binding protein (RBP) of Escherichia coli , located in the periplasm, binds to ribose and mediates transport and chemotaxis. The regions on the tertiary structure of RBP that interact with the membrane permease, an ABC transporter, were genetically probed by screening a mutation using the chimeric receptor Trz. Trz is a hybrid protein between the periplasmic domain of chemoreceptor Trg and the cytoplasmic portion of osmosensor EnvZ, which provides a system for monitoring the chemotactic interaction of RBP on MacConkey agar plates when coupled with a reporter lacZ fused to an ompC gene. The expression of ompC can be increased by an interaction of ribose-bound RBP with Trz. A transport defect, either in the binding protein or in the membrane permease, causes a signalling-constitutive Lac⁺ phenotype of Trz even in the absence of ribose. This appears to be due to the presence of a small amount of ribose, which is normally taken up by the high-affinity transport system. By taking advantage of this, we have designed a system for genetic screening that permits a selection for mutations in the binding protein, causing specific defects in permease interaction but not in tactic interaction. Mutant RBPs that were isolated were unable to perform normal ribose uptake and to utilize ribose as a carbon source, while other functions such as taxis and sugar-binding properties were not substantially affected. The mutational changes were repeatedly found in several residues of RBP, concentrating on three surface regions and comprising two domains of the tertiary structure. We suggest that the two regions, including residues 52 and 166, are specifically involved in the permease interaction while the third region, including residues 72, 134, and others, recognizes both the permease and the chemosensory receptor.     

Molecular cloning and characterization of genes required for ribose transport and utilization in Escherichia coli K-12.

We isolated spontaneous and transposon insertion mutants of Escherichia coli K-12 that were specifically defective in utilization or in high-affinity transport of D-ribose (or in both). Cotransduction studies located all of the mutations near ilv, at the same position as previously identified mutations causing defects in ribokinase ( rbsK ) or ribose transport ( rbsP ). Plasmids that complemented the rbs mutations were isolated from the collection of ColE1 hybrid plasmids constructed by Clarke and Carbon. Analysis of those plasmids as well as of fragments cloned into pBR322 and pACYC184 allowed definition of the rbs region. Products of rbs genes were identified by examination of the proteins produced in minicells containing various rbs plasmids. We identified four rbs genes: rbsB , which codes for the 29-kilodalton ribose-binding protein; rbsK , which codes for the 34-kilodalton ribokinase ; rbsA , which codes for a 50-kilodalton protein required for high-affinity transport; and rbsC , which codes for a 27-kilodalton protein likely to be a transport system component. Our studies showed that these genes are transcribed from a common promoter in the order rbsA rbsC rbsB rbsK . It appears that the high-affinity transport system for ribose consists of the three components, ribose-binding protein, the 50-kilodalton RbsA protein, and the 27-kilodalton RbsC protein, although a fourth, unidentified component could exist. Mutants defective in this transport system, but normal for ribokinase , are able to grow normally on high concentrations of the sugar, indicating that there is at least a second, low-affinity transport system for ribose in E. coli K-12.     

Molecular interactions in ribose transport: the binding protein module symmetrically associates with the homodimeric membrane transporter.

The Escherichia coli high-affinity ribose transporter is composed of the periplasmic ribose-binding protein (RBP or RbsB), the membrane component (RbsC) and the ATP-binding protein (RbsA). In order to dissect the molecular interactions initiating the transport process, RbsC suppressors for transport-defective rbsB mutations were isolated. These suppressors are localized in two regions of RbsC, which are allele-specific to N- or C-terminal domain mutations of RBP, suggesting that there are two distinct regions of RbsC, each interacting with one of the two domains of RBP. To demonstrate that these two regions provide a homodimeric binding surface for RBP we constructed a dimeric rbsC in which two genes are joined tandemly from head to tail with the addition of a linker. The dimeric RbsC protein is stable and functional in growth and ribose uptake. By exploiting the allele specificity between the domain-specific mutations and their suppressors, we generated all mutation-suppressor combinations in a single rbsB plus the dimeric rbsC genes. Their phenotypes are consistent with the proposal that the binding protein module interacts symmetrically with homodimeric RbsC. The mode of association proposed here for the ribose transport components could be extended to other ABC transporters with similar structural organizations.     

Reconstitution of binding protein dependent ribose transport in spheroplasts derived from a binding protein negative escherichia coli K12 mutant and from salmonella typhimurium

Highly purified ribose-binding protein from Escherichia coli has been used to reconstitute a binding-protein-dependent ribose transport in spheroplasts derived from a binding-protein-deficient mutant of E coli K 12, and in spheroplasts derived from Salmonella typhimurium. The cross-species reconstitution was nearly as efficient as the reconstitution of the E coli strain from which the binding protein was derived. Antibody raised against the ribose binding protein completely prevented reconstitution, whereas it had no effect on whole cells. The reconstitution procedure has been improved by generating spheroplasts from cells grown in a rich medium and by reducing the background uptake in spheroplasts through a special washing procedure. Rapid purification of ribose binding protein by high pressure liquid chromatography is also described.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver. Factors affecting the activation of the binding protein by adenosine 5'-triphosphate.

A cyclic AMP-adenosine binding protein, whose binding sites are activated by preincubation in the presence of Mg⁺-ATP, has been purified to apparent homogeneity from mouse liver (P.M. Ueland and S.O. Døskeland, 1977, J. Biol. Chem.,252, 677–686). The degree of activation of both the cyclic AMP binding site and a high-affinity site for adenosine depends on the concentration of ATP during the preincubation. The velocity and the degree of activation are dependent on the temperature and the presence of Mg²⁺ and K⁺. The NH₄⁺ ion can be substituted for K⁺, whereas Na⁺ is inefficient. Low pH promotes the conversion from the inactive to the active form. The apparent affinity for adenosine to the high-affinity site for this adenine derivative and the affinity for cyclic AMP to the site specific for this nucleotide are independent of the degree of activation as judged from the slope of Scatchard plots. The activation of the cyclic AMP binding site by ATP (6 mm) was determined at pH 7 in the presence of 10 μm cyclic AMP, AMP, ADP, or adenosine. Adenosine specifically inhibits the activation and does not promote the inactivation of the binding protein. The possibility that the apparent inhibition of activation was effected by interference with cyclic AMP binding by adenosine was ruled out.     

Effect of sulfhydryl reagents on nucleoside transport in cultured mammalian cells

Incubation of Novikoff rat hepatoma cells; mouse L929, P388 and L1210 cells; and Chinese hamster ovary cells with sulfhydryl reagents, such as p-hydroxymercuribenzoate or p-hydroxymercuribenzenesulfonate, reduced the zero-trans influx of uridine in a concentration-dependent manner. The sensitivity of uridine transport to inhibition varied somewhat for the cell lines, Chinese hamster ovary cells being the most sensitive. Maximum inhibition by p-hydroxymercuribenzoate occurred in 10–20 min of incubation at 37 °C, and was associated with a decrease in maximum transport velocity without significant change in substrate affinity of the carrier. The development of inhibition of uridine influx correlated with binding of [¹⁴C]p-hydroxymercuribenzoate to the cells. Inhibition of transport also roughly correlated with a decreased binding of 6-nitrobenzylthioinosine to high-affinity binding sites on the cells (presumably representing the nucleoside transporter) without affecting binding affinity. Treatment of cells with p-hydroxymercuribenzenesulfonate reduced uridine influx and efflux to a similar extent. Inhibition of uridine transport and binding of [¹⁴C]p-hydroxymercuribenzoate were readily reversed by incubation of the cells with dithiothreitol. The results indicate that sulfhydryl groups are essential for the functioning of the nucleoside transporter, perhaps for the binding of substrate. Blockage of the sulfhydryl groups results in a reversible inactivation of the carrier. Treatment of the cells with the sulfhydryl reagents also caused a concentration-dependent increase in cell volume, which was readily reversed by incubation of the cells with dithiothreitol but seemed unrelated to the inhibition of nucleoside transport.     

A New Mechanism for High-Affinity Uptake of C4-Dicarboxylates in Bacteria Revealed by the Structure of Rhodopseudomonas palustris MatC (RPA3494), a Periplasmic Binding Protein of the Tripartite Tricarboxylate Transporter (TTT) Family

C4-dicarboxylates play a central role in cellular physiology as key metabolic intermediates. Under aerobic conditions, they participate in the citric acid cycle, while in anaerobic bacteria, they are important in energy-conserving fermentation and respiration processes. Ten different families of secondary transporters have been described to participate in C4-dicarboxylate movement across biological membranes, but only one of these utilizes an extracytoplasmic solute binding protein to achieve high-affinity uptake. Here, we identify the MatBAC system from the photosynthetic bacterium Rhodopseudomonas palustris as the first member of the tripartite tricarboxylate transport family to be involved in C4-dicarboxylate transport. Tryptophan fluorescence spectroscopy showed that MatC, the periplasmic binding protein from this system, binds to l- and d-malate with K_d values of 27 and 21 nM, respectively, the highest reported affinity to date for these C4-dicarboxylates, and to succinate (K_d = 110 nM) and fumarate (K_d = 400 nM). The 2.1-Å crystal structure of MatC with bound malate shows a high level of substrate coordination, with participation of two water molecules that bridge hydrogen bonds between the ligand proximal carboxylic group and the main chain of two conserved loops in the protein structure. The substrate coordination in MatC correlates with the binding data and explains the protein's selectivity for different substrates and respective binding affinities. Our results reveal a new function in C4-dicarboxylate transport by members of the poorly characterized tripartite tricarboxylate transport family, which are widely distributed in bacterial genomes but for which details of structure–function relationships and transport mechanisms have been lacking.     

Role of D-ribose as a cometabolite in D-xylose metabolism by Saccharomyces cerevisiae.

The influence of D-ribose as a cosubstrate on the uptake and metabolism of the non-growth substrate D-xylose by Saccharomyces cerevisiae ATCC 26602 was investigated. Xylose was taken up by means of low- and high-affinity glucose transport systems. In cells exposed for 2 days to a mixture of xylose and ribose, only the high-affinity system could be detected. Glucose strongly inhibited the transport of xylose by both systems. Starvation or exposure to either xylose or ribose resulted in inactivation of xylose transport, which did not occur in the presence of a mixture of ribose and xylose. A constitutive non-glucose-repressible NADPH2-dependent xylose reductase with a specific activity of ca. 5 mU/mg of protein that converted xylose to xylitol was present in a glucose-grown culture. No activity converting xylitol to xylulose or vice versa was found in crude extracts. Both xylose and ribose were converted to their corresponding polyols, xylitol and ribitol, as indicated by 13C nuclear magnetic resonance spectroscopy. Furthermore, ethanol was detected, and this implied that pathways for the complete catabolism of xylose and ribose exist. However, the NADPH2 required for the conversion of xylose to xylitol is apparently not supplied by the pentose phosphate pathway since the ethanol produced from D-[1-13C]xylose was labelled only in the C-2 position. Acetic acid was produced from ribose and may assist in the conversion of xylose to xylitol by cycling NADPH2.     

Bacterial chemotaxis to saccharides is governed by a trade-off between sensing and uptake

To swim up gradients of nutrients, E. coli senses nutrient concentrations within its periplasm. For small nutrient molecules, periplasmic concentrations typically match extracellular concentrations. However, this is not necessarily the case for saccharides, such as maltose, which are transported into the periplasm via a specific porin. Previous observations have shown that, under various conditions, E. coli limits maltoporin abundance so that, for extracellular micromolar concentrations of maltose, there are predicted to be only nanomolar concentrations of free maltose in the periplasm. Thus, in the micromolar regime, the total uptake of maltose from the external environment into the cytoplasm is limited not by the abundance of cytoplasmic transport proteins but by the abundance of maltoporins. Here, we present results from experiments and modeling suggesting that this porin-limited transport enables E. coli to sense micromolar gradients of maltose despite having a high-affinity ABC transport system that is saturated at these micromolar levels. We used microfluidic assays to study chemotaxis of E. coli in various gradients of maltose and methyl-aspartate and leveraged our experimental observations to develop a mechanistic transport-and-sensing chemotaxis model. Incorporating this model into agent-based simulations, we discover a trade-off between uptake and sensing: although high-affinity transport enables higher uptake rates at low nutrient concentrations, it severely limits the range of dynamic sensing. We thus propose that E. coli may limit periplasmic uptake to increase its chemotactic sensitivity, enabling it to use maltose as an environmental cue.     

Irreversible inhibition of folate transport in Lactobacillus casei by covalent modification of the binding protein with carbodiimide-activated folate

Activated folate formed by reaction of folic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide irreversibly inhibits the folate transport system of Lactobacillus casei. Complete inhibition of both folate binding to the carrier protein and folate transport was achieved by pretreatment of the cells at low temperature (4 °C) and at neutral pH with 200 nm activated folate. Fifty percent inhibition of binding and transport occurred at 35 and 40 nm activated folate, respectively. Specificity was demonstrated by the fact that excess nonactivated folate added during the pretreatment step afforded complete protection of the binding protein against inhibition, and that activated folate had no effect on the binding or transport of thiamine. Rapid measurements at 4 °C were employed to show that, prior to the appearance of irreversible inhibition, activated folate (K_i = 15 nM) interacted reversibly with the binding site for folate (K_d = 0.8 nM). Cells treated with activated [³H]folate incorporated 1 mol of folate per mole of binding protein. Purification of the labeled protein followed by digestion with Pronase led to the isolation of a compound identified as ?-N-folyl lysine. The ?-amino group of a lysyl residue thus appears to be the nucleophilic group at the binding site that reacts with activated folate.     

Binding properties of the 5-methyltetrahydrofolate/methotrexate transport system in L1210 cells

A binding component with a high affinity for 5-methyltetrahydrofolate (K_D = 0.11μm) is present on the external surface of L1210 cells. The amount of binder (1 pmol/mg protein) corresponds to 8 × 10⁴ sites per cell. The participation of this component in the high-affinity 5-methyltetrahydrofolate/methotrexate transport system is supported by similarities in the K_D values for 5-methyltetrahydrofolate and methotrexate binding and the K_t values of these compounds for transport. Relative affinities for other folate substrates (aminopterin, 5-formyltetrahydrofolate, and folate) and various competitive inhibitors (thiamine pyrophosphate, ADP, AMP, arsenate, and phosphate) are also similar for both the binding component and the transport system. The measured binding activity does not represent low-temperature transport of substrate into cells, since it is readily saturable with time and is eliminated by either washing the cells with buffer or by the addition of excess unlabeled substrate.     

Transport of uridine in Escherichia coli B and A showdomycin-resistant mutant

The mechanism of uridine transport in Escherichia coli B cells was studied using experimental approaches designed to limit possible ambiguities in interpretation of data obtained previously. For this purpose, the transport of [2-¹⁴C]uridine and [U-¹⁴C]uridine was determined in E. coli B and an E. coli B mutant which is resistant to the inhibitory effects of the nucleoside antibiotic, showdomycin.The majorty of the uridine transported as the intact nucleoside is cleaved to uracil and ribose l-phosphate. The uracil, in large part, is excreted, while ribose l-phosphate is retained. In addition, uridine is also rapidly cleaved to uracil and ribose l-phosphate in the periplasmic space. The uracil moiety may enter the cell, whereas ribose l-phosphate is not transported. The showdomycin-resistant mutant transports the intact nucleoside inefficiently, or not at all, but retains its ability to convert uridine to uracil in the periplasmic space.     

Ligand-induced conformational changes in a thermophilic ribose-binding protein

Background

Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments.

Results

Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile Thermotoga maritima in its ribose-bound and unliganded state. The T. maritima RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation ( ^app T _m value is 108°C) than the mesophilic Escherichia coli homolog (ecRBP) ( ^app T _m value is 56°C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved.

Conclusion

Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different mechanisms to form a ligand-bound state.     

Temperature-sensitive periods and autonomy of pleiotropic effects of l(1)Nts1, a conditional notch lethal in Drosophila.

Analysis of temperature-shift experiments using strains homo- and/or hemizygous for a temperature-sensitive (ts) mutation of the Notch locus, l(1)N^ts1, has permitted us to localize temperature-sensitive periods (TSPs) both for lethality and for adult ectodermal morphology defects. Discrete TSPs for lethality are localized to the first half of the embryonic period, to the second larval instar, to the third larval instar, and to a 15 hr period immediately after pupation. TSPs for adult morphology defects are localized to the second and third larval instars for eyeless-headless and duplicated antenna, to the third larval instar for small and rough (spl-like) eye, eye scar, fused leg segments, shortened tarsal leg segments, Notch wings, and extra macrochaetae, and to the early pupal period for extra and missing microchaetae, fa^g-like rough eye and thick wing vein defects. Within the third larval instar, distinct patterns of eye, wing, and leg defects are observed. There is a striking similarity between the adult morphology defects and TSPs of l(1)N^ts1 and those of the larval and adult locomotor mutant, shi^ts1 (C. A. Poodry, L. Hall, and D. T. Suzuki, 1973, Develop. Biol.32, 373–386). Expression of l(1)N^ts1 also has been studied in genetic mosaics, in which we find that the pleiotropic effects of l(1)N^ts1 are autonomously expressed.     

Conformational Changes of Three Periplasmic Receptors for Bacterial Chemotaxis and Transport: The Maltose-, Glucose/Galactose- and Ribose-binding Proteins.

Small-angle X-ray scattering experiments were carried out for the maltose-, glucose/galactose- and ribose-binding proteins of Gram negative bacteria. All were shown to be monomers that decrease in radius of gyration on ligand binding.The results obtained for the maltose-binding protein agree well with crystal structures of the closed, ligand-bound, and open, ligand-free protein, suggesting that these are indeed the primary forms in solution. The closed form is stabilized by protein – sugar interactions, while the open conformation is stabilized by close contacts between the two domains. Since it is the proper spacial relationship of the domains in the closed form that is most important for interaction with chemotaxis and transport partners, the stabilization of the open form would help keep ligand-free molecules from interfering in function.The scattering results also provide evidence that a large conformational change takes place in association with ligand binding to the glucose/galactose- and ribose-binding proteins, and that the two changes are similar. Modeling suggests that the open forms resemble those found in the related leucine and leucine/isoleucine/valine-binding proteins, but are different from those observed for the maltose-binding protein and the related purine repressor.     

Fast Induction of High-Affinity HCO3− Transport in Cyanobacteria

The induction of a high-affinity state of the CO₂-concentration mechanism was investigated in two cyanobacterial species, Synechococcus sp. strain PCC7002 and Synechococcus sp. strain PCC7942. Cells grown at high CO₂ concentrations were resuspended in low-CO₂ buffer and illuminated in the presence of carbonic anhydrase for 4 to 10 min until the inorganic C compensation point was reached. Thereafter, more than 95% of a high-affinity CO₂-concentration mechanism was induced in both species. Mass-spectrometric analysis of CO₂ and HCO₃⁻ fluxes indicated that only the affinity of HCO₃⁻ transport increased during the fast-induction period, whereas maximum transport activities were not affected. The kinetic characteristics of CO₂ uptake remained unchanged. Fast induction of high-affinity HCO₃⁻ transport was not inhibited by chloramphenicol, cantharidin, or okadaic acid. In contrast, fast induction of high-affinity HCO₃⁻ transport did not occur in the presence of K252a, staurosporine, or genistein, which are known inhibitors of protein kinases. These results show that induction of high-affinity HCO₃⁻ transport can occur within minutes of exposure to low-inorganic-C conditions and that fast induction may involve posttranslational phosphorylation of existing proteins rather than de novo synthesis of new protein components.     

Properties of the galactose binding protein of Salmonella typhimurium and Escherichia coli.

The galactose binding protein implicated in transport and in chemotaxis has been purified to homogeneity from the shock fluids of Salmonella typhimurium and Escherichia coli. Both proteins are monomers of molecular weight 33 000 and exhibit cross-reactivity with antibody. The Salmonella galactose receptor showed binding of 1 mol of [14C]galactose or 1 mol of [14C]glucose at saturation. The dissociation constants were 0.38 and 0.17 muM, respectively. In light of the previously published report that the E. coli protein contains two binding sites with two different affinities, the binding characteristics of this protein were reexamined. Using highly purified radiolabeled substrate and homogeneous protein, a single binding site and single binding affinity were seen galactose (KD = 0.48 muM) or for glucose (KD = 0.21 muM). The competition between glucose and galactose for the same site is intriguing in view of the competition between ribose and galactose at the receptor level.