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Journal of Bacteriology, February 2005, p. 1210-1218, Vol. 187, No. 4
0021-9193/05/$08.00+0     doi:10.1128/JB.187.4.1210-1218.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Zélia Silva,1,{dagger} Maria-Manuel Sampaio,2,{dagger} Anke Henne,3 Alex Böhm,4 Ruben Gutzat,4 Winfried Boos,4 Milton S. da Costa,1 and Helena Santos2*

Centro de Neurociências e Biologia Celular, Departamento de Zoologia, Universidade de Coimbra, Coimbra,1 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal,2 Göttingen Genomics Laboratory, Institut für Mikrobiologie und Genetik, Göttingen,3 Department of Biology, University of Constance, Constance, Germany4

Received 31 August 2004/ Accepted 1 November 2004


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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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INTRODUCTION
 
The superfamily of ATP-binding cassette (ABC) transport systems comprises a large diversity of primary pumps that use ATP hydrolysis to translocate substrates across biological membranes. Prokaryotic ABC importers typically consist of an extracytoplasmic or membrane-anchored binding protein that provides the recognition site for the substrate, a translocation complex formed by two membrane components, and two copies of an ATP-hydrolyzing protein. In the archetypal maltose/maltodextrin transporter of Escherichia coli, MalE designates the substrate binding protein, MalF and MalG are the two membrane components of the translocation complex, and two MalK subunits form the ATP-hydrolyzing complex. The encoding genes form a cluster on the chromosome of E. coli where malE/malF/malG constitutes an operon with an orientation opposite to malK (4).

Binding protein-dependent ABC transporters are ubiquitous among sequenced prokaryotic genomes, leading to the speculation that these systems were selected very early in the evolutionary process. Curiously, PEP-dependent phosphotransferase systems (PTS) have not been encountered thus far in (hyper)thermophiles, an observation in line with the concept of a hot origin of life, as this observation seems to imply that the PTS only evolved later in mesophiles.

We were the first to report the characterization of a high-affinity binding protein-dependent transporter in a hyperthermophile: i.e., the maltose/trehalose transport system of the hyperthermophilic archeaon Thermococcus litoralis (21, 45). Subsequently, this transporter has been thoroughly characterized at the molecular and biochemical levels (11, 12, 18, 19, 21). Interestingly, the organization of the corresponding operon in this archaeon is very similar to that of the maltose/maltodextrin protein-dependent ABC transporter of the model bacterium, E. coli (4).

Sugar transport mediated by binding protein-dependent ABC transporters has been characterized to different extents only in a few (hyper)thermophiles: in the hyperthermophilic archaeon Sulfolobus solfataricus (2, 26); in the hyperthermophilic bacterium Thermotoga maritima (31, 42); and in the thermophilic bacteria Thermoanaerobacterium thermosulfurigenes (33), Alicyclobacillus acidocaldarius (22), and Thermoanaerobacter ethanolicus (24, 25).

The members of the genus Thermus had a notable historical contribution to our present knowledge of thermophilic organisms, and this genus probably comprises the best representatives of aerobic thermophilic bacteria. The species of this genus have an optimum growth temperature in the range of 70 to 75°C and typically have been isolated from continental hydrothermal freshwater springs; accordingly, they are unable to grow in media containing more than 1% (wt/vol) NaCl. The strains of the species Thermus thermophilus are, however, commonly isolated from marine hot springs and can grow in media containing up to about 6% NaCl (8). The halotolerant strains accumulate trehalose and/or mannosylglycerate in response to increasing salinity of the growth medium, and the combination of these two solutes is required for growth at NaCl concentrations above 3% (1, 32, 40).

Mannosylglycerate is a compatible solute largely restricted to bacteria and archaea that colonize hot marine environments (35). Trehalose, however, is a ubiquitous compatible solute, found in a wide variety of organisms from all three domains of life. In bacteria and yeast, there is a wealth of evidence for the dual function of this disaccharide as a reserve compound and as a protective metabolite accumulating in response to osmotic or thermal stress (3, 15). Recently, the role of trehalose in osmotic adaptation of T. thermophilus RQ-1 was firmly established (40). An RQ-1 mutant in which the otsA and otsB genes have been knocked out is impaired in its ability to grow under salt stress conditions, but that ability is restored by supplementation of the growth medium with trehalose (40). These results also provided clear evidence for the presence of a functional trehalose transport system in T. thermophilus.

In this work, we studied the transport of trehalose in T. thermophilus HB27. The properties of the transport system were analyzed in whole cells. Moreover, two genes with homology to malE genes of different organisms were expressed in E. coli, followed by purification and characterization of their encoded binding proteins. The gene composition of the maltose/trehalose/sucrose/palatinose transport system was identified by constructs with disruptions of the respective elements. Despite the sequence homology with the maltose-binding protein of E. coli, the second system did not recognize maltose or maltodextrins.


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MATERIALS AND METHODS
 
14C-labeled compounds. For transport assays, [U-14C]maltose and [U-14C]sucrose with specific activities of 591 and 660 mCi/mmol (Amersham), respectively, were used. [U-14C]trehalose with a specific activity of 600 mCi/mmol was purchased from Trenzyme Biotechnology GmbH. [14C]maltotriose was obtained from Biotrend Chemikalen GmbH.

Strains and culture conditions. T. thermophilus strain HB27 (DSM7039) was obtained from the Deutsche Sammlung von Mikroorganismen and Zelkulturen, Braunschweig, Germany. The organism was grown in Thermus medium (43), which is composed of Castenholtz salts base (7), with the addition of 1.0-g/liter tryptone and 1.0-g/liter yeast extract, or in defined medium (TD), which contains basal salts of Degryse medium 162 (10), 2.0-g/liter tryptone, and a vitamin solution (37). TD medium lacked yeast extract, which is a source of trehalose. To study the expression of the transport system, cells were grown in TD medium supplemented with trehalose, maltose, or sucrose at a final concentration of 0.26 mM. To test carbon source utilization, cells were grown in a minimal medium containing basal salts of Degryse medium 162, 1.0-g/liter (NH4)2SO4, biotin (0.1 mg/liter), thiamine (1.0 mg/liter), and 2 g of the disaccharides per liter.

Transport assays in whole cells. Cells were grown in Thermus medium to mid-exponential phase (optical density at 600 nm [OD600] = 0.5 to 0.7), harvested by centrifugation (6,000 x g, 20°C, 7 min), washed twice with a solution with the same composition of the growth medium but without a carbon source, and suspended in the same solution. To measure the transport of trehalose, maltose, or sucrose, a cell suspension with an OD600 of 0.05 to 0.06 (corresponding to 12 to 15 µg of protein per ml) was used. To 3 ml of cell suspension, prewarmed for 1 min at 70°C, [14C]trehalose (28 nM), [14C]maltose (28 nM), or [14C]sucrose (40 nM) was added. For higher concentrations, cold substrate was mixed with radioactive substrate (using always the same concentration of labeled compound). At each time point (20, 40, 60, 90, and 120 s), 0.5 ml of cell suspension was filtered through Millipore filters (pore size, 0.45 µm) with a rapid filtration apparatus. The filters were counted in a toluene-based scintillation fluid. In transport competition assays, unlabeled carbohydrates (100 µM final concentration) were incubated with the cells prior to the addition of labeled substrate. Linear correlations of the number of counts versus time were obtained.

Trehalose-binding assay. Cells were grown to mid-exponential phase in Thermus medium. After centrifugation, cells were washed, suspended in 50 mM Tris-HCl (pH 7.6), and passed twice through a French pressure cell, resulting in a crude extract fraction. [14C]trehalose (10 µl; final concentration, 0.1 µM) was added to 100 µl of extract (prewarmed for 1 min at 70°C). This mixture was then added to 2 ml of ice-cold 50 mM Tris-HCl (pH 7.6) saturated with ammonium sulfate and incubated on ice for 10 min. Samples were then passed through a nitrocellulose filter and washed with additional Tris-ammonium sulfate solution. Radioactivity present in the filters was counted.

Standard DNA methods. Chromosomal DNA from T. thermophilus HB27 was purified as described in reference 27. Plasmids were extracted from E. coli with the Mini- or Midi-plasmid kit (QIAGEN GmbH, Hilden, Germany). Digestions by endonucleases, ligations, and PCR were performed by standard procedures (28, 34). Proofreading DNA polymerase (Pwo or Pfu) was used for all PCR applications.

Production of recombinant MalE proteins in E. coli. A PCR fragment encompassing the gene encoding glutathione S-transferase (GST) from Schistosoma japonicum was synthesized with primers 5'-GCGCCATGGCCCCTATACTAGGTTATTGGAAAATTAAG-3' (NcoI site underlined) and 5'-GCGGGATCCCCCTCCGCCACCTCCATCCGATTTTGGAGGATGGTC-3' (BamHI site underlined), and the GST-containing PEG(KG) plasmid as a template (29). This fragment was cloned into NcoI and BamHI sites of pTRC99A expression vector (Pharmacia Biotech), yielding pTRCGE. The malE1 gene lacking the signal peptide-encoding fragment was amplified from HB27 chromosomal DNA by PCR, using the primers 5'-GCGGGATCCGACGATGACGATAAGCAGTCCGGCCCCGTG-3' (BamHI site underlined and enterokinase cleavage site in boldface) and 5'-GCGTCTAGACTAGCGCAGGATGCG-3' (XbaI site underlined). The truncated malE gene was cloned into BamHI/XbaI sites of pTRCGE. The resulting plasmid (pTRCGEMal1) bears a fusion between GST and the malE1 gene without its predicted signal sequence. This construct was used to transform the BL21– Rosetta strain (Novagen).

The malE2 gene lacking the signal peptide-encoding fragment was amplified from the chromosomal DNA of HB27, using the forward primer 5'-GCGGGATCCCAGGGAAAGATCACCGTCTGG-3' (BamHI site underlined) and the reverse primer 5'-GCGTCTAGATTACCTGCCGATGGC-3' (XbaI site underlined). The plasmid PEG(KG) was digested with SacI and XbaI to obtain the GST tag and the thrombin cleavage site. This fragment was then ligated to pTRC99A digested with the same enzymes, yielding pTRCGT. The partial PCR-amplified malE2 gene was cloned into BamHI and XbaI sites of pTRCGT, yielding pTRCGTMal2. The resulting plasmid was used to transform E. coli DH5{alpha} (20).

Construction of T. thermophilus deletion mutants. pRGGE7 vector, containing malEFG1, was generated in two steps. First a malFG1 fragment that had been amplified from HB27 genomic DNA with the primers 5'-ACCCCAAGCATATGCTCACCCTAAGACAGGTCCGTTTG-3' (NdeI site underlined) and 5'-CGAATTCCTGCCCTTCACCGCCCCCG-3' (EcoRI site underlined), was cloned in pET24a (Novagen). A second PCR-amplified malEF1 fragment that had been amplified with the primers 5'-GAAGATCTAGTATAACAGAAACCTTAAGGCCCGAC-3' (XbaI site underlined) and 5'-AGGACCAGGATCCAGGCCAAACG-3' (BamHI site underlined) was inserted by employing a naturally occurring BamHI site at the beginning of the malF1 gene and the XbaI site in pET24. The same BamHI site was used to insert the thermostable kanamycin cassette that had been excised from pMK18 (9) with BamHI, yielding plasmid pRG7kan. A similar approach was used to construct the malF2::kan malG2-carrying plasmid. First, malFG2 was PCR amplified with primers 5'-GGAATTCCATATGAAGCACCCTCCCGGACTCAAAGGCTTCCTCC-3' (NdeI site underlined) and 5'-GGAATTCCCTTCTTTCACGGCCCCCGCGGTGTAGCCGGA-3' (EcoRI site underlined) and cloned into pET24a. This plasmid was then used to insert the same thermostable kanamycin cassette described above into the naturally occurring BamHI site in malF2 (both malF1 and malF2 contain a BamHI site). The resulting plasmid was called pRG7kan.

Two plasmids harboring malK1 and malK2, with a kanamycin resistance cassette in the middle of each one of the genes, were constructed. For the construction of the malK1::kan vector, pRG1kan, gene malK1 was PCR amplified with the primers 5'-CGGGATCCCATGGCCAAGGTCAGGCTGGAACAC-3' (BamHI site underlined) and 5'-CCCAAGCTTGGCGATCCCGATGACGAAGG-3' (HindIII site underlined) and inserted into pCJ30 (30). The malK2::kan plasmid, pRGUC1kan, was generated by inserting a PCR-generated fragment amplified with the primers 5'-GGGTTCGAATCAGGCGAGGGCCTTCCC-3' (HindIII site underlined) and 5'-AACTGCAGATGGCCGGGATCCGCG-3' (PvuI site underlined) into the pUC19 polylinker. In both constructions after the malK gene had been cloned, a thermostable kanamycin resistance cassette from pMK18 was inserted into a BglII site (both malK1 and malK2 possess a unique BglII site), after being PCR amplified with primers 5'-GAAGATCTAGTATAACAGAAACCTTAAGGCCCGAC-3' (BglII site underlined) and 5'-GAAGATCTCATCTGTGCGGTATTTCACACC-3' (BglII site underlined). Transformation of the naturally competent HB27 cells was carried out by adding 200 ng of plasmid DNA to a 5-ml aerated mid-log-phase liquid culture and further incubation for 2 h. Subsequently, aliquots were plated on selective Thermus agar plates containing kanamycin at a final concentration of 30 mg/liter.

Purification of the recombinant proteins. BL21– Rosetta strain, containing pTRCGEMal1, was cultivated at 37°C in Luria-Bertani medium containing 100-µg/ml ampicillin and 50-µg/ml chloramphenicol. Strain DH5{alpha}, containing pTRCGTMal2, was cultivated in the same medium with 100-µg/ml ampicillin. When an OD610 of 1.0 was reached, the expression of GST-MalE1 or GST-MalE2 fusion proteins was induced by adding 0.5 mM IPTG (isopropyl-ß-D-thiogalactopyranoside). After 3 h of incubation, the cells were harvested (7,000 x g, 10 min, 4°C); suspended in phosphate-buffered saline (pH 7.3) containing (per milliliter of suspension) DNase I (10 µg) and the protease inhibitors phenylmethylsulfonyl fluoride (80 µg), leupeptin (20 µg), and antipain (20 µg); and ruptured by sonication. To remove cell debris, the extracts were centrifuged (18,000 x g, 1 h, 4°C) and filtered through 0.22-µm-pore-size-filters (Schleicher & Schuell). The extracts were applied to a GSTprepFF16/10 column and eluted with 50 mM Tris-HCl-10 mM glutathione (Sigma) (pH 8.0). The fractions containing GST-MalE1 or GST-MalE2 were treated with enterokinase (Novagen) or thrombin (Amersham Biosciences), respectively, for 16 h at 22°C. To separate GST from MalE1 or MalE2, the samples were applied to a MonoQ fast-flow column equilibrated with 20 mM Tris-HCl (pH 7.6). Elution was carried out with a linear NaCl gradient (0.0 to 1.0 M). The fractions containing MalE1 or MalE2 were eluted before applying the gradient. Those fractions were concentrated, and the purity of the samples was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Determination of dissociation constants (Kd). To measure the Kd values, a method based on the retention of ligand by the binding proteins was used (38). Small dialysis tubing (diameter, 1 cm) open on one end was tightly fit with the open end onto a bluntly cut plastic pipetting tip of a 1-ml automatic pipette. Four hundred microliters of pure binding protein solution (0.316, 0.181, 0.632, and 0.905 µM for trehalose, maltose, sucrose, and maltotriose assays, respectively) was mixed with the substrates and introduced into the dialysis tubing, which was immersed into a 1-liter Erlenmeyer flask filled with 20 mM Tris-HCl, pH 7.6. Final substrate concentrations were 400 nM for maltose and trehalose, 300 nM for sucrose, and 800 nM for maltotriose. The buffer in the Erlenmeyer flask was kept at 70°C and gently stirred during the assay. Aliquots of 20 µl were removed from the bag at different time intervals, and the radioactivity was counted.

The same procedure was repeated for the substrates but in the absence of the protein (control assays). The time to release half of the substrate from the bag was greater by a factor of 1 + (P/Kd) in the presence of molar concentrations of binding protein P (assuming one binding site) than in its absence. Kd was obtained in a molar concentration.

DSC. Differential scanning calorimetry (DSC) scans were performed on a MicroCal VP-DSC MicroCalorimeter controlled by the VP-viewer program. Calibrations of temperature and heat flow were carried out according to the MicroCal instructions. Stock solutions of MalE1 and MalE2 were prepared in 50 mM phosphate buffer, pH 7.0, and extensively washed with the same buffer in a Centricon tube (10-kDa cutoff). A diluted protein solution was prepared from the stock protein to achieve a final concentration of 0.5 mg/ml. This solution was extensively dialyzed against 50 mM phosphate buffer, pH 7.0, containing 2.3 M guanidinium chloride and was used to fill up the calorimeter cell. The reference cell was filled with buffer containing 2.3 M guanidinium chloride. The sample and the reference solutions were degassed for 8 min. DSC scans were performed from 20 to 110°C with a constant heating rate of 1°C/min. An overpressure of about 2 atm was applied to the calorimeter cells to prevent bubble formation during heating. To assess reversibility of the transition, a second scan was run with the same protein solution used in the first scan. The thermograms were analyzed with the MicroCal Origin software supplied with the instrument to determine the protein melting temperature Tm.

Extraction and determination of intracellular solutes. Cells were harvested by centrifugation (7,000 x g, 10 min, 4°C) during the mid-exponential phase of growth and washed twice with an NaCl solution identical in concentration to that of the growth medium. Cell pellets were extracted twice with boiling 80% ethanol as described previously (40). Freeze-dried extracts were dissolved in D2O and analyzed by proton nuclear magnetic resonance. Spectra were acquired in a Bruker AMX300 as described earlier (39).

Protein determinations. The protein content of cells was determined, after sonication, by the Bradford microprotein assay (6).

The concentration of purified protein was determined from the quantification of amino acids in protein hydrolysates, taking into account the deduced amino acid composition. Amino acids were determined in a Pico-Tag amino acid analysis system (Waters, Milford, Mass.). Serine, histidine, alanine, isoleucine, leucine, and threonine were used for protein determination.

Computer-aided sequence analysis. Protein homology searches were done with BLAST (http://www.ncbi.nlm.nih.gov/BLAST), and multiple sequence alignments were done with CLUSTALW (http://www.ebi.ac.uk/clustalw). For the prediction of signal peptide sequence, we used SIGNAL P (http://www.cbs.dtu.dk/services/SignalP).


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RESULTS
 
T. thermophilus HB27 transports maltose, trehalose, and sucrose with high affinity. Cells of HB27 grown in Thermus medium showed high rates of transport for trehalose, maltose, and sucrose. The dependence of the transport rate on substrate concentration was plotted, and a Michaelis-Menten type of dependence was observed (Fig. 1). The Km values were 109 nM for trehalose, 114 nM for maltose, and 424 nM for sucrose; the Vmax was 12.7 nmol/min · mg of protein–1 for trehalose, 13.5 nmol/min · mg of protein–1 for maltose, and 12.6 nmol/min · mg of protein–1 for sucrose. Trehalose transport at 28 nM was strongly inhibited by maltose, sucrose, maltotriose, and glucose (Fig. 2A); maltose transport was inhibited by trehalose, sucrose, maltotriose, and glucose (Fig. 2B); and a similar competitive pattern was observed for sucrose transport, when trehalose, maltose, maltotriose, and glucose were used (Fig. 2C). Other sugars, namely D-galactose, D-fucose, L-rhamnose, L-arabinose, and D-mannose, were tested as putative competitors but did not exhibit inhibitory effect (results not shown). The observed inhibition by maltotriose was due to maltose, a contaminant (1%) of the commercial maltotriose sample, as the mutant with a disruption of malF1 showed no growth defect on maltotriose (described below).



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  		FIG. 1. Rates of disaccharide transport in resting cells of T. thermophilus HB27 grown in Thermus medium at 70°C. The kinetic parameters for trehalose (A) are Km = 109 nM and Vmax = 10 nmol/min · mg of protein–1, those for maltose (B) are Km = 150 nM and Vmax = 11 nmol/min · mg of protein–1, and those for sucrose (C) are Km = 300 nM and Vmax = 12 nmol/min · mg of protein–1.



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  		FIG. 2. Effect of competing sugars on trehalose/maltose/sucrose transport. (A) Trehalose transport; (B) maltose transport; (C) sucrose transport. Assays were done in the presence of a 100 µM concentration of the competing sugar at 70°C.

To evaluate the effect of the carbon source on sugar transport rates, HB27 was grown in defined TD medium supplemented with trehalose, maltose, or sucrose. There was no difference between the rates of sugar transport in control cells (grown in TD medium) and those in cells grown in the presence of trehalose, maltose, or sucrose, showing that there was no induction of transport by any of these sugars (results not shown).

Trehalose-binding activity in cell extracts of T. thermophilus HB27. To determine whether HB27 possesses a trehalose-binding protein as one of the components of its high-affinity transport system, cell extracts of cultures grown in Thermus medium were incubated at 70°C with radiolabeled trehalose and precipitated with ammonium sulfate. After extensive washing, radioactivity remained in the filter, showing that the extract contained a trehalose-binding protein. To verify whether trehalose binding was specific, competition studies were done by adding an excess of unlabeled sugars (10 mM final concentration) to the binding assay. Trehalose binding was nearly abolished by the addition of unlabeled trehalose, and a similar effect was observed with maltose or sucrose (results not shown), indicating that the trehalose-binding protein also binds maltose and sucrose.

Identification of two gene clusters encoding putative maltose ABC transporters in T. thermophilus HB27. To identify the genes that encode the trehalose/maltose/sucrose ABC transporter, we used the well-characterized maltose/maltodextrin transporter from E. coli and the maltose/trehalose transporter of T. litoralis and searched the T. thermophilus genome sequence at the Göttingen Genomics Laboratory server (http://www.g2l.bio.uni-goettingen.de/BLAST/blast_search.html) for homologues. Meanwhile, the full sequence of T. thermophilus HB27 has been published (http://www.ncbi.nlm.nih.gov/genomes). Individual BLAST searches were carried out with the MalE (TMBP), MalF, MalG, and MalK amino acid sequences from E. coli and T. litoralis. The T. thermophilus genome appeared to possess two predicted clusters encoding proteins with considerable similarity to the MalE (or TMBP), MalF, and MalG proteins from both E. coli and T. litoralis and two monocistronic genes that encode proteins that are homologous to E. coli MalK and T. litoralis MalK. We will refer to those as malEFG1, malEFG2, malK1, and malK2. The similarities and identities of the deduced amino acid sequences to the T. litoralis and E. coli homologs are shown in Table 1 along with the respective genome annotation numbers.


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  		TABLE 1. Similarities and identities of putative binding and inner membrane proteins of two maltose transport systems found by BLAST search in the genome of T. thermophilus HB27

The individual genes in the clusters malEFG1 (TTC1627 to -1629) and malEFG2 (TTC1288 to -1286) are organized similarly to the corresponding genes in several other organisms (Fig. 3). In the genome of HB27, malK1 (TTC0211) and malK2 (TTC0611) are distant from the malEFG clusters. Upstream of the malE1 region we found promoter elements –10 (TATAAT) and –35 (TTGACG) and a ribosome-binding site (AGGA) located 6 nucleotides upstream of the malE1 translation start codon. Between malE1 and malF1, there is an intergenic region composed of 56 nucleotides, and an intergenic region (59 nucleotides) was also found between malE2 and malF2. From sequence analysis, it is tempting to speculate that the two malEFG clusters constitute operons.



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  		FIG. 3. Schematic representation of the organization of the genes encoding two ABC transport systems in T. thermophilus HB27 with sequence homology to known maltose transport systems. Transport system 1 has been fully characterized in the present work. The organization of the genes encoding maltose/maltodextrin and maltose/trehalose ABC transport systems in selected hyperthermophiles (on the left) and mesophiles (on the right) is also shown.

As expected for periplasmic proteins, the two putative MalEs show signal peptides of secretory precursor proteins. In MalE1, the predicted cleavage site was VRAQS and the signal peptide corresponds to the first 27 amino acids. The cleavage site predicted for MalE2 is ALAQG, resulting in a signal peptide of 18 amino acids.

Cloning, expression, and purification of putative binding proteins. Our attempts to purify the binding protein from cell extracts of HB27 by affinity chromatography with agarose-coupled amylose were unsuccessful. Thus, to find out whether malEFG1 or malEFG2 encodes the binding protein of the detected maltose/trehalose/sucrose transport system, we cloned and expressed signal peptide-deficient Thermus MalE1 and MalE2 in E. coli. The cloning strategy involved the creation of GST fusion proteins to facilitate protein purification. Single bands with the expected molecular masses were obtained in SDS-PAGE of the final preparations (not shown). As a result of the cleavage with different enzymes (enterokinase for MalE1 and thrombin for MalE2), the recombinant MalE1 is identical to the original sequence whereas the recombinant MalE2 has two extra amino acids (glycine and serine) at its N terminus.

Binding affinity of the recombinant proteins. The substrate retention behavior was used as a way to measure the binding affinity of the recombinant proteins MalE1 and MalE2, because, in contrast with fluorescence measurements, this technique is insensitive to the presence of bound unlabeled substrate. The Kd values for binding of maltose, trehalose, and sucrose to MalE1 were 103, 67, and 401 nM, respectively (Fig. 4). Glucose and maltotetraose were also examined as putative substrates, but binding of these sugars was not detected. An apparent Kd value of approximately 890 nM was determined for maltotriose, which was actually due to binding of maltose, a contaminant present in the maltotriose preparation. A considerable effort was invested to identify the substrates for MalE2, but we failed to detect binding with any of the sugars examined. Binding of trehalose, sucrose, maltose, maltotriose, maltotetraose, and glucose was probed with the dialysis assay with radiolabeled substrates, and binding of arabinose was investigated with fluorescence measurements (results not shown).



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  		FIG. 4. Binding affinity of the recombinant MalE1 binding protein as measured by substrate retention. Shown is exit of substrate (trehalose [A], maltose [B], or sucrose [C]) from a dialysis bag containing purified recombinant protein (0.316 µM for the assay with trehalose, 0.181 µM for that with maltose, and 0.632 µM for that with sucrose) (closed symbols) or substrate only (open symbols). Samples of 20 µl were removed from the dialysis bag at different time intervals, and radioactivity was counted in a scintillation counter. The half-life of internal substrate was calculated after the rate of exit had become first order. The temperature was kept constant at 70°C. The half-life in the presence of the protein (66, 77, and 50 min, respectively) was larger by the factor of 1 + (P/Kd) than that in the absence of protein (16.3, 20.9, and 19.5 min, respectively). P is the concentration of protein expressed in the molar concentration of binding sites. (One binding site per polypeptide chain was assumed.)

Thermal stability of recombinant MalE1 and MalE2. The Tm of the recombinant proteins was determined by DSC. Both MalE1 and MalE2 showed extremely high stability. Under nondenaturing conditions, melting did not occur over the range of temperatures tested (20 to 110°C). In the presence of 2.3 M guanidinium hydrochloride, the Tm value for MalE1 was 90°C and that for MalE2 was 105°C (Fig. 5). The denaturation process was irreversible.



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  		FIG. 5. Thermal stability of recombinant proteins MalE1 and MalE2 of T. thermophilus HB27 determined by DSC. Solid line, MalE1; dashed line, MalE2. Protein solutions (0.5 mg/ml) were prepared in 50 mM phosphate buffer, pH 7.0, containing 2.3 M guanidinium chloride. DSC scans were performed from 20 to 110°C with a constant heating rate of 1°C/min.

MalK1 is the ABC subunit of the MalEFG1 system, and palatinose is another substrate of this transporter. To confirm that malEFG1 encodes the binding protein and the two transmembrane subunits of the trehalose/maltose/sucrose ABC transporter and to ascertain whether either of the two malK genes encodes the ATP binding cassette of this transporter, we used a genetic approach: malEFG1, malEFG2, malK1, and malK2 were cloned into E. coli plasmid vectors. These plasmids were used to disrupt the open reading frames of malF1, malF2, malK1, and malK2 by inserting a thermostable kanamycin cassette. The constructs were linearized and transformed into T. thermophilus HB27. Stable transformants that had inserted the kanamycin cassette into the chromosome by a double homologous recombination event were selected on complex Thermus medium supplemented with kanamycin. The gene disruptions were confirmed by analytical PCR (not shown), and the growth phenotypes of the insertion mutants were compared to that of wild-type HB27 on minimal medium plates (see Materials and Methods) containing different sugars as the sole source of carbon. Neither the malF1::kan mutant nor the malK1::kan mutant grew on sucrose, trehalose, or maltose plates, while both mutants grew on plates supplemented with glucose. However, it must be noted that the malK1::kan mutant appeared to grow more slowly, as assessed by colony size, than the wild-type control on glucose minimal medium, while it displayed no growth defect on rich medium. The reason for this partial growth defect is unclear. The growth of the malF1 mutant on glucose, fructose, galactose, mannose, maltotriose, or maltopentaose was comparable to that of the wild-type strain. The malF2 and malK2 mutants showed no growth defect on any of the carbon sources examined (i.e., glucose, fructose, galactose, mannose, maltose, trehalose, sucrose, palatinose, maltotriose, or maltopentaose). Thus, the substrate for the ABC transporter encoded by the malEFG2 gene cluster remains unknown.

Since all three disaccharides that were found to be a substrate of the MalEFG1 transporter are {alpha}-glucosidic disaccharides, we reasoned that another {alpha}-glucoside, palatinose (6-O-{alpha}-D-glucopyranosyl-D-fructose), might also be a substrate and found indeed that the malF1::kan and the malK1::kan mutants did not grow on palatinose, while the wild-type control grew well on this carbon source. To confirm that the growth defects of the insertion mutants are really associated with the inactivation of malF1 or malK1, we performed complementation experiments with both mutants by transformation with linearized malEFG1 or malK1 plasmids. Complementation, as indicated by growth on trehalose and sucrose minimal plates, was only successful when the malF1::kan mutant was transformed with a linearized plasmid harboring malEFG1 and when the malK1::kan mutant was transformed with a linearized plasmid harboring malK1; transformation with various control plasmids did not yield any transformants. Stable revertants were found to have lost their kanamycin resistance, thus indicating that a true reversion event with a loss of the kanamycin cassette had occurred.

Together with the results from the binding assays with purified MalE1, we conclude from these data that the malEFG1 cluster together with malK1 encodes the binding protein-dependent maltose/trehalose/sucrose/palatinose ABC transporter from T. thermophilus HB27.

Trehalose is not an osmolyte in T. thermophilus HB27. As we found an efficient transport system for trehalose in HB27, we were interested to know whether trehalose is accumulated by this organism in response to osmotic stress. The strain was grown in TD medium, TD medium supplemented with trehalose, or Thermus medium in the presence of 2% NaCl. Irrespective of the growth medium used, mannosylglycerate was the only organic osmolyte detected in the cytoplasm of HB27 (Fig. 6).



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  		FIG. 6. Accumulation of compatible solutes by T. thermophilus HB27, grown in the presence of 2% (wt/vol) NaCl in different growth media. TD, TD medium; TD+tre, TD medium supplemented with 0.26 mM trehalose; Thermus, Thermus medium. See Materials and Methods for details. Bars represent mannosylglycerate content.


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DISCUSSION
 
The genome sequence of T. thermophilus HB27 contains two gene clusters that show sequence similarity to the maltose/trehalose transport operon of T. litoralis and to the maltose/maltodextrin operon in E. coli (Table 1). These clusters were annotated as the trehalose/maltose transport system (TTC1627, -1628, and -1630), referred here as system I (malEFG1) and the maltose/maltodextrin transport system (TTC1286 to -1288), referred here as system II (malEFG2). Two genes showing homology with malK genes of T. litoralis and E. coli were also found, but their localization in the genome is distant from the malEFG clusters. Both malK genes were assigned as sugar-binding transport ATP-binding proteins, and are referred to here as malK1 (TTC0211) and malK2 (TTC0611). The existence of two putative binding protein-dependent maltose transport systems was not a surprise as it had been observed in other prokaryotes (13, 23, 31). The hyperthermophilic archaeon Pyrococcus furiosus possesses two gene clusters that are apparently involved in the transport and metabolism of maltose. One of these gene clusters is an almost exact copy of the maltose/trehalose cluster of T. litoralis (21), and its presence was attributed to a lateral gene transfer phenomenon (13). In the genome of the hyperthermophilic bacterium T. maritima, two putative maltose-binding proteins sharing very high sequence similarity (around 80%) were found (31). A similar redundancy was found in Sinorhizobium meliloti, where two maltose/trehalose/sucrose ABC transporters were characterized (23, 44). One of them (encoded by thuEFGK) is primarily targeted toward trehalose, whereas the second one (encoded by aglEFGAK) is induced primarily by sucrose and to a lesser extent by trehalose (23).

The genetic organization of malEFG in HB27 is commonly found in ABC transport systems, but the gene encoding the ATP-binding subunit (malK) is distantly located in the genome, and this organization is relatively rare (22, 33, 36). It is not known whether this organization has some physiological relevance, but in Streptomyces spp., the distant location of the ATPase subunit (MsiK) has been associated with the function of assisting two different transport systems for maltose and cellobiose (36).

The need to confirm the physiological role of proteins whose function had been assigned on the basis of sequence homology is generally recognized (17). The two gene clusters coding for the putative maltose transport systems in strain HB27 were sufficiently different to warrant the expression and characterization of the two putative maltose-binding proteins, MalE1 and MalE2. The set of experimental data comprises transport and competition assays, affinity measurements in pure binding proteins, and growth tests with mutants disrupted in several elements of the transport systems and led to the conclusion that the disaccharides maltose, trehalose, sucrose, and palatinose are substrates of a single transporter, which is composed of the proteins encoded by malEFG1 and by the ATP-binding subunit encoded by malK1. Competition assays also showed that glucose was an efficient inhibitor of the transport of the other sugars, and this result led us to think that glucose was an additional substrate for this transporter. However, this deduction was not corroborated in binding assays with the pure binding protein (MalE1), and a malF1 insertion mutant did not show a growth defect when glucose was used as the sole carbon source. The reason for the inhibitory effect of glucose on the uptake of other sugars by resting cells is elusive, but it is acceptable to speculate that this is a manifestation of carbon catabolite regulation via an inducer exclusion mechanism. In mesophilic bacteria, this type of regulation is generally associated with PTS protein components (41). T. thermophilus HB27, like other (hyper)thermophiles, lacks this type of transporters, but regulatory mechanisms that operate independently of the sugar PTS have been reported in the hyperthermophilic bacterium Thermotoga neapolitana (16).

The transport system identified in HB27 shows a broader range of substrate specificity than most of the maltose transport systems studied thus far, the presence of a 1-linked glucosyl unit being a structural feature common to all the sugar substrates used. The homologous system from T. litoralis takes up maltose and trehalose with very high affinity but does not recognize maltodextrins (21). The canonical maltose/maltodextrin transporter of E. coli is specific for these substrates, and similar substrate specificities are found, for example, in the hyperthermophilic archaeon S. solfataricus and in the thermophilic bacterium Alicyclobacillus acidocaldarius (5, 14, 22).

Two binding protein-dependent maltose transport systems have been annotated in the genome sequence of the hyperthermophilic bacterium T. maritima (Fig. 3). One of the maltose-binding proteins was extensively characterized and binds trehalose and maltotriose, in addition to maltose, with dissociation constants in the nanomolar-to-micromolar range (42). The binding protein MalE1 from T. thermophilus HB27 shows even broader substrate specificity, recognizing sucrose and palatinose besides maltose and trehalose. Binding protein-dependent systems that are able to recognize maltose and trehalose are not restricted to thermophiles or hyperthermophiles. In S. meliloti, the binding protein encoded by thuE has high affinity for maltose, trehalose and sucrose, while a second transporter, encoded by aglEFGK, recognizes cellobiose in addition to those three disaccharides (23). Whether the proteins from S. meliloti are also able to bind palatinose, as MalE1 from HB27, is not known since this substrate was not tested (23). However, it is interesting that ThuE shares a high sequence homology with MalE1 from HB27 (45% identity; 63% similarity) whereas the homology with equivalent proteins from T. litoralis or E. coli is much lower (Table 1).

Despite the sequence similarity between MalE1 and MalE2 (40%), the latter protein did not bind any of the substrates examined. The disruption of the malEFG1 cluster or of the malK1 gene produced HB27 mutant strains unable to grow on trehalose, maltose, sucrose, and palatinose. Conversely, disruption of malF2 or malK2 genes did not affect growth on these sugars or any other sugar tested. The results showed unequivocally that system I is responsible for trehalose, maltose, sucrose, and palatinose transport and that malK1 encodes the ATP binding subunit of this transport system. The function of system II remains unclear, but we showed that it is not a maltose/maltodextrin transporter as assigned on the basis of sequence similarity.

Several T. thermophilus strains rely on the accumulation of trehalose to cope with supraoptimal NaCl concentrations in the growth medium (40). Given that HB27 has an efficient trehalose transport system and lacks the trehalose biosynthetic genes (otsA, otsB, and treS), it came as a surprise to find that this common osmolyte was not accumulated in this organism. Irrespective of the growth medium used, under salt stress conditions, HB27 depended solely on the accumulation of mannosylglycerate. Consistent with this result is the observation that trehalose transport rates in HB27 were not affected by salinity or temperature of the growth medium (results not shown). Additionally, the present work shows that HB27 can grow on trehalose as the sole carbon source. Therefore, it appears that trehalose taken up from the external medium is directed exclusively to metabolism, probably because this organism lacks the osmoregulatory machinery required for trehalose accumulation. In fact, other T. thermophilus strains impaired in the synthesis of trehalose are able to import and accumulate this osmolyte (1).

In conclusion, we demonstrated that the apparent redundancy with respect to binding protein-dependent maltose transporters in the genome sequence of T. thermophilus HB27 does not exist. One of the putative maltose transport systems efficiently takes up maltose, trehalose, sucrose, and palatinose, whereas the second system does not recognize any of these disaccharides nor maltotriose, maltotetraose, or maltopentaose. Maltose/trehalose ABC transporters with narrower substrate specificity have been found in the hyperthermophiles T. litoralis and T. maritima, but any temptation to establish phylogenetic or evolutionary correlations from these results is untimely.


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ACKNOWLEDGMENTS
 
This research was funded by the European Commission (projects QLK3-CT-2000-00640 and COOP-CT-2003-508644) and FCT/FEDER (project POCTI/BIO/42331/2001). Z. Silva acknowledges a Ph.D. scholarship from PRAXIS XXI (BD/21669/99). M.-M. Sampaio is the recipient of Ph.D. grant SFRH/BD/1180/2000 from Fundação para a Ciência e a Tecnologia. The work in Constance was supported by the Deutsche Forschungsgemeinschaft (SFB/TRG 11).

We thank Tiago Faria for carrying out the DSC measurements and Susana Alarico for technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780-156 Oeiras, Portugal. Phone: 351-214469828. Fax: 351-214428766. E-mail: santos{at}itqb.unl.pt. Back

{dagger} Z.S. and M.-M.S. made equal contributions to this work. Back


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Journal of Bacteriology, February 2005, p. 1210-1218, Vol. 187, No. 4
0021-9193/05/$08.00+0     doi:10.1128/JB.187.4.1210-1218.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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