The High-Affinity Maltose/Trehalose ABC Transporter in the Extremely Thermophilic Bacterium Thermus thermophilus HB27 Also Recognizes Sucrose and Palatinose

ABSTRACT We have studied the transport of trehalose and maltose in the thernophilic bacterium Thermus thermophilus HB27, which grows optimally in the range of 70 to 75°C. The Km values at 70°C were 109 nM for trehalose and 114 nM for maltose; also, a high Km (424 nM) was found for the uptake of sucrose. Competition studies showed that a single transporter recognizes trehalose, maltose, and sucrose, while d-galactose, d-fucose, l-rhamnose, l-arabinose, and d-mannose were not competitive inhibitors. In the recently published genome of T. thermophilus HB27, two gene clusters designated malEFG1 (TTC1627 to -1629) and malEFG2 (TTC1288 to -1286) and two monocistronic genes designated malK1 (TTC0211) and malK2 (TTC0611) are annotated as trehalose/maltose and maltose/maltodextrin transport systems, respectively. To find out whether any of these systems is responsible for the transport of trehalose, the malE1 and malE2 genes, lacking the sequence encoding the signal peptides, were expressed in Escherichia coli. The binding activity of pure recombinant proteins was analyzed by equilibrium dialysis. MalE1 was able to bind maltose, trehalose, and sucrose but not glucose or maltotetraose (Kd values of 103, 67, and 401 nM, respectively). Mutants with disruptions in either malF1 or malK1 were unable to grow on maltose, trehalose, sucrose, or palatinose, whereas mutants with disruption in malK2 or malF2 showed no growth defect on any of these sugars. Therefore, malEFG1 encodes the binding protein and the two transmembrane subunits of the trehalose/maltose/sucrose/palatinose ABC transporter, and malK1 encodes the ATP-binding subunit of this transporter. Despite the presence of an efficient transporter for trehalose, this compound was not used by HB27 for osmoprotection. MalE1 and MalE2 exhibited extremely high thermal stability: melting temperatures of 90°C for MalE1 and 105°C for MalE2 in the presence of 2.3 M guanidinium chloride. The latter protein did not bind any of the sugars examined and is not implicated in a maltose/maltodextrin transport system. This work demonstrates that malEFG1 and malK1 constitute the high-affinity ABC transport system of T. thermophilus HB27 for trehalose, maltose, sucrose, and palatinose.

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Proton gradient-dependent renal transport of glycine: evidence for vesicle studies

AJP Renal Physiology ◽

10.1152/ajprenal.1990.258.2.f388 ◽

1990 ◽

Vol 258 (2) ◽

pp. F388-F396 ◽

Cited By ~ 2

Author(s):

H. Roigaard-Petersen ◽

H. Jessen ◽

S. Mollerup ◽

K. E. Jorgensen ◽

C. Jacobsen ◽

...

Keyword(s):

Transport System ◽

Membrane Vesicles ◽

Proton Gradient ◽

Transport Systems ◽

Rabbit Kidney ◽

Renal Transport ◽

High Affinity ◽

Luminal Membrane ◽

Pars Recta ◽

Pars Convoluta

The characteristics of renal transport of glycine by luminal membrane vesicles isolated from either proximal convoluted part (pars convoluta) or proximal straight part (pars recta) of rabbit proximal tubule were investigated. In vesicles from pars convoluta two transport systems have been characterized: a Na(+)-dependent system with intermediate affinity (half-saturation 3.64 mM) and a Na(+)-independent system that, in the presence of H+ gradient (extravesicular greater than intravesicular), can accelerate the transport of glycine into these vesicles. This is the first demonstration of H(+)-glycine cotransport across the luminal membrane of rabbit kidney proximal convoluted tubule. By contrast, in membrane vesicles from pars recta, transport of glycine was strictly dependent on Na+ and occurred via a dual transport system, namely a high-affinity (half-saturation 0.34 mM) and a low-affinity system (half-saturation 8.56 mM). The demonstration of competition between the H(+)-gradient dependent uptake of glycine, L-alanine, and L-proline, but insignificant inhibition with L-phenylalanine in vesicles from pars convoluta suggests that glycine, L-proline, and L-alanine probably share a common proton gradient-dependent transport system. In vesicles from pars recta, the Na(+)-dependent uptake of glycine was inhibited by low concentrations of L-alanine and L-phenylalanine, whereas addition of L-proline to the incubation medium did not significantly alter the uptake of glycine, suggesting that the Na(+)-dependent high-affinity system for glycine located in pars recta is shared with the high-affinity L-alanine and L-phenylalanine but not L-proline transport system.

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The d-Xylose-Binding Protein, XylF, from Thermoanaerobacter ethanolicus 39E: Cloning, Molecular Analysis, and Expression of the Structural Gene

Journal of Bacteriology ◽

10.1128/jb.180.14.3570-3577.1998 ◽

1998 ◽

Vol 180 (14) ◽

pp. 3570-3577 ◽

Cited By ~ 10

Author(s):

Milutin Erbeznik ◽

Herbert J. Strobel ◽

Karl A. Dawson ◽

Chris R. Jones

Keyword(s):

Amino Acid ◽

Binding Proteins ◽

Binding Protein ◽

Xylose Isomerase ◽

Thermophilic Bacterium ◽

Transport Systems ◽

Uptake System ◽

Calculated Molecular Mass ◽

Thermoanaerobacter Ethanolicus ◽

High Affinity

ABSTRACT Immediately downstream from the Thermoanaerobacter ethanolicus xylAB operon, comprising genes that encoded-xylose isomerase and d-xylulose kinase, lies a 1,101-bp open reading frame that exhibits 61% amino acid sequence identity to the Escherichia coli d-xylose binding periplasmic receptor, XylF, a component of the high-affinity binding-protein-dependent d-xylose transport. The 25-residue N-terminal fragment of the deduced T. ethanolicus XylF has typical features of bacterial leader peptides. The C-terminal portion of this leader sequence matches the cleavage consensus for lipoproteins and is followed by a 22-residue putative linker sequence rich in serine, threonine, and asparagine. The putative mature 341-amino-acid-residue XylF (calculated molecular mass of 37,069 Da) appears to be a lipoprotein attached to the cell membrane via a lipid anchor covalently linked to the N-terminal cysteine, as demonstrated by metabolic labelling of the recombinant XylF with [14C]palmitate. The induced E. coli avidly bound d-[14C]xylose, yielding additional evidence that T. ethanolicus XylF is thed-xylose-binding protein. On the basis of sequence comparison of XylFs to other monosaccharide-binding proteins, we propose that the sequence signature of binding proteins specific for hexoses and pentoses be refined as (KDQ)(LIVFAG)3IX3(DN)(SGP)X3(GS)X(LIVA)2X2A. Transcription of the monocistronic 1.3-kb xylF mRNA is inducible by xylose and unaffected by glucose. Primer extension analysis indicated that xylF transcription initiates from two +1 sites, both situated within the xylAB operon. Unlike in similar transport systems in other bacteria, the genes specifying the membrane components (e.g., ATP-binding protein and permease) of the high-affinity d-xylose uptake system are not located in the vicinity of xylF in T. ethanolicus. This is the first report of a gene encoding a xylose-binding protein in a gram-positive or thermophilic bacterium.

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High-Affinity Vanadate Transport System in the Cyanobacterium Anabaena variabilis ATCC 29413

Journal of Bacteriology ◽

10.1128/jb.188.2.464-468.2006 ◽

2006 ◽

Vol 188 (2) ◽

pp. 464-468 ◽

Cited By ~ 26

Author(s):

Brenda S. Pratte ◽

Teresa Thiel

Keyword(s):

Transport System ◽

Azotobacter Vinelandii ◽

Nitrogenase Activity ◽

Rhodopseudomonas Palustris ◽

Methanosarcina Barkeri ◽

Anabaena Variabilis ◽

Transport Systems ◽

Bacterial Strains ◽

High Affinity ◽

Major Cluster

ABSTRACT High-affinity vanadate transport systems have not heretofore been identified in any organism. Anabaena variabilis, which can fix nitrogen by using an alternative V-dependent nitrogenase, transported vanadate well. The concentration of vanadate giving half-maximum V-nitrogenase activity when added to V-starved cells was about 3 × 10−9 M. The genes for an ABC-type vanadate transport system, vupABC, were found in A. variabilis about 5 kb from the major cluster of genes encoding the V-nitrogenase, and like those genes, the vupABC genes were repressed by molybdate; however, unlike the V-nitrogenase genes the vanadate transport genes were expressed in vegetative cells. A vupB mutant failed to grow by using V-nitrogenase unless high levels of vanadate were provided, suggesting that there was also a low-affinity vanadate transport system that functioned in the vupB mutant. The vupABC genes belong to a family of putative metal transport genes that include only one other characterized transport system, the tungstate transport genes of Eubacterium acidaminophilum. Similar genes are not present in the complete genomes of other bacterial strains that have a V-nitrogenase, including Azotobacter vinelandii, Rhodopseudomonas palustris, and Methanosarcina barkeri.

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Evidence of a calcium-ion-transport system in mitochondria isolated from flight muscle of the developing sheep blowfly Lucilia cuprina

Biochemical Journal ◽

10.1042/bj1460601 ◽

1975 ◽

Vol 146 (3) ◽

pp. 601-608 ◽

Cited By ~ 27

Author(s):

F L Bygrave ◽

A A Daday ◽

F A Doy

Keyword(s):

Transport System ◽

Flight Muscle ◽

Specific Binding ◽

Liver Mitochondria ◽

Ruthenium Red ◽

Lucilia Cuprina ◽

Transport Systems ◽

Rat Liver Mitochondria ◽

High Affinity ◽

Energy Saturation

The EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-Ruthenium Red-quench technique (Reed & Bygrave, 1974a) was used to measure initial rates of Ca-2+ transport in mitochondria from flight muscle of the blowfly Lucilia cuprina. Evidence is provided for the existence in these mitochondria of a Ca-2+-transport system that has many features in common with that known to exist in rat liver mitochondria. These include requirement for energy, saturation at high concentrations of Ca-2+, a sigmoidal relation between initial rates of Ca-2+ transport and Ca-2+ concentration, a high affinity for free Ca-2+ (Km approx. 5 muM) and high affinity for the Ca-2+-transport inhibitoy, Ruthenium Red (approx. 0.03 nmol of carrier-specific binding-sites/mg of protein; Ki approx. 1.6 × 10- minus 8 M). Controlled respiration can be stimulated by Ca-2+ after a short lag-period provided the incubation medium contains KCl and not sucrose. The ability of Lucilia mitochondria to transport Ca-2+ critically depends on the stage of mitochondrial development; Ca-2+ transport is minimal in mitochondria from pharate adults, is maximal between 0 and 2h post-emergence and thereafter rapidly declines to reach less than 20% of the maximum value by about 2-3 days post-emergence. Respiration in mitochondria from newly emerged flies does not respond to added Ca-2+; that from 3-5-day-old flies is stimulated approx. 50%. Whereas very low concentrations of Ca-2+ inhibit ADP-stimulated respiration and oxidative phosphorylation in mitochondria from newly emerged flies (Ki approx. 60 ng-ions of Ca-2+/mg of protein); much higher concentrations (approx. 200 ng-ion/mg of protein) are needed to inhibit these processes in those from older flies. The potential of this system for studying the function and development of metabolite transport systems in mitochondria is discussed.

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Characterization of Two Inducible Phosphate Transport Systems in Rhizobium tropici

Applied and Environmental Microbiology ◽

10.1128/aem.66.1.15-22.2000 ◽

2000 ◽

Vol 66 (1) ◽

pp. 15-22 ◽

Cited By ~ 13

Author(s):

Lina M. Botero ◽

Thamir S. Al-Niemi ◽

Timothy R. McDermott

Keyword(s):

Transport System ◽

Response Regulator ◽

Insertion Site ◽

Dry Weight ◽

Type Systems ◽

Phosphate Transport ◽

Transport Systems ◽

Atpase Inhibitor ◽

Rhizobium Tropici ◽

High Affinity

ABSTRACT Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropiciassociation is sensitive to the availability of phosphate (Pi). To better understand phosphorus movement between the bacteroid and the host plant, Pi transport was characterized in R. tropici. We observed two Pitransport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. TheKm and V max values for the low-affinity system were estimated to be 34 ± 3 μM Pi and 118 ± 8 nmol of Pi · min−1 · mg (dry weight) of cells−1, respectively, and the Km andV max values for the high-affinity system were 0.45 ± 0.01 μM Pi and 86 ± 5 nmol of Pi · min−1 · mg (dry weight) of cells−1, respectively. Both systems were inducible by Pi starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but Pi transport through both systems was eliminated by the ATPase inhibitor N,N′-dicyclohexylcarbodiimide; the Pi transport rate was correlated with the intracellular ATP concentration. Also, Pi movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both Pi transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology withkdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.

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Cellobiose Uptake in the Hyperthermophilic ArchaeonPyrococcus furiosus Is Mediated by an Inducible, High-Affinity ABC Transporter

Journal of Bacteriology ◽

10.1128/jb.183.17.4979-4984.2001 ◽

2001 ◽

Vol 183 (17) ◽

pp. 4979-4984 ◽

Cited By ~ 31

Author(s):

Sonja M. Koning ◽

Marieke G. L. Elferink ◽

Wil N. Konings ◽

Arnold J. M. Driessen

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Abc Transporter ◽

Transport System ◽

Abc Transporters ◽

Pyrococcus Furiosus ◽

Binding Protein ◽

Hyperthermophilic Archaeon ◽

High Affinity ◽

Dependent Transport

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosuscan utilize different β-glucosides, like cellobiose and laminarin. Cellobiose uptake occurs with high affinity (K m = 175 nM) and involves an inducible binding protein-dependent transport system. The cellobiose binding protein (CbtA) was purified from P. furiosusmembranes to homogeneity as a 70-kDa glycoprotein. CbtA not only binds cellobiose but also cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, and sophorose. The cbtAgene was cloned and functionally expressed in Escherichia coli. cbtA belongs to a gene cluster that encodes a transporter that belongs to the Opp family of ABC transporters.

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Molecular Mechanisms of Phosphate Sensing, Transport and Signalling in Streptomyces and Related Actinobacteria

International Journal of Molecular Sciences ◽

10.3390/ijms22031129 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1129

Author(s):

Juan Francisco Martín ◽

Paloma Liras

Keyword(s):

Transport System ◽

Inorganic Phosphate ◽

Molecular Mechanisms ◽

Phosphate Transport ◽

Transport Systems ◽

Outer Side ◽

Phosphate Binding ◽

Streptomyces Species ◽

High Affinity ◽

Sugar Phosphates

Phosphorous, in the form of phosphate, is a key element in the nutrition of all living beings. In nature, it is present in the form of phosphate salts, organophosphates, and phosphonates. Bacteria transport inorganic phosphate by the high affinity phosphate transport system PstSCAB, and the low affinity PitH transporters. The PstSCAB system consists of four components. PstS is the phosphate binding protein and discriminates between arsenate and phosphate. In the Streptomyces species, the PstS protein, attached to the outer side of the cell membrane, is glycosylated and released as a soluble protein that lacks its phosphate binding ability. Transport of phosphate by the PstSCAB system is drastically regulated by the inorganic phosphate concentration and mediated by binding of phosphorylated PhoP to the promoter of the PstSCAB operon. In Mycobacterium smegmatis, an additional high affinity transport system, PhnCDE, is also under PhoP regulation. Additionally, Streptomyces have a duplicated low affinity phosphate transport system encoded by the pitH1–pitH2 genes. In this system phosphate is transported as a metal-phosphate complex in simport with protons. Expression of pitH2, but not that of pitH1 in Streptomyces coelicolor, is regulated by PhoP. Interestingly, in many Streptomyces species, three gene clusters pitH1–pstSCAB–ppk (for a polyphosphate kinase), are linked in a supercluster formed by nine genes related to phosphate metabolism. Glycerol-3-phosphate may be transported by the actinobacteria Corynebacterium glutamicum that contains a ugp gene cluster for glycerol-3-P uptake, but the ugp cluster is not present in Streptomyces genomes. Sugar phosphates and nucleotides are used as phosphate source by the Streptomyces species, but there is no evidence of the uhp gene involved in the transport of sugar phosphates. Sugar phosphates and nucleotides are dephosphorylated by extracellular phosphatases and nucleotidases. An isolated uhpT gene for a hexose phosphate antiporter is present in several pathogenic corynebacteria, such as Corynebacterium diphtheriae, but not in non-pathogenic ones. Phosphonates are molecules that contains phosphate linked covalently to a carbon atom through a very stable C–P bond. Their utilization requires the phnCDE genes for phosphonates/phosphate transport and genes for degradation, including those for the subunits of the C–P lyase. Strains of the Arthrobacter and Streptomyces genera were reported to degrade simple phosphonates, but bioinformatic analysis reveals that whole sets of genes for putative phosphonate degradation are present only in three Arthrobacter species and a few Streptomyces species. Genes encoding the C–P lyase subunits occur in several Streptomyces species associated with plant roots or with mangroves, but not in the laboratory model Streptomyces species; however, the phnCDE genes that encode phosphonates/phosphate transport systems are frequent in Streptomyces species, suggesting that these genes, in the absence of C–P lyase genes, might be used as surrogate phosphate transporters. In summary, Streptomyces and related actinobacteria seem to be less versatile in phosphate transport systems than Enterobacteria.

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Evidence that cycloleucine affects the high-affinity systems of amino acid uptake in cultured human fibroblasts

Biochemical Journal ◽

10.1042/bj2240309 ◽

1984 ◽

Vol 224 (1) ◽

pp. 309-315 ◽

Cited By ~ 3

Author(s):

M Feneant ◽

N Moatti ◽

J Maccario ◽

M Gautier ◽

S Guerroui ◽

...

Keyword(s):

Amino Acid ◽

Transport System ◽

Human Fibroblasts ◽

Diffusion Constant ◽

Amino Acid Uptake ◽

Cell Uptake ◽

Transport Systems ◽

Acid Uptake ◽

High Affinity ◽

Cultured Human Fibroblasts

The influence of cycloleucine on kinetic parameters of uptake of L-alanine, L-proline and L-leucine into cultured human fibroblasts was examined under initial-rate conditions with substrate concentrations of 0.05-10 mM and 5 mM-cycloleucine. Kinetic data obtained by computer analysis showed that, in the absence of cycloleucine, cell uptake was heterogeneous for each amino acid. L-Alanine and L-leucine entered by two transport systems with different affinities; L-proline was taken up by one saturable transport system plus a diffusion-like process. This heterogeneity disappeared in the presence of cycloleucine, since the high-affinity systems were no longer detectable. The remaining process had the same kinetic constants as the low-affinity system for alanine and leucine and a KD similar to the diffusion constant for proline. The influence of cycloleucine on the amino acid uptake was not specific either to the amino acid concerned or to a particular transport system, since the three neutral amino acid-transport systems, A, ASC and L, were involved in these experiments. This influence was shown to be unaffected by the absence of Na+ (for leucine uptake). ATP content of the cells was identical in the presence or in the absence of cycloleucine.

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Renal transport of neutral amino acids. Tubular localization of Na+-dependent phenylalanine- and glucose-transport systems

Biochemical Journal ◽

10.1042/bj2200015 ◽

1984 ◽

Vol 220 (1) ◽

pp. 15-24 ◽

Cited By ~ 29

Author(s):

U Kragh-Hansen ◽

H Røigaard-Petersen ◽

C Jacobsen ◽

M I Sheikh

Keyword(s):

Glucose Transport ◽

Transport System ◽

Membrane Vesicles ◽

Transport Systems ◽

Rabbit Kidney ◽

Outer Cortex ◽

High Affinity ◽

Pars Recta ◽

Pars Convoluta ◽

Phenylalanine Transport

The transport properties for phenylalanine and glucose in luminal-membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit kidney were studied by a spectrophotometric method. Uptake of phenylalanine as well as of glucose by the two types of membrane vesicles was found to be Na+-dependent, electrogenic and stereospecific. Na+-dependent transport of L-phenylalanine by outer-cortical membrane vesicles could be accounted for by one transport system (KA congruent to 1.5 mM). By contrast, in the outer-medullary preparation, L-phenylalanine transport occurred via two transport systems, namely a high-affinity system with K1A congruent to 0.33 mM and a low-affinity system with K2A congruent to 7 mM respectively. Na+-dependent uptake of D-glucose by pars convoluta and pars recta membrane vesicles could be described by single, but different, transport systems, namely a low-affinity system with KA congruent to 3.5 mM and a high-affinity system with KA congruent to 0.30 mM respectively. Attempts to calculate the stoichiometry of the different Na+/D-glucose transport systems by using Hill-type plots revealed that the ratio of the Na+/hexose co-transport probably is 1:1 in the case of pars convoluta and 2:1 in membrane vesicles from pars recta. The Na+/L-phenylalanine stoichiometry of the pars convoluta transporter probably is 1:1. Both the high-affinity and the low-affinity Na+-dependent L-phenylalanine transport system of pars recta membrane vesicles seem to operate with a 1:1 stoichiometry. The physiological importance of the arrangement of low-affinity and high-affinity transport systems along the kidney proximal tubule is discussed.

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Role of Periplasmic Trehalase in Uptake of Trehalose by the Thermophilic Bacterium Rhodothermus marinus

Journal of Bacteriology ◽

10.1128/jb.01616-07 ◽

2008 ◽

Vol 190 (6) ◽

pp. 1871-1878

Author(s):

Carla D. Jorge ◽

Luís L. Fonseca ◽

Winfried Boos ◽

Helena Santos

Keyword(s):

Kinetic Parameters ◽

Glucose Transport ◽

Transport System ◽

Glucose Transporter ◽

Thermophilic Bacterium ◽

Transport Systems ◽

Uptake System ◽

Rhodothermus Marinus ◽

Whole Cells ◽

Physiological Relevance

ABSTRACT Trehalose uptake at 65°C in Rhodothermus marinus was characterized. The profile of trehalose uptake as a function of concentration showed two distinct types of saturation kinetics, and the analysis of the data was complicated by the activity of a periplasmic trehalase. The kinetic parameters of this enzyme determined in whole cells were as follows: Km = 156 ± 11 μM and V max = 21.2 ± 0.4 nmol/min/mg of total protein. Therefore, trehalose could be acted upon by this periplasmic activity, yielding glucose that subsequently entered the cell via the glucose uptake system, which was also characterized. To distinguish the several contributions in this intricate system, a mathematical model was developed that took into account the experimental kinetic parameters for trehalase, trehalose transport, glucose transport, competition data with trehalose, glucose, and palatinose, and measurements of glucose diffusion out of the periplasm. It was concluded that R. marinus has distinct transport systems for trehalose and glucose; moreover, the experimental data fit perfectly with a model considering a high-affinity, low-capacity transport system for trehalose (Km = 0.11 ± 0.03 μM and V max = 0.39 ± 0.02 nmol/min/mg of protein) and a glucose transporter with moderate affinity and capacity (Km = 46 ± 3 μM and V max = 48 ± 1 nmol/min/mg of protein). The contribution of the trehalose transporter is important only in trehalose-poor environments (trehalose concentrations up to 6 μM); at higher concentrations trehalose is assimilated primarily via trehalase and the glucose transport system. Trehalose uptake was constitutive, but the activity decreased 60% in response to osmotic stress. The nature of the trehalose transporter and the physiological relevance of these findings are discussed.

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