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Journal of Bacteriology, October 2006, p. 7123-7131, Vol. 188, No. 20
0021-9193/06/$08.00+0     doi:10.1128/JB.00757-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular and Biochemical Characterization of {alpha}-Glucosidase and {alpha}-Mannosidase and Their Clustered Genes from the Thermoacidophilic Archaeon Picrophilus torridus{dagger}

Angel Angelov,1 Mateusz Putyrski,1,2 and Wolfgang Liebl1*

Institute of Microbiology and Genetics, University of Goettingen, Grisebachstrasse 8, D-37077 Goettingen, Germany,1 Institute of Microbiology, Department of Bacterial Genetics, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland2

Received 26 May 2006/ Accepted 30 July 2006


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ABSTRACT
 
The genes encoding a putative {alpha}-glucosidase (aglA) and an {alpha}-mannosidase (manA) appear to be physically clustered in the genome of the extreme acidophile Picrophilus torridus, a situation not found previously in any other organism possessing aglA or manA homologs. While archaeal {alpha}-glucosidases have been described, no {alpha}-mannosidase enzymes from the archaeal kingdom have been reported previously. Transcription start site mapping and Northern blot analysis revealed that despite their colinear orientation and the small intergenic space, the genes are independently transcribed, both producing leaderless mRNA. aglA and manA were cloned and overexpressed in Escherichia coli, and the purified recombinant enzymes were characterized with respect to their physicochemical and biochemical properties. AglA displayed strict substrate specificity and hydrolyzed maltose, as well as longer {alpha}-1,4-linked maltooligosaccharides. ManA, on the other hand, hydrolyzed all possible linkage types of {alpha}-glycosidically linked mannose disaccharides and was able to hydrolyze {alpha}3,{alpha}6-mannopentaose, which represents the core structure of many triantennary N-linked carbohydrates in glycoproteins. The probable physiological role of the two enzymes in the utilization of exogenous glycoproteins and/or in the turnover of the organism's own glycoproteins is discussed.


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INTRODUCTION
 
Picrophilus torridus is an extremophilic organism belonging to the euryarchaeal phylum in which there has been considerable interest in the last few years due to its astonishing ability to live under highly acidic conditions, at pH values close to and even below pH 0. Strains of this species were first isolated from a dry solfataric field in northern Japan (28). In such geothermally heated habitats, the acidity is due to the sulfuric acid formed by the oxidation of volcanic sulfur to SO3, which reacts with water to produce H2SO4, and the acid concentration can be further increased by water evaporation. P. torridus grows optimally at 60°C and pH 0.7 and requires oxygen and complex organic compounds. The specialization of Picrophilus strains for growth in extremely acidic habitats is evident from their inability to grow at pH values above 4.0 and their unusually low intracellular pH (pH 4.6), which distinguishes them from other thermoacidophilic organisms, which maintain internal pH values close to neutral (35, 39). The complete genome sequence of P. torridus has been reported previously (11), and it provides a basis for obtaining a better understanding of the mechanisms that enable survival under such extreme acid conditions.

{alpha}-Glucosidases are enzymes that typically catalyze the hydrolysis of terminal, nonreducing, 1,4-linked D-glucose residues. In contrast to glucoamylases (glucan 1,4-{alpha}-glucosidases), {alpha}-glucosidases favor oligosaccharides as substrates, while polysaccharides are hydrolyzed relatively slowly or not at all. Numerous {alpha}-glucosidases from bacteria and eukaryotes have been characterized, and the majority of them have been from mesophilic organisms. The previously reported archaeal representatives are the enzymes from Sulfolobus solfataricus (23), Pyrococcus furiosus (3), and Thermococcus sp. (21), and only the S. solfataricus {alpha}-glucosidase has been produced recombinantly (24).

Eukaryal {alpha}-mannosidases are membrane-bound or cytosolic proteins with important biological functions in glycoprotein processing and catabolism (5). In bacteria, a few enzymes with {alpha}-mannosidase activity have been described; examples include a secreted 1,2-{alpha}-mannosidase from Bacillus sp. (17), a cytoplasmic enzyme from Mycobacterium tuberculosis (22), and a thermoactive cytosolic {alpha}-mannosidase from Thermotoga maritima (18). An interesting bacterial representative of these enzymes has been found in Escherichia coli, and this enzyme is presumably involved in the utilization of the compatible solute 2-{alpha}-mannosyl-D-glycerate (27). Several archaeal sequences annotated as {alpha}-mannosidase can be found in the public databases; most of them originated from whole-genome sequencing projects. However, an archaeal {alpha}-mannosidase has not been characterized previously, and the physiological role of {alpha}-mannosidases in the domain Archaea remains obscure.


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MATERIALS AND METHODS
 
Strains and growth conditions. P. torridus DSM 9790 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen and was grown aerobically at 60°C and pH 0.7 in Brock's medium supplemented with 0.2% (vol/vol) yeast extract, as described by Schleper et al. (28). This medium contained (per liter) 1.32 g (NH4)2SO4, 0.28 g KH2PO4, 0.25 g MgSO4 · 7H2O, 0.07 g CaCl2 · 2H2O, 0.02 g FeCl3 · 6H2O, 1.8 mg MnCl2 · 4H2O, 4.5 mg Na2B4O7 · 10H2O, 0.22 mg ZnSO4 · 7H2O, 0.05 mg CuCl2 · 2H2O, 0.03 mg Na2MoO4 · 2H2O, 0.03 mg VOSO4 · 2H2O, and 0.01 mg CoSO4. The pH was adjusted with concentrated H2SO4.

E. coli XL1-Blue was used as a general host for DNA manipulations. For expression of the recombinant AglA and ManA enzymes, an E. coli strain was constructed by introducing the 4.7-kb plasmid conferring streptomycin resistance [obtained from E. coli BL21-CodonPlus(DE3)-RIPL (Stratagene)] into E. coli BL21 (Novagen) to obtain E. coli BL21RS. This strain was transformed with each of five chaperone-encoding plasmids (TAKARA BIO). The resulting strains were cultivated in Luria-Bertani medium at 37°C, and when necessary, 50 µg/ml ampicillin and/or 34 µg/ml chloramphenicol, 25 µg/ml kanamycin, and 50 µg/ml streptomycin were added to the medium to maintain the plasmids.

Sequence and phylogenetic analyses. The sequences of glycoside hydrolase family 31 (GHF 31) members used for phylogenetic tree construction (Fig. 1) were selected based on the available biochemical data (substrate specificity), with the exception of the enzymes from Thermoplasma acidophilum, Thermoplasma volcanium, Thermoplasma thermophilus, and Bacillus thermoamyloliquefaciens. Multiple-sequence alignments were constructed using the program ClustalW v1.83 (available at http://www.ebi.ac.uk/clustalw/) with default parameters. The phylogenetic tree in Fig. 1 was constructed using the Mega2 program (15) and distance neighbor joining based on pairwise distances between amino acid sequences and global gap removal. Confidence limits for branch points were estimated from 1,000 bootstrap replications.


Figure 1
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  		FIG. 1. Phylogenetic tree of selected members of glycoside hydrolase family 31. The EC numbers for protein sequences with known substrate specificities are given in parentheses; the numbers at the nodes are bootstrap confidence values. The following abbreviations are used (accession numbers are in parentheses): PTO-AglA, P. torridus AglA (AAT42677); SSO-MalA, S. solfataricus MalA (AAK43151); SSO-XylS, S. solfataricus XylS (AAK43123); TAC, T. acidophilum (CAC11443); TVO, T. volcanium (BAB60467); BTH, B. thermoamyloliquefaciens (BAA76396); TTH, T. thermophilus (AAS82549); ATH-Aglu1, Arabidopsis thaliana Aglu1 (AAB82656); ATH-Xyl1, A. thaliana Xyl1 (AAD05539); HSA-GAA, Homo sapiens GAA (CAA68763); HSA-SI, H. sapiens SI (CAA45140); BTA-Agl, Bos taurus Agl (AAF81637); RNO-SI, Rattus norvegicus SI (AAA65097); GLE-Glq1, Gracilariopsis lemaneiformis Glq1 (CAB51910); ECO-YihQ, E. coli YihQ (AAB03011); ECO-YicI, E. coli YicI (AAA62009).

Cloning of the P. torridus {alpha}-glucosidase and {alpha}-mannosidase genes and expression in E. coli. The P. torridus open reading frames (ORFs) PTO0091 and PTO0092 encode putative {alpha}-mannosidase and {alpha}-glucosidase and were designated manA and aglA, respectively (11). Cloning of the P. torridus ORF PTO0092 was accomplished by amplifying the genome region surrounding the aglA gene with primers 985reg_for (5'-TGCGGAATACCATTCGGCAGCAT-3') and 985reg_rev (5'-TCAATACGGCCGCACCAACAAGT-3') using genomic DNA as the template. The 2,996-bp product contained a 500-bp DNA sequence upstream of the gene's start codon (ATG) and a 500-bp sequence downstream of its stop codon (amber) and was cloned in pCR4-TOPO using a TOPO cloning kit (Invitrogen). The vector obtained (pCR-aglA reg) was cut with BspHI (NcoI compatible) and EagI (NotI compatible), and the fragment containing the aglA gene was ligated with pET24d (Novagen) digested with NcoI and NotI. For cloning of the manA gene, primers 984.F-nco (5'-CACCATGGTAAACATTAAAAGAAAGC-3') and 984.R (5'-ATTCAGGTTTAACATTGGCATC-3') were used to amplify the P. torridus ORF PTO0091 with genomic DNA as the template. The PCR product obtained was cloned into pDrive (QIAGEN PCR cloning kit), and the 3,084-bp NcoI-SacI fragment containing the manA gene (NcoI was introduced with the 984.F-nco primer; the restriction site is underlined) was cloned in pET24d digested with the same enzymes. The resulting recombinant plasmids, p24-aglA and p24-manA, were transformed in E. coli BL21RS, carrying each of five different plasmids for homologous chaperones (TAKARA BIO). Expression of the aglA gene in E. coli was induced by adding isopropyl-ß-D-thiogalactopyranoside (IPTG) at a concentration of 0.1 mM after the cell culture optical density reached 0.5. The manA gene was expressed without addition of IPTG.

Purification of recombinant AglA and ManA and molecular weight determination. The recombinant AglA and ManA enzymes were purified from E. coli BL21RS grown in 2 liters of Luria-Bertani medium. The cells were harvested by centrifugation (15 min, 6,000 x g), washed with 50 mM Tris-HCl buffer (pH 8.0) for the ManA samples or with 50 mM acetate buffer (pH 5.0) for the AglA samples, and lysed by twofold passage through a French press. AglA was purified further by heat treatment (15 min at 67°C), by anion-exchange chromatography in 50 mM Tris-HCl (pH 8.0) on a Source Q30 column (Amersham Biosciences) using a linear NaCl gradient (0 to 1.0 M), and finally by gel filtration on a Superdex 200 column (Amersham Biosciences) in Tris-HCl (pH 8.0) containing 150 mM NaCl. For purification of ManA, 1 M (NH4)2SO4 was added to the cell lysate before hydrophobic interaction chromatography on a HighLoad Phenyl Sepharose column (Amersham Biosciences), which was followed by anion-exchange (Source Q30) and gel filtration (Superdex 200) steps using the same conditions that were used for AglA. After each purification step the fractions obtained were evaluated for specific enzyme activity and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and the most active fractions were pooled, concentrated (Amicon Ultra columns; Millipore), and dialyzed against the corresponding buffer.

Gel filtration chromatography (Superdex 200 equilibrated with 50 mM Tris-HCl [pH 8.0]-150 mM NaCl) was used to determine the oligomerization state of the recombinant enzymes. The following proteins, purchased from Boehringer Mannheim, were used as standards: cytochrome c (12.5 kDa), bovine albumin (68 kDa), catalase (240 kDa), and ferritin (450 kDa).

Enzyme assays and kinetics. (i) Standard assay. In the standard assay, {alpha}-glucosidase and {alpha}-mannosidase activities were determined with p-nitrophenyl-{alpha}-D-glucopyranoside and p-nitrophenyl-{alpha}-D-mannopyranoside (pNP-Man), respectively, purchased from Sigma. The reaction mixtures (total volume, 50 µl) contained 50 mM acetate buffer (pH 5.0), 10 mM p-nitrophenyl substrate, and enzyme solutions. When {alpha}-mannosidase activity was assayed, the reaction buffer was supplemented with 1 mM CdCl2. After a 10-min incubation at 85°C for AglA or at 70°C for ManA, the reactions were stopped by addition of 100 µl 1 M Na2CO3, and the absorbance at 420 nm was determined. Specific activity was expressed in µmol of p-nitrophenol released (molar absorption coefficient, 1.12 x 104 M–1 cm–1) per min per mg of protein under the conditions specified above.

(ii) Alternative assays. The activities of the two enzymes with natural substrates were analyzed by thin-layer chromatography (TLC) on silica gel plates (Silica Gel 60 F254; Merck), using 1-propanol-water-ethyl acetate (6:3:1, vol/vol/vol) as the mobile phase. The reaction products were detected by spraying the chromatograms with aniline-diphenylamine reagent (1% [wt/vol] diphenylamine and 1% [vol/vol] aniline in acetone mixed with 0.1 volume of 87% [vol/vol] phosphoric acid prior to use) and baking them for 10 min at 160°C.

The {alpha}-glucosidase and {alpha}-mannosidase activities with natural substrates were measured quantitatively by determining the amounts of monosaccharides (glucose and mannose, respectively) released after enzyme treatment. The assays were performed like the standard assay but with longer incubation times (15 min), and the reactions were stopped by heating the samples at 100°C for 15 min. The amount of glucose formed was estimated with a glucose oxidase/peroxidase-coupled assay (Sigma procedure no. 510), and the mannose concentration was determined with a coupled assay utilizing the enzymes hexokinase (Roche), phosphomannose isomerase (Sigma), phosphoglucose isomerase (Sigma), and glucose-6-phosphate dehydrogenase (Sigma) essentially as described by Etchison and Freeze (10). The substrates tested with ManA were {alpha}-1,2-, {alpha}-1,3-, methyl-O-{alpha}-1,4-, and {alpha}-1,6-mannobiose and {alpha}-Man-(1->3)({alpha}-Man-[1->6])-{alpha}-Man-(1->6)({alpha}-Man-[1->3])-Man (referred to as {alpha}3,{alpha}6-mannopentaose below) (Sigma).

Transcription analysis. P. torridus RNA was prepared from 10 ml of a culture grown to the end of the exponential phase using either an RNeasy mini kit (QIAGEN) or the RNAgents total RNA isolation system (Promega) as described by the manufacturers. The cells (approximately 4 x 108 cells) were centrifuged (6,000 x g), washed in 50 mM acetate buffer (pH 5.0), and lysed by resuspending them in 100 µl of 50 mM Tris-HCl (pH 8.0).

Northern blot analysis. For Northern blot preparation, total P. torridus RNA was separated on 1.2% agarose gels in the presence of 0.2 M formaldehyde in morpholinepropanesulfonic acid (MOPS) buffer, followed by capillary transfer to a Hybond XL membrane (Amersham Biosciences), as described by Sambrook et al. (26) except that hybridization was performed with UltraHyb buffer (Ambion) at 50°C. The radioactive DNA probe was generated by random primer synthesis with a HexaLabel DNA labeling kit (Fermentas) and {alpha}-32P-labeled deoxynucleoside triphosphates using a 583-bp fragment of the aglA gene (PstI-HindIII fragment of pCR-aglA reg) as the template.

Mapping of aglA and manA transcription start sites. RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) was used to determine the transcription start sites of the aglA and manA genes (FirstChoice RLM-RACE kit; Ambion), using the protocol of the manufacturer. Two independent replications of the 5' RLM-RACE procedure were carried out, including a control without tobacco acid pyrophosphatase treatment. The cDNA obtained was subjected to nested PCR with SuperTaq Plus DNA polymerase (Ambion) using the primers listed in Table 1, and the PCR products were column purified (QIAquick PCR purification; QIAGEN) and cloned using a StrataClone PCR cloning kit (Stratagene). A total of 10 colonies per gene were analyzed, and the insert was sequenced in both directions (ABI 3700; Applied Biosystems).


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  		TABLE 1. Primers used in this study

Quantitative RT-PCR. The aglA and manA transcripts were quantified by reverse transcription (RT) with RevertAid Moloney murine leukemia virus reverse transcriptase (Fermentas), followed by absolute real-time PCR (for primer sequences see Table 1) using SYBR green (qPCR MasterMix Plus for SYBR green I with fluorescein; Eurogentec) with an iCycler (Bio-Rad). External standard curves were generated for both genes with recombinant RNAs (recRNAs) covering the range from 1 x 104 to 1 x 1010 molecules per RT reaction mixture. Recombinant RNAs were synthesized for both genes in in vitro T7 promoter-directed transcription reactions with linearized p24-manA and p24-aglA as the templates (T7 RNA polymerase; Fermentas). After purification by phenol-chloroform extraction, the recRNAs obtained were serially diluted in water, and 2 ng of a total E. coli RNA preparation was added to each tube in order to compensate for background effects and mimic a natural RNA distribution like that in native total RNA (20). The recRNA dilutions were subjected in duplicate to RT and quantitative PCR, and plots of cycle threshold versus log recRNA copy number were generated. The recRNA external standard curves showed PCR efficiencies of 108.8% (R2 = 0.978) for the manA gene and 100.8% (R2 = 0.993) for the aglA gene over the range tested. Quantification of the native aglA and manA transcripts was performed in duplicate for three dilutions of the total P. torridus RNA, and the intraassay and interassay coefficients of variation were estimated.


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RESULTS
 
Phylogenetic analysis of P. torridus {alpha}-glucosidase and {alpha}-mannosidase. The P. torridus ORF PTO0092 translation product (AglA), annotated as {alpha}-glucosidase (EC 3.2.1.20), displayed a high level of amino acid sequence similarity (38.6% identity) to the previously characterized ortholog from S. solfataricus, MalA (9, 24). The 645-residue protein could be classified as a member of GHF 31 based on the presence of the conserved active site pattern [GF]-[LIVMF]-W-x-D-M-[NSA]-E characteristic of this family (Carbohydrate Active Enzymes database [http://www.cazy.org]) (4) (see Fig. 1S in the supplemental material). At present, this family contains numerous enzymes of organisms belonging to all major branches of the phylogenetic tree, including 10 enzymes of archaeal origin having several known activities, such as {alpha}-glucosidase (EC 3.2.1.20), sucrase-isomaltase (EC 3.2.1.48 and EC 3.2.1.10), {alpha}-xylosidase (EC 3.2.1.-), {alpha}-glucan lyase (EC 4.2.2.13), and isomaltosyltransferase (EC 2.4.1.-). Phylogenetic analysis of the AglA sequence performed by the neighbor-joining tree method (15) revealed that this sequence is more similar to orthologs from members of the genus Sulfolobus than to sequences from members of the phylogenetically closely related genus Thermoplasma (Fig. 1).

The P. torridus {alpha}-mannosidase gene (manA, ORF PTO0091) codes for a 952-amino-acid protein and is located immediately upstream of aglA. Analysis of the protein sequence indicated that ManA is a member of GHF 38, which contains enzymes with {alpha}-mannosidase activity (EC 3.2.1.24) (see Fig. S2 in the supplemental material). GHF 38 currently includes some archaeal representatives (6 enzymes), many bacterial representatives (75 enzymes), and many eukaryal representatives (76 enzymes). Extensive phylogenetic analyses have shown that the currently available {alpha}-mannosidase sequences form three distinct groups (7, 11). The archaeal representatives fall into clade III of the {alpha}-mannosidase phylogeny, which is the most ancient and taxonomically diverse group of {alpha}-mannosidases (12), and these representatives seem to be restricted to the orders Sulfolobales (S. solfataricus and Sulfolobus tokodaii), Thermoplasmatales (P. torridus and T. volcanium), and Thermococcales (Pyrococcus horikoshii). Interestingly, BLAST analysis revealed that the ManA protein and its homologs from T. volcanium and S. tokodaii contain a distinct N-terminal domain which is not found in the rest of the available {alpha}-mannosidase sequences. So far, no archaeal {alpha}-mannosidase enzyme has been characterized.

Both AglA and ManA lacked detectable signal peptide sequences (SignalP 3.0) (1). Also, the native {alpha}-glucosidase and {alpha}-mannosidase activities measured in fractionated P. torridus cell extracts were associated with the soluble fraction (not shown), suggesting that the two enzymes are located in the P. torridus cytosol.

Purification of recombinant P. torridus {alpha}-glucosidase and {alpha}-mannosidase, enzyme properties, and substrate specificity. Both enzymes were found to be expressed in the active form in E. coli only after the host was cotransformed with plasmids containing the genes for host chaperones and for tRNAs for codons which are rare in E. coli. Therefore, E. coli BL21 derivatives were constructed that harbored a plasmid for rare E. coli tRNAs (E. coli BL21RS) (see Materials and Methods) and each of five different plasmids encoding E. coli chaperones. The highest activities were observed after coexpression of the P. torridus {alpha}-mannosidase gene (manA) with groES-groEL and after coexpression of the {alpha}-glucosidase gene (aglA) with groEL-groES-tig (data not shown). These cells were used for overexpression and purification of the recombinant enzymes. Both enzymes were purified to electrophoretic homogeneity by use of heat treatment (for AglA), followed by hydrophobic interaction, anion-exchange, and size exclusion chromatography (Table 2 and Fig. 2). It is worth noting that the E. coli expression strain described here has proved to be useful for obtaining several other P. torridus enzymes in the native form, which were otherwise misfolded or not expressed at all (unpublished data).


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  		TABLE 2. Purification of AglA and ManA


Figure 2
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  		FIG. 2. SDS-PAGE analysis of the steps in the purification of recombinant P. torridus {alpha}-glucosidase (A) and {alpha}-mannosidase (B). (A) Lane 1, molecular weight marker; lane 2, E. coli BL21RS::p24-aglA cellular extract (8 µg); lane 3, heat-treated extract (5 µg); lane 4, AglA pooled fractions after anion-exchange chromatography (2 µg); lane 5, AglA pooled fractions after gel filtration chromatography (2 µg). (B) Lane 1, molecular weight marker; lane 2, E. coli BL21RS::p24-manA cellular extract (8 µg); lane 3, ManA pooled fractions after hydrophobic interaction chromatography (5 µg); lane 4, ManA pooled fractions after anion-exchange chromatography (3 µg); lane 5, ManA pooled fractions after gel filtration chromatography (0.7 µg).

AglA. The apparent size of the purified recombinant AglA enzyme estimated by SDS-PAGE (approximately 74 kDa) is in good agreement with the size calculated from the primary structure (75.4 kDa). The size of the native enzyme, determined by analytical gel permeation chromatography, was calculated to be 440 kDa, which suggests that AglA is active as a hexamer.

Using a 10-min assay, the purified recombinant {alpha}-glucosidase was found to be most active at 87°C when it was tested with p-nitrophenyl {alpha}-D-glucopyranoside in 50 mM acetate buffer at pH 5.0. Remarkably, this temperature is 27°C above the optimum growth temperature of P. torridus and more than 20°C above its maximum growth temperature. At 60°C the enzyme showed 23% of its maximal activity. Also, AglA retained considerable activity (81%) after preincubation of the enzyme for 120 min at 80°C. The kinetics of heat inactivation at temperatures between 70°C and 90°C appeared to be first order (data not shown). The enzyme displayed significant activity over a broad pH range, with maximum activity at pH 5.0. The optimum pH for AglA activity corresponds well to the reported intracellular pH of Picrophilus cells, pH 4.6 (35).

The substrate specificity of the enzyme was tested with a panel of oligosaccharides (maltose, isomaltose, maltotriose, maltotetraose, panose, trehalose, melibiose, sucrose, raffinose, turanose, melizitose, and {alpha}-, ß- and {gamma}-cyclodextrins) and polysaccharides (dextrin 10, amylose, starch, glycogen, and dextran), as well as with synthetic aryl glycosides (p-nitrophenyl-linked {alpha}- and ß-glucopyranosides, {alpha}- and ß-galactopyranosides, {alpha}- and ß-xylopyranosides, and {alpha}-mannopyranoside). AglA showed the highest activity with maltose, and it hydrolyzed p-nitrophenyl {alpha}-D-glucopyranoside with a lower efficiency (Table 3). Strict specificity was observed both for the glycone residue of synthetic substrates (only glucose was accepted) and for the type of the glycosidic bond (only {alpha}-1,4 bonds were cleaved). The rate of maltooligosaccharide hydrolysis decreased with increasing number of maltose units in the substrate. Still, considerable activity was observed with maltotetraose (54% of the activity with maltose) or dextrin 10 (27%). In contrast to the closely related homolog from S. solfataricus or {alpha}-glucosidases of mammalian origin, AglA was unable to release glucose from polymeric substrates, like glycogen (24). Thin-layer chromatography analyses of the reaction products of AglA showed that only glucose was formed when maltooligosaccharides with different chain lengths were used as substrates and that no transferase activity was evident (Fig. 3, A). This substrate utilization spectrum indicates that AglA is a typical maltase enzyme (EC 3.2.1.20).


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  		TABLE 3. Characteristics of AglA and ManA


Figure 3
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  		FIG. 3. Thin-layer chromatography analysis of the reaction products of P. torridus {alpha}-glucosidase (A) and {alpha}-mannosidase (B). (C) Schematic diagram of the structure of {alpha}3,{alpha}6-mannopentaose. The common core mannotriose structure found in N-glycans is indicated by boldface type. The reactions were carried out in 20 µl of 50 mM acetate buffer (pH 5.0) with 0.2% substrate and with (+) or without (–) 3.6 µg of purified AglA enzyme at 85°C (A) or with (+) or without (–) 0.3 µg of purified ManA at 65°C (B) for 4 h. (A) Lane 1, G1 to G7 maltooligosaccharide standards; lanes 2 and 3, maltose; lanes 4 and 5, maltotriose; lanes 6 and 7, maltopentaose; lanes 8 and 9, maltohexaose. (B) Lane 1, mannose; lanes 2 and 3, 1,2-{alpha}-mannobiose; lanes 4 and 5, 1,3-{alpha}-mannobiose; lanes 6 and 7, methyl-O-1,4-{alpha}-mannobiose; lanes 8 and 9, 1,6-{alpha}-mannobiose; lanes 10 and 11, {alpha}3,{alpha}6-mannopentaose.

ManA. The elution profile of the recombinant P. torridus {alpha}-mannosidase in gel permeation chromatography under native conditions suggested that the size of this enzyme is 220 kDa, which corresponds to a dimer consisting of 111.5-kDa subunits. A small fraction of ManA activity could also be detected at approximately 120 kDa, most probably reflecting single subunits.

The maximum activity of ManA was observed at 70°C and pH 5.2 when pNP-Man was used as the substrate. ManA displayed an unusual metal requirement for activity; Cd2+ at a concentration of 1 mM was found to be the most potent activator (12.5-fold activation compared to the apoenzyme), followed by Co2+ (8.3-fold activation at a concentration of 10 mM), Fe2+ (4.5-fold activation at a concentration of 10 mM), and Mn2+ (3.8-fold activation at a concentration of 10 mM). EDTA abolished this activation completely when it was supplied in equimolar amounts or in excess. Zn2+, Ca2+, Mg2+, Ba2+, K+, and Li+ had no significant effect on ManA activity when they were added at a concentration of 1 mM or 10 mM in the assay, while Ag+, Ni2+, Cu2+, and Fe3+ were strong inhibitors at these concentrations. In addition, the resistance of ManA to heat inactivation (at 80°C) was increased considerably by the presence of Co2+ in the incubation buffer, and this effect was also neutralized by EDTA (Fig. 4). An inactive apoenzyme was obtained by dialysis of the purified ManA against 50 mM acetate buffer (pH 5.6) containing 5 mM EDTA, followed by a second dialysis against the same buffer without EDTA. Cd2+, Co2+, and Fe2+ at a concentration of 5 mM were able to completely restore the activity of the EDTA-treated enzyme, while incubation of the apoenzyme with Zn2+, Ca2+, Mg2+, and Ba2+ did not lead to measurable restoration of activity.


Figure 4
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  		FIG. 4. Temperature inactivation kinetics of recombinant P. torridus {alpha}-mannosidase at 80°C. The purified enzyme (0.1 mg/ml) was incubated at 80°C in 50 mM acetate buffer (pH 5.0) with different additives, samples were withdrawn, and the residual activities were determined as described in Materials and Methods. Relative activity is expressed as the percentage of the activity without heat treatment.

The specificity of P. torridus {alpha}-mannosidase was examined with mannobiose substrates having all possible types of {alpha}-linkages (1->2, 1->3, 1->4, and 1->6), as well as with {alpha}3,{alpha}6-mannopentaose, which is the core structure of some triantennary N-linked carbohydrates in glycoproteins. TLC analysis of the reaction products after incubation of ManA with these substrates revealed that the enzyme has broad specificity and released mannose from all of the substrates (Fig. 3B and C). In addition, ManA was equally active with these mannobiose substrates when the released mannose was quantified, resulting in a specific activity of approximately 6.6 U/mg under optimal conditions. In order to examine the possible role of P. torridus {alpha}-mannosidase in the turnover of mannose-containing compatible solutes, the enzyme was incubated with 2-{alpha}-mannosyl-D-glycerate. No free mannose was detected after incubation of ManA for 12 h with this substrate either by TLC or by an enzymatic coupled assay.

Inhibition profile of ManA. Eukaryal {alpha}-mannosidases display different behaviors in the presence of the specific inhibitors 1-deoxymannojirimycin and swainsonine depending on their class affiliation. Class I enzymes are generally inhibited by 1-deoxymannojirimycin but not by swainsonine, while class II and III {alpha}-mannosidases have the opposite inhibition profile (6, 8, 29). As no archaeal representatives have been characterized previously, we examined the effects of these inhibitors on P. torridus {alpha}-mannosidase. Deoxymannojirimycin had no effect on ManA at concentrations up to 500 µM, while swainsonine showed competitive inhibition when pNP-Man was used as a substrate (Fig. 5A and B). In Fig. 5C the results for inhibition by swainsonine at two different substrate concentrations are presented as a Dixon plot, which revealed a Ki of 1.22 µM.


Figure 5
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  		FIG. 5. Inhibition profile of P. torridus {alpha}-mannosidase. The enzyme (0.26 µg) was incubated for 15 min at 70°C in 50 mM acetate buffer (pH 5.0) with pNP-Man and different concentrations of the inhibitors deoxymannojirimycin (A) and swainsonine (B). (C) Dixon plot of the inhibition by swainsonine at two different substrate concentrations, resulting in a Ki of 1.22 µM.

Genetic organization and transcription analysis of P. torridus manA and aglA genes. The manA and aglA genes are situated colinearly in the P. torridus genome and are separated by a 42-bp intergenic region. This functional cluster was found only in P. torridus; it was found in none of the organisms that possess orthologs of one of the two ORFs. Sequence analysis of the upstream gene regions revealed the presence of a nearly canonical TATA box sequence at position –28 relative to the start codon of the aglA gene and an adenine-rich region at positions –35 to –30, corresponding to an archaeal BRE element, while at –17 bp upstream of the manA gene a TA-rich sequence with only limited similarity to a TATA box was detected. Also, a poly(T) region was present 25 bp after the manA stop codon, possibly representing a termination signal. This finding pointed to the possibility that despite the colinear organization and the presumed physiological relatedness of the two genes, they might be transcribed separately. Therefore, we investigated whether the manA and aglA genes are cotranscribed. Initial RT-PCR experiments suggested that the two genes may be transcribed as a single message (not shown). However, further transcript analysis by Northern blotting yielded an approximately 2.1-kb fragment when the aglA DNA sequence was used as a probe, and no signal pointing to cotranscription (expected size, ~5 kb) was observed (Fig. 6B). In addition, 5' RLM-RACE experiments showed the presence of a primary aglA transcript and revealed the aglA and manA transcription start sites. Therefore, we concluded that the RT-PCR products spanning the manA-aglA intergenic region that were obtained initially were most probably due to RNA polymerase readthrough (inefficient termination at end of the upstream gene). The aglA transcription start was mapped by 5' RLM-RACE at the adenine of the start codon of the ORF (in plasmids from 10 individual clones analyzed), while two forms of the manA transcript were detected, starting at a guanine at position –8 (in 4 of 10 plasmids analyzed) and at an adenine at position 1 (in 6 of 10 plasmids analyzed) (Fig. 6A). Thus, both genes appeared to produce leaderless transcripts (transcripts lacking a 5' untranslated region [5'-UTR]). The possibility that the manA and aglA genes are cotranscribed and processed further (cleaved) to generate two messages can be excluded because the 5' RLM-RACE procedure distinguishes between primary (genuine) and processed transcripts based on their 5' phosphorylation states (2). It has to be mentioned that in this case, transcript analysis by 5' RLM-RACE is more informative than primer extension or S1 nuclease mapping, with which such discrimination cannot be made. The results from Northern hybridization and 5' RLM-RACE indicate that the aglA gene is transcribed separately from its upstream neighbor manA and lacks a 5'-UTR.


Figure 6
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  		FIG. 6. (A) Genome region containing the manA and aglA genes (gray arrows). Start and stop codons are underlined, putative TATA box regions are enclosed in boxes, and the transcription start sites identified are indicated by bent arrows. (B) Northern blot analysis of the aglA gene. Lane 1 contained 2.5 µg total P. torridus RNA, and lanes 2 and 3 contained 5 µg total RNA isolated by two different methods (see Materials and Methods). The radioactive probe used for hybridization is indicated by a solid bar in panel A.

Furthermore, we performed absolute aglA and manA mRNA quantification analyses with total P. torridus RNA preparations by using quantitative RT-PCR. The results obtained for the manA transcripts (4.14 x 106 molecules/reaction mixture; interassay coefficient of variation, 3.2%) and for the aglA transcripts (6.63 x 107 molecules/reaction mixture; interassay coefficient of variation, 2.7%) show that there was an approximately 16-fold excess of aglA mRNA compared to the amount of manA mRNA. These data are also consistent with independent transcription initiation of the downstream aglA gene, since normally internal operon genes are transcribed less efficiently.


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DISCUSSION
 
Phylogenetic and biochemical analysis. Phylogenetic analysis clustered P. torridus {alpha}-glucosidase together with sequences of members of the distantly related crenarchaeal genus Sulfolobus rather than with the polypeptides from the closely related genus Thermoplasma (Fig. 1). It has been hypothesized that extensive lateral gene transfer occurred between these two lineages (10, 25). Therefore, it was not surprising to observe that AglA displayed properties similar to those of the previously characterized S. solfataricus homolog MalS (23, 24). The remarkable stability of the enzyme against thermoinactivation and its "optimum temperature" (27°C above the optimal growth temperature of P. torridus, measured under the standard assay conditions) are in agreement with the phylogenetic allocation of the AglA sequence and support the hypothesis that there was a gene transfer event. In this respect it would be interesting to examine the currently uncharacterized AglA homologs found in other euryarchaea, like T. acidophilum and T. volcanium (accession numbers CAC11443 and BAB60467, respectively). Most probably, the P. torridus {alpha}-glucosidase plays an important role in the pathway for utilization of {alpha}-glucans like starch (30) and of maltooligosaccharides. No ortholog of a maltodextrin phosphorylase was found in the P. torridus genome, suggesting that maltose (maltodextrin) hydrolysis is the only means of linking these substrates to glycolysis.

To our knowledge, the P. torridus {alpha}-mannosidase enzyme described here is the first characterized archaeal member of GHF 38. Together, the observed broad substrate spectrum, the lack of a signal peptide sequence, the inhibition profile, and the unusual metal requirements of ManA suggest that it is a cytosolic enzyme belonging to the class III {alpha}-mannosidases. A role of P. torridus {alpha}-mannosidase in the utilization and/or turnover of compatible solutes like 2-{alpha}-D-mannosyl-glycerate can be ruled out, as ManA was inactive with this compound. In agreement, the genome of P. torridus does not contain genes for key enzymes necessary for 2-{alpha}-D-mannosyl-glycerate synthesis (e.g., mannosyl-3-phosphoglycerate synthase and mannosyl-3-phosphoglycerate phosphatase). Another possible role for ManA is a contribution to the utilization of exogenous glycoproteins and/or to the turnover of the organism's own glycoproteins, specifically in metabolizing the glycone moiety, which was shown to be a substrate for ManA. Indeed, in the closely related organism T. acidophilum, it has been reported that there are glycoproteins with high mannose contents (37), and a functional glycosylation system (dolichyl-phosphomannose synthase) has been described (38). Protein glycosylation in P. torridus has not been studied biochemically. Analysis of the P. torridus genome revealed at least two possible routes for metabolizing the released mannose, through a mannose dehydrogenase and mannonate dehydratase (PTO0149), yielding 2-keto-3-deoxygluconate (KDG), which is part of the Entner-Doudoroff pathway, or via a mannose-6-phosphate isomerase (PTO0939), yielding fructose-6-phosphate, which can be channeled into glycolysis.

Molecular analysis. Transcription initiation in members of the Archaea has been extensively studied (32). However, most of the data comes from experiments with representatives of the halophilic Archaea (Haloferax), methanogens (Methanococcus), and Crenarchaeota (Sulfolobus). In contrast, information about the mode of transcription initiation and the regulatory elements that govern it in species of the Thermoplasma group is very limited.

The experiments aimed at elucidating the mode of transcription of the P. torridus {alpha}-mannosidase and {alpha}-glucosidase genes credibly showed that these genes have separate transcription initiation sites despite the colinear orientation and the small 42-bp intergenic region. A cotranscript could not be detected by Northern blot analysis or 5' RLM-RACE. A PCR product spanning the intergenic region which was found in RT-PCR experiments was most probably due to inefficient termination at the end of the upstream gene (manA).

The presence of leaderless mRNA has been described in very diverse taxa (Bacteria, Archaea, and Eukarya), and there is experimental evidence that such mRNA is capable of initiating translation (reference 36 and references therein). In the Archaea, such an unusual transcription start site has been computationally predicted for S. solfataricus (34) and has been experimentally shown for 10 genes in Pyrobaculum aerophilum (31); both of these organisms are members of the Crenarchaeota. Also experimentally, transcription has been shown to initiate at the adenine in the first codon of the S. solfataricus lacS (ß-glycosidase) gene (14). Notably, the P. torridus aglA gene was shown to produce a leaderless transcript, and the manA gene was found to have two distinct transcription initiation sites, one of which also produced an mRNA lacking a 5'-UTR. The 5' RLM-RACE method used in the present study did not allow quantification of the alternative transcripts and did not allow us to address whether growth on different media has an effect on the manA transcription start site selection. However, the results of the experiments provide additional and independent proof for the presence of primary (5'-triphosphate-containing), leaderless mRNA in archaea, as the currently available evidence was derived from primer extension and S1 nuclease mapping assays.

Currently, the mechanism for translation of leaderless RNA is not completely clear. One hypothesis proposes the existence of downstream sequence elements that interact with rRNA (16, 33). Another possibility, which does not necessarily exclude the first possibility, is participation of trans-acting elements like initiation factor 2 (13).

Whole-genome computational analyses have suggested that in Crenarchaeota two distinct gene groups can be defined based on conserved sequences in their upstream regions. One group has well-defined Shine-Dalgarno (SD) sequences and consists of internal operon genes, whereas the second group lacks detectable SD sequences and includes single or operon-initiating genes (34). Since SD sequences are readily detected in several of the sequenced euryarchaeal genomes (19), it remains to be determined whether the leaderless transcription reported here is a general phenomenon or an exception in P. torridus and in representatives of the closely related genus Thermoplasma.


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ACKNOWLEDGMENTS
 
We thank E. Galinski (Bonn, Germany) for providing 2-{alpha}-mannosyl-D-glycerate and Mechthild Bömeke for excellent technical support.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany. Phone: 49-551-393795. Fax: 49-551-393793. E-mail: wliebl{at}gwdg.de. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Journal of Bacteriology, October 2006, p. 7123-7131, Vol. 188, No. 20
0021-9193/06/$08.00+0     doi:10.1128/JB.00757-06
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