Biochemical evidence for the presence of two alpha-glucoside ABC-transport systems in the hyperthermophilic archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus can utilize different carbohydrates, such as starch, maltose and trehalose. Uptake of alpha-glucosides is mediated by two different, binding protein-dependent, ATP-binding cassette (ABC)-type transport systems. The maltose transporter also transports trehalose, whereas the maltodextrin transport system mediates the uptake of maltotriose and higher malto-oligosaccharides, but not maltose. Both transport systems are induced during growth on their respective substrates.     

The high-affinity maltose/trehalose ABC transporter in the extremely thermophilic bacterium Thermus thermophilus HB27 also recognizes sucrose and palatinose

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 degrees C. The K(m) values at 70 degrees C were 109 nM for trehalose and 114 nM for maltose; also, a high K(m) (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 (K(d) 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 degrees C for MalE1 and 105 degrees 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.     

High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis.

The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.     

A Novel Sinorhizobium meliloti Operon Encodes an α-Glucosidase and a Periplasmic-Binding-Protein-Dependent Transport System for α-Glucosides

The most abundant carbon source transported into legume root nodules is photosynthetically produced sucrose, yet the importance of its metabolism by rhizobia in planta is not yet known. To identify genes involved in sucrose uptake and hydrolysis, we screened a Sinorhizobium meliloti genomic library and discovered a segment of S. meliloti DNA which allows Ralstonia eutropha to grow on the alpha-glucosides sucrose, maltose, and trehalose. Tn5 mutagenesis localized the required genes to a 6.8-kb region containing five open reading frames which were named agl, for alpha-glucoside utilization. Four of these (aglE, aglF, aglG, and aglK) appear to encode a periplasmic-binding-protein-dependent sugar transport system, and one (aglA) appears to encode an alpha-glucosidase with homology to family 13 of glycosyl hydrolases. Cosmid-borne agl genes permit uptake of radiolabeled sucrose into R. eutropha cells. Analysis of the properties of agl mutants suggests that S. meliloti possesses at least one additional alpha-glucosidase as well as a lower-affinity transport system for alpha-glucosides. It is possible that the Fix+ phenotype of agl mutants on alfalfa is due to these additional functions. Loci found by DNA sequencing to be adjacent to aglEFGAK include a probable regulatory gene (aglR), zwf and edd, which encode the first two enzymes of the Entner-Doudoroff pathway, pgl, which shows homology to a gene encoding a putative phosphogluconolactonase, and a novel Rhizobium-specific repeat element.     

Role of trehalose transport and utilization in Sinorhizobium meliloti--alfalfa interactions

Genes thuA and thuB in Sinorhizobium meliloti Rm1021 code for a major pathway for trehalose catabolism and are induced by trehalose but not by related structurally similar disaccharides like sucrose or maltose. S. meliloti strains mutated in either of these two genes were severely impaired in their ability to grow on trehalose as the sole source of carbon. ThuA and ThuB show no homology to any known enzymes in trehalose utilization. ThuA has similarity to proteins of unknown function in Mesorhizobium loti, Agrobacterium tumefaciens, and Brucella melitensis, and ThuB possesses homology to dehydrogenases containing the consensus motif AGKHVXCEKP. thuAB genes are expressed in bacteria growing on the root surface and in the infection threads but not in the symbiotic zone of the nodules. Even though thuA and thuB mutants were impaired in competitive colonization of Medicago sativa roots, these strains were more competitive than the wild-type Rml021 in infecting alfalfa roots and forming nitrogen-fixing nodules. Possible reasons for their increased competitiveness are discussed.     

A binding protein-dependent transport system in Streptococcus mutans responsible for multiple sugar metabolism.

An 11-kilobase gene region of Streptococcus mutans has been identified which contains eight contiguous genes involved with the uptake and metabolism of multiple sugars (the msm system). Sequence analysis of this region indicates that several of these genes specify proteins with strong homology to components of periplasmic binding protein-dependent transport systems of Gram-negative bacteria. Additionally, this operon is controlled by a regulatory gene (msmR) that acts as a positive effector. The proteins specified by the structural genes of the msm operon include alpha-galactosidase (aga), a "periplasmic-like" sugar-binding protein (msmE), two membrane proteins (msmF, msmG), sucrose phosphorylase (gtfA), an ATP-binding protein (msmK), and dextran glucosidase (dexB). Insertional inactivation of each of these genes along with uptake data indicate that this system is responsible for the uptake of melibiose, raffinose, and isomaltotriose and the metabolism of melibiose, sucrose, and isomaltosaccharides.     

Sugar transport in Sulfolobus solfataricus is mediated by two families of binding protein-dependent ABC transporters

The extreme thermoacidophilic archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 3 and uses a variety of sugars as sole carbon and energy source. Glucose transport in this organism is mediated by a high-affinity binding protein-dependent ATP-binding cassette (ABC) transporter. Sugar-binding studies revealed the presence of four additional membrane-bound binding proteins for arabinose, cellobiose, maltose and trehalose. These glycosylated binding proteins are subunits of ABC transporters that fall into two distinct groups: (i) monosaccharide transporters that are homologous to the sugar transport family containing a single ATPase and a periplasmic-binding protein that is processed at an unusual site at its amino-terminus; (ii) di- and oligosaccharide transporters, which are homologous to the family of oligo/dipeptide transporters that contain two different ATPases, and a binding protein that is synthesized with a typical bacterial signal sequence. The latter family has not been implicated in sugar transport before. These data indicate that binding protein-dependent transport is the predominant mechanism of transport for sugars in S. solfataricus.     

MsiK-dependent trehalose uptake in Streptomyces reticuli

During cultivation in the presence of trehalose Streptomyces reticuli expresses an inducible, highly specific trehalose uptake system that is absent in Streptomyces lividans. A palmitated trehalose-binding protein was identified in the cytoplasmic membrane of mycelia, extracted with the detergent Triton X-100 and purified using a trehalose affinity matrix. Immunological studies showed that within S. reticuli the synthesis of the ATP-binding protein MsiK is induced by trehalose. The data suggest that MsiK assists the trehalose ABC transporter, like the previously described ABC transport systems for maltose and cellobiose/cellotriose, respectively.     

Overlapping substrate specificity for sucrose and maltose of two binding protein-dependent sugar uptake systems in Streptococcus mutans

Sugar metabolism by Streptococcus mutans is associated with tooth decay. The most abundant sugars in the human diet are sucrose and maltose, a derivative of starch. Previously, we reported a binding protein-dependent transport system (msm) in S. mutans that transports sucrose and maltose, but its associated enzymes do not metabolize maltose. By searching the S. mutans genomic sequence for a maltose system (mal), we found a gene cluster encoding proteins with homology to those of msm and the Escherichia coli maltose system. Mutants were constructed by deleting msm or mal, or both, and tested for sugar utilization. Deletion of the mal system diminished the ability of S. mutans to ferment maltose, but deletion of only the mal transporter genes or msm showed reduced utilization of chromogenic maltosides. Maltose, sucrose, glucose, fructose, mannose, and N-acetyl glucosamine inhibited utilization of chromogenic maltosides by the wild-type strain and mutants. In conclusion, the two binding protein-dependent systems in S. mutans appear to transport collaboratively their common substrate sugars, notably sucrose and maltose.     

TreT, a novel trehalose glycosyltransferring synthase of the hyperthermophilic archaeon Thermococcus litoralis

The gene cluster in Thermococcus litoralis encoding a multicomponent and binding protein-dependent ABC transporter for trehalose and maltose contains an open reading frame of unknown function. We cloned this gene (now called treT), expressed it in Escherichia coli, purified the encoded protein, and identified it as an enzyme forming trehalose and ADP from ADP-glucose and glucose. The enzyme can also use UDP- and GDP-glucose but with less efficiency. The reaction is reversible, and ADP-glucose plus glucose can also be formed from trehalose and ADP. The rate of reaction and the equilibrium favor the formation of trehalose. At 90 degrees C, the optimal temperature for the enzymatic reaction, the half-maximal concentration of ADP-glucose at saturating glucose concentrations is 1.14 mm and the V(max) is 160 units/mg protein. In the reverse reaction, the half-maximal concentration of trehalose at saturating ADP concentrations is 11.5 mm and the V(max) was estimated to be 17 units/mg protein. Under non-denaturating in vitro conditions the enzyme behaves as a dimer of identical subunits of 48 kDa. As the transporter encoded in the same gene cluster, TreT is induced by trehalose and maltose in the growth medium.     

Molecular adaptation strategies to high temperature and thermal denaturation mechanism of the D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose uptake system. In the hyperthermophilic archaeon T. litoralis this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds both trehalose and maltose with high affinity. In this work we used differential scanning calorimetry studies together with a detailed analysis, at the molecular level, of the three-dimensional protein structure to shed light on the basis of the high thermostability exhibited by the recombinant TMBP from the archaeon T. litoralis. The obtained data suggest that the presence of trehalose does not change the overall mechanism of the denaturation of this protein but it selectively modifies the stability of the TMBP structural domains.     

Further study of the Hansenula polymorpha MAL locus: characterization of the alpha-glucoside permease encoded by the HpMAL2 gene

The HpMAL2 gene of the MAL gene cluster of Hansenula polymorpha codes for a permease similar to yeast maltose and alpha-glucoside transporters. Genomic disruption of HpMAL2 resulted in an inability of cells to grow on maltose, sucrose, trehalose, maltotriose and turanose, as well as a lack of p-nitrophenyl-alpha-D-glucopyranoside (PNPG) transport. PNPG uptake was competitively inhibited by all these substrates, with Ki values between 0.23 and 1.47 mM. Transport by HpMal2p was sensitive to pH and a protonophore carbonyl cyanide-m-chlorophenylhydrazone (CCCP), revealing its energization by the proton gradient over the cell membrane. Although HpMAL2 was responsible for trehalose uptake, its expression was not induced during trehalose growth. A maltase disruption mutant did not grow on maltotriose and turanose, whereas it showed normal growth on trehalose, demonstrating the dispensability of maltase for intracellular hydrolysis of trehalose. In a Genolevures clone pBB0AA011B12, the promoter region and the N-terminal fragment of the putative transactivator of MAL genes is located adjacent to HpMAL2. A reporter gene assay showed that expression from that promoter was induced by maltose and sucrose, repressed by glucose, and derepressed during glycerol and trehalose growth. Therefore, we presume that the gene encodes a functional regulator.     

The Sinorhizobium meliloti nutrient-deprivation-induced tyrosine degradation gene hmgA is controlled by a novel member of the arsR family of regulatory genes

The regulation of the nutrient-deprivation-induced Sinorhizobium meliloti homogentisate dioxygenase (hmgA) gene, involved in tyrosine degradation, was examined. hmgA expression was found to be independent of the canonical nitrogen regulation (ntr) system. To identify regulators of hmgA, secondary mutagenesis of an S. meliloti strain harboring a hmgA-luxAB reporter gene fusion (N4) was carried out using transposon Tn1721. Two independent Tn1721 insertions were found to be located in a positive regulatory gene (nitR), encoding a protein sharing amino acid sequence similarity with proteins of the ArsR family of regulators. NitR was found to be a regulator of S. meliloti hmgA expression under nitrogen deprivation conditions, suggesting the presence of a ntr-independent nitrogen deprivation regulatory system. nitR insertion mutations were shown not to affect bacterial growth, nodulation of Medicago sativa (alfalfa) plants, or symbiotic nitrogen fixation under the physiological conditions examined. Further analysis of the nitR locus revealed the presence of open reading frames encoding proteins sharing amino acid sequence similarities with an ATP-binding phosphonate transport protein (PhnN), as well as transmembrane efflux proteins.     

Characterization of a Snorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake

The symbiotic soil bacterium Sinorhizobium meliloti uses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli (ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed in hut mutants (hutX and hutH2). Expression analysis of the hut operon determined using a hutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.     

Selective inhibition of carbohydrate transport by the local anesthetic procaine in Escherichia coli.

Maltose and lactose transport systems have been used to investigate the action of procaine on insertion and activity of membrane proteins and translocation of exported proteins in Escherichia coli. Procaine mildly inhibited growth on lactose. The level of inhibition was consistent with the small reduction observed in active and facilitated transport functions of the lac permease. However, procaine caused a severe reduction of growth rate on maltose, as well as an inhibition of induction of maltose regulon activities. In both constitutive and inducible strains, the synthesis of both maltose transport activity (malB operon) and amylomaltase activity (malA operon) was inhibited. Coordinate inhibition of soluble and membrane products was not observed with the lac operon. beta-Galactosidase synthesis proceeded normally during growth on procaine, whereas, the appearance of new transport activity was reduced. Regardless of carbon source, procaine specifically inhibited the appearance of ompF protein in the membrane fraction.