Synergistic production of L-arabinose from arabinan by the combined use of thermostable endo- and exo-arabinanases from Caldicellulosiruptor saccharolyticus.

The optimum conditions for the production of L-arabinose from debranched arabinan were determined to be pH 6.5, 75°C, 20 g l(-1) debranched arabinan, 42 Um l(-1) endo-1,5-α-L-arabinanase, and 14 U ml(-1) α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus and the conditions for sugar beet arabinan were pH 6.0, 75°C, 20 g l(-1) sugar beet arabinan, 3 U ml(-1) endo-1,5-α-L-arabinanase, and 24 U ml(-1) α-L-arabinofuranosidase. Under the optimum conditions, 16 g l(-1)l-arabinose was obtained from 20 g l(-1) debranched arabinan or sugar beet arabinan after 120 min, with a hydrolysis yield of 80% and a productivity of 8 g l(-1)h(-1). This is the first reported trial for the production of L-arabinose from the hemicellulose arabinan by the combined use of endo- and exo-arabinanases.

X-ray crystal structures of Moorella thermoacetica FprA. Novel diiron site structure and mechanistic insights into a scavenging nitric oxide reductase.

Several members of a widespread class of bacterial and archaeal metalloflavoproteins, called FprA, likely function as scavenging nitric oxide reductases (S-NORs). However, the only published X-ray crystal structure of an FprA is for a protein characterized as a rubredoxin:dioxygen oxidoreductase (ROO) from Desulfovibrio gigas. Therefore, the crystal structure of Moorella thermoacetica FprA, which has been established to function as an S-NOR, was solved in three different states: as isolated, reduced, and reduced, NO-reacted. As is the case for D. gigas ROO, the M. thermoacetica FprA contains a solvent-bridged non-heme, non-sulfur diiron site with five-coordinate iron centers bridged by an aspartate, and terminal glutamate, aspartate, and histidine ligands. However, the M. thermoacetica FprA diiron site showed four His ligands, two to each iron, in all three states, whereas the D. gigas ROO diiron site was reported to contain only three His ligands, even though the fourth His residue is conserved. The Fe1-Fe2 distance within the diiron site of M. thermoacetica FprA remained at 3.2-3.4 A with little or no movement of the protein ligands in the three different states and with conservation of the two proximal open coordination sites. Molecular modeling indicated that each open coordination site can accommodate an end-on NO. This relatively rigid and symmetrical diiron site structure is consistent with formation of a diferrous dinitrosyl as the committed catalytic intermediate leading to formation of N(2)O. These results provide new insight into the structural features that fine-tune biological non-heme diiron sites for dioxygen activation vs nitric oxide reduction.

Time-dependent XAS studies of trapped enzyme-substrate complexes of alcohol dehydrogenase from Thermoanaerobacter brockii.

The understanding of structure-function relationships in proteins has been significantly advanced with the advent of the biotechnological revolution. A goal yet to be realized for many metalloenzyme systems is to characterize the dynamic changes in structure that bridge the static endpoints provided by crystallography. We present here a series of edge and EXAFS spectra of the metalloenzyme alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH) complexed with its substrate. The enzyme-substrate complexes were trapped by fast freezing at various times, following their enzyme activity. Our edge and EXAFS analyses both reveal the time-dependent changes in the structure of the active site of TbADH.

Cloning, sequencing and expression in Escherichia coli of the primary alcohol dehydrogenase gene from Thermoanaerobacter ethanolicus JW200.

The structural gene, adhA, for a thermostable primary alcohol dehydrogenase was cloned from Thermoanaerobacter ethanolicus JW200. Constitutive expression from its own promoter was observed in Escherichia coli. The nucleotide sequence of adhA corresponded to an open reading frame of 1197 bp, encoding a polypeptide of 399 amino acids with a calculated Mr of 43 192. Amino acid sequence analysis showed 67-69% identity with alcohol dehydrogenases from two archaeal species and 29-37% identity with bacterial type III alcohol dehydrogenases. This represents the first reported cloning of an alcohol dehydrogenase from a bacterial species that is both thermostable and active against primary long-chain alcohols.

Cloning, sequencing, and characterization of the bifunctional xylosidase-arabinosidase from the anaerobic thermophile thermoanaerobacter ethanolicus.

The gene for the bifunctional xylosidase-arabinosidase (xarB) from the thermophilic anaerobe Thermoanaerobacter ethanolicus JW200 was cloned, sequenced, and expressed in Escherichia coli (Genebank Accession No. AF135015). Analysis of the recombinant enzyme revealed activity against multiple substrates with the highest affinity towards p-nitrophenyl beta-D-xylopyranoside (pNPX) and highest activity against p-nitrophenyl alpha-L-arabinopyranoside (pNPAP), respectively. Thus, we classify this enzyme as a bifunctional xylosidase-arabinosidase. Even though both sequences are 96% identical on the amino acid level, excluding the amino-terminal end, a frame-shift mutation in the 5' region of the gene in T. brockii ATCC 33075 and a deletion in a downstream open reading frame in T. ethanolicus seem to have occurred through evolutionary divergence of these two species. This represents an interesting phenomenon of molecular evolution of bacterial species, as PCR analysis of the region around the deletion indicates that the deletion is not present in T. brockii ssp. finnii and T. brockii ssp. brockii type strain HTD4.

A beta-1,4-endoglucanase-encoding gene from Cellulomonas pachnodae.

A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50-55 degrees C. The recombinant endoglucanase Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262-319 and 448-473, which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding.

Oligomeric integrity--the structural key to thermal stability in bacterial alcohol dehydrogenases.

Principles of protein thermostability have been studied by comparing structures of thermostable proteins with mesophilic counterparts that have a high degree of sequence identity. Two tetrameric NADP(H)-dependent alcohol dehydrogenases, one from Clostridium beijerinckii (CBADH) and the other from Thermoanaerobacter brockii (TBADH), having exceptionally high (75%) sequence identity, differ by 30 degrees in their melting temperatures. The crystal structures of CBADH and TBADH in their holo-enzyme form have been determined at a resolution of 2.05 and 2.5 A, respectively. Comparison of these two very similar structures (RMS difference in Calpha = 0.8 A) revealed several features that can account for the higher thermal stability of TBADH. These include additional ion pairs, "charged-neutral" hydrogen bonds, and prolines as well as improved stability of alpha-helices and tighter molecular packing. However, a deeper structural insight, based on the location of stabilizing elements, suggests that enhanced thermal stability of TBADH is due mainly to the strategic placement of structural determinants at positions that strengthen the interface between its subunits. This is also supported by mutational analysis of structural elements at critical locations. Thus, it is the reinforcement of the quaternary structure that is most likely to be a primary factor in preserving enzymatic activity of this oligomeric bacterial ADH at elevated temperatures.

Chemical modification of lysine side chains of cyclodextrin glycosyltransferase from Thermoanaerobacter causes a shift from cyclodextrin glycosyltransferase to alpha-amylase specificity.

Cyclodextrin glycosyltransferases and alpha-amylases are two groups of enzymes with related secondary structures. However, cyclodextrin glycosyltransferases display transferase activities not present in alpha-amylases, probably derived from the existence of two more domains and different amino acid sequences. The hydrolytic activity of cyclodextrin glycosyltransferases is generally quite low, except for two cyclodextrin glycosyltransferases from termophiles. In this work, we have carried out the chemical modification (with acetic anhydride) of the amino groups of cyclodextrin glycosyltransferase from Thermoanaerobacter to assess their contributions to protein function. The acetylated cyclodextrin glycosyltransferase showed a significant reduction of its cyclization, coupling and disproportionation activities. Surprisingly, the hydrolytic (saccharifying) activity was slightly enhanced. These results suggest the participation of one or more lysine side chains in the interactions contributing to the transferase activity, either in any of the S11 subsites or in the acceptor binding site.

Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme.

Several models have been proposed to explain the high temperatures required to denature enzymes from thermophilic organisms; some involve greater maximum thermodynamic stability for the thermophile, and others do not. To test these models, we reversibly melted two analogous protein domains in a two-state manner. E2cd is the isolated catalytic domain of cellulase E2 from the thermophile Thermomonospora fusca. CenAP30 is the analogous domain of the cellulase CenA from the mesophile Cellulomonas fimi. When reversibly denatured in a common buffer, the thermophilic enzyme E2cd had a temperature of melting (Tm) of 72.2 degrees C, a van't Hoff enthalpy of unfolding (DeltaHVH) of 190 kcal/mol, and an entropy of unfolding (DeltaSu) of 0.55 kcal/(mol*K); the mesophilic enzyme CenAP30 had a Tm of 56.4 degrees C, a DeltaHVH of 107 kcal/mol, and a DeltaSu of 0. 32 kcal/(mol*K). The higher DeltaHVH and DeltaSu values for E2cd suggest that its free energy of unfolding (DeltaGu) has a steeper dependence on temperature at the Tm than CenAP30. This result supports models that predict a greater maximum thermodynamic stability for thermophilic enzymes than for their mesophilic counterparts. This was further explored by urea denaturation. Under reducing conditions at 30 degrees C, E2cd had a concentration of melting (Cm) of 5.2 M and a DeltaGu of 11.2 kcal/mol; CenAP30 had a Cm of 2.6 M and a DeltaGu of 4.3 kcal/mol. Under nonreducing conditions, the Cm and DeltaGu of CenAP30 were increased to 4.5 M and 10.8 kcal/mol at 30 degrees C; the Cm for E2cd was increased to at least 7.4 M at 32 degrees C. We were unable to determine a DeltaGu value for E2cd under nonreducing conditions due to problems with reversibility. These data suggest that E2cd attains its greater thermal stability (DeltaTm = 15.8 degrees C) through a greater thermodynamic stability (DeltaDeltaGu = 6.9 kcal/mol) compared to its mesophilic analogue CenAP30.

A kinetic locking-on strategy for bioaffinity purification: further studies with alcohol dehydrogenase.

The kinetic locking-on strategy improves the selectivity of protein purification procedures based on immobilized cofactor derivatives through use of enzyme-specific substrate analogues in irrigants to promote biospecific adsorption. This paper describes the development and application of this strategy to the one-chromatographic step affinity purification of NAD(P)+-dependent alcohol dehydrogenases using 8'-azo-linked immobilized NAD(P)+, S6-linked and N6-linked immobilized NAD+, and N6-linked immobilized NADP+ derivatives. These studies were carried out using alcohol dehydrogenases from Saccharomyces cerevisiae (YADH, EC 1.1.1.1), equine liver (HLADH, EC 1.1.1.1), and Thermoanaerobium brockii (TBADH, EC 1.1.1.2). The results reveal that the factors which require careful consideration before development of a truly biospecific system based on the locking-on strategy include: (i) the stability of the immobilized cofactor derivative; (ii) the spacer-arm composition of the affinity derivative; (iii) the accessible immobilized cofactor concentration; (iv) the soluble locking-on ligand concentration; (v) the dissociation constant of locking-on ligand, and (vi) the identification and elimination of nonbiospecific interference. The S6-linked immobilized NAD+ derivative (synthesized with a hydrophilic spacer arm) proved to be the most suitable of the affinity adsorbents investigated in the present study for use with the locking-on strategy. This conclusion was based primarily on the observations that this affinity adsorbent was stable, retained cofactor activity with the "test" enzymes under study, and was not prone to nonbiospecific interactions. Using this immobilized derivative in conjunction with the locking-on strategy, alcohol dehydrogenase from Saccharomyces cerevisiae was purified to electrophoretic homogeneity in a single affinity chromatographic step.

Crystal structure of a thermophilic alcohol dehydrogenase substrate complex suggests determinants of substrate specificity and thermostability.

The crystal structure of a thermophilic alcohol dehydrogenase (TBAD) from Thermoanaerobacter brockii has been determined in a binary complex with sec-butanol as substrate to a resolution of 3.0 A. Van der Waals interactions of the carbon C1 atom of sec-butanol with atoms in His59, Ala85, Trp110, Asp150, and Leu294 account for the substrate preference of this enzyme for secondary over primary alcohols. A crevice from the surface to the active site provides access for substrates and products. This opening is lined with the hydrophobic residues Ile49, Leu107, Trp110, Tyr267, Leu294 as well as Cys283 and Met285 from another molecule within the tetrameric assembly. This might explain the tolerance of this enzyme toward organic solvents. The zinc ion occupies a position in the active site, which is too remote for direct interaction with the alcohol group. A mechanism is suggested whereby the introduction of NADP would trigger a displacement of the zinc ion to its catalytic site. Features important for the unusually high melting temperature of 98 degrees C are suggested by comparison to the crystal structure of a highly homologous mesophilic alcohol dehydrogenase from Clostridium beijerinckii (CBAD). The thermophilic enzyme has a more hydrophilic exterior, a more hydrophobic interior, a smaller surface area, more prolines, alanines, and fewer serines than CBAD. Furthermore, in the thermophilic enzyme the number of all types of intersubunit interactions in these tetrameric enzymes is increased: more salt bridges, hydrogen bonds, and hydrophobic interactions. All these effects combined can account for the higher melting temperature of the thermophilic enzyme.

NADP-dependent bacterial alcohol dehydrogenases: crystal structure, cofactor-binding and cofactor specificity of the ADHs of Clostridium beijerinckii and Thermoanaerobacter brockii.

We have determined the X-ray structures of the NADP(H)-dependent alcohol dehydrogenase of Clostridiim beijerinckii (CBADH) in the apo and holo-enzyme forms at 2.15 A and 2.05 A resolution, respectively, and of the holo-alcohol dehydrogenase of Thermoanaerobacter brockii (TBADH) at 2.5 A. These are the first structures of prokaryotic alcohol dehydrogenase to be determined as well as that of the first NADP(H)-dependent alcohol dehydrogenase. CBADH and TBADH 75% have sequence identity and very similar three-dimensional structures. Both are tetramers of 222 symmetry. The monomers are composed of two domains: a cofactor-binding domain and a catalytic domain. These are separated by a deep cleft at the bottom of which a single zinc atom is bound in the catalytic site. The tetramers are composed of two dimers, each structurally homologous to the dimer of alcohol dehydrogenases of vertebrates. The dimers form tetramers by means of contacts between surfaces opposite the interdomain cleft thus leaving it accessible from the surface of the tetramer. The tetramer encloses a large internal cavity with a positive surface potential. A molecule of NADP(H) binds in the interdomain cleft to the cofactor-binding domain of each monomer. The specificity of the two bacterial alcohol dehydrogenases toward NADP(H) is determined by residues Gly198, Ser199, Arg200 and Tyr218, with the latter three making hydrogen bonds with the 2'-phosphate oxygen atoms of the cofactor. Upon NADP(H) binding to CBADH, Tyr218 undergoes a rotation of approximately 120 degrees about chi1 which facilitates stacking interactions with the adenine moiety and hydrogen bonding with one of the phosphate oxygen atoms. In apo-CBADH the catalytic zinc is tetracoordinated by side-chains of residues Cys37, His59, Asp150 and Glu60; in holo-CBADH, Glu60 is retracted from zinc in three of the four monomers whereas in holo-TBADH, Glu60 does not participate in Zn coordination. In both holo-enzymes, but not in the apo-enzyme, residues Ser39 and Ser113 are in the second coordination sphere of the catalytic zinc. The carboxyl group of Asp150 is oriented with respect to the active carbon of NADP(H) so as to form hydrogen bonds with both pro-S and pro-R hydrogen atoms.

Enhanced thermal stability of Clostridium beijerinckii alcohol dehydrogenase after strategic substitution of amino acid residues with prolines from the homologous thermophilic Thermoanaerobacter brockii alcohol dehydrogenase.

A comparison of the three-dimensional structures of the closely related mesophilic Clostridium beijerinckii alcohol dehydrogenase (CBADH) and the hyperthermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) suggested that extra proline residues in TBADH located in strategically important positions might contribute to the extreme thermal stability of TBADH. We used site-directed mutagenesis to replace eight complementary residue positions in CBADH, one residue at a time, with proline. All eight single-proline mutants and a double-proline mutant of CBADH were enzymatically active. The critical sites for increasing thermostability parameters in CBADH were Leu-316 and Ser-24, and to a lesser degree, Ala-347. Substituting proline for His-222, Leu-275, and Thr-149, however, reduced thermal stability parameters. Our results show that the thermal stability of the mesophilic CBADH can be moderately enhanced by substituting proline at strategic positions analogous to nonconserved prolines in the homologous thermophilic TBADH. The proline residues that appear to be crucial for the increased thermal stability of CBADH are located at a beta-turn and a terminating external loop in the polypeptide chain. Positioning proline at the N-caps of alpha-helices in CBADH led to adverse effects on thermostability, whereas single-proline mutations in other positions in the polypeptide had varying effects on thermal parameters. The finding presented here support the idea that at least two of the eight extra prolines in TBADH contribute to its thermal stability.

Construction of an efficient Bacillus subtilis system for extracellular production of heterologous proteins.

An efficient expression/secretion vector, designated pM2Veg, was constructed for extracellular production of heterologous proteins in Bacillus subtilis. To construct pM2Veg, a synthetic cassette, the Veg cassette carrying: (1) the strong vegetative vegI promoter from B. subtilis, (2) the Escherichia coli lac operator, (3) the B. subtilis consensus ribosome-binding site, (4) the Staphylococcal protein A leader sequence, (5) a cloning region for insertion of foreign genes, (6) translational stop codons in all three reading frames, and (7) the gnt transcriptional terminator, was cloned into a derivative of the stable pRB373 B. subtilis/E. coli shuttle plasmid, the pM2 vector. The application of pM2Veg to effect secretory production of heterologous proteins was illustrated using two widely different proteins: the endoglucanase (Eng) encoded by the cenA gene of Cellulomonas fimi and human epidermal growth factor (hEGF). Levels of Eng and hEGF measured in culture supernatant samples of B. subtilis transformants harboring recombinant constructs formed between pM2Veg and the cenA and hEGF genes were 8.3 U ml-1 and 7.0 mg l-1, respectively. The Eng activity is more than four times higher than the yield from the best cenA recombinant construct previously reported, and the hEGF data represents the first successful expression of the factor in B. subtilis.

Insights into transition state stabilization of the beta-1,4-glycosidase Cex by covalent intermediate accumulation in active site mutants.

The catalytic mechanism of 'retaining' beta-glycosidases has been the subject of considerable interest and debate for many years. The visualization of a covalent glycosyl enzyme intermediate by X-ray crystallography was first accomplished with a saccharide substrate substituted with fluorine at its 2-position. The structure implicated major roles for residue His 205 and for the 2-hydroxyl position of the proximal saccharide in binding and catalysis. Here we have studied the kinetic behavior of various His 205 mutants. One of these mutants, a double mutant H205N/E127A, has been used to stabilize a covalent glycosyl-enzyme intermediate involving an unsubstituted sugar, permitting crystallographic analysis of the interactions between its 2-hydroxyl group and the enzyme.

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases.

The effects of temperature on the kinetic parameters kcat and Km, for three isolates of the highly conserved monomeric enzyme 3-phosphoglycerate kinase (PGK), were investigated in detail using a rapid automated kinetics apparatus. PGK was purified from the thermophilic bacterium Thermoanaerobacter sp. Rt8.G4 (optimum growth temperature 68 degrees C), the mesophile Zymomonas mobilis (optimum growth temperature 32 degrees C) and a second, unidentified, soil mesophile designated unid A (optimum growth temperature 27 degrees C). The kinetic behaviour with temperature of each PGK preparation was distinct, despite the conserved nature of the enzyme. The kcat values increased with temperature, but not as rapidly exponentially, as might be expected from the Arrhenius equation. Maximum kcat values were at much higher temperatures than the optimum growth temperatures for the mesophiles, but for the thermophile the temperature of maximum kcat was close to its optimum growth temperature. Km values were in general nearly constant through the lower temperature ranges, but increased substantially as the optimum temperature (highest kcat) was passed. Thermal irreversible denaturation of the PGK proteins was also investigated by measuring loss of activity over time. In a dilute buffer, Arrhenius plots for denaturation were linear, and the calculated apparent energy of activation (Eact) for denaturation for the thermophilic PGK was 600 kJ.mol-1, whereas for the mesophilic enzymes the values were 200-250 kJ.mol-1. In the presence of substrates, a considerable stabilization occurred, and in the case of the Z. mobilis enzyme, the apparent Eact was increased to 480 kJ.mol-1. A theoretical explanation for these observations is presented. Comparing the kinetics data with irreversible denaturation rates determined at relevant temperatures, it was clear that kcat values reached a maximum, and then decreased with higher temperature before irreversible denaturation had any significant influence.

The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins.

Allicin (thio-2-propene-1-sulfinic acid S-allyl ester) is the main biologically active component of garlic clove extracts. Its biological activity was attributed to either antioxidant activity or thiol disulfide exchange. Antioxidant properties of both allicin and its precursor, alliin (+S-allyl-L-cysteine sulfoxide), were investigated in the Fenton oxygen-radical generating system [H2O2-Fe(II)]. Using the spin trapping technique and ESR, it was found that both compounds possessed significant antioxidant activity. The reaction between allicin and L-cysteine was studied by 1H and 13C-NMR, and a S-thiolation product, S-allylmercaptocysteine, was identified. Allicin irreversibly inhibited SH-protease papain, NADP(+)-dependent alcohol dehydrogenase from Thermoanaerobium brockii (TBAD), and the NAD(+)-dependent alcohol dehydrogenase from horse liver (HLAD). All the three enzymes could be reactivated with thiol containing compounds. Papain could be reactivated with glutathione, TBAD with dithiothreitol or 2-mercaptoethanol (2-ME) but not by glutathione, while HLAD could be reactivated only with 2-ME. This study demonstrates that in addition to its antioxidant activity, the major biological effect of allicin should be attributed to its rapid reaction with thiol containing proteins.

Stability and oligosaccharide binding of the N1 cellulose-binding domain of Cellulomonas fimi endoglucanase CenC.

Differential scanning calorimetry has been used to study the thermal stability and oligosaccharide-binding thermodynamics of the N-terminal cellulose-binding domain of Cellulomonas fimi beta-1,4-glucanase CenC (CBDN1). CBDN1 has a relatively low maximum stability (delta Gmax = 33 kJ/mol = 216 J/residue at 1 degree C and pH 6.1) compared to other small single-domain globular proteins. The unfolding is fully reversible between pH 5.5 and 9 and in accordance with the two-state equilibrium model between pH 5.5 and 11. When the single disulfide bond in CBDN1 is reduced, the protein remains unfolded at all conditions, as judged by NMR spectroscopy. This indicates that the intramolecular cross-link makes a major contribution to the stability of CBDN1. The measured heat capacity change of unfolding (delta Cp = 7.5 kJ mol-1 K-1) agrees well with that calculated from the predicted changes in the solvent accessible nonpolar and polar surface areas upon unfolding. Extrapolation of the specific enthalpy and entropy of unfolding to their respective convergence temperature indicates that per residue unfolding energies for CBDN1, an isolated domain, are in accordance with those found by Privalov (1) for many single-domain globular proteins. DSC thermograms of the unfolding of CBDN1 in the presence of various concentrations of cellopentaose were fit to a thermodynamic model describing the linkage between protein-ligand binding and protein unfolding. A global two-dimensional minimization routine is used to regress the binding enthalpy, binding constant, and unfolding thermodynamics for the CBDN1-cellopentaose system. Extrapolated binding constants are in quantitative agreement with those determined by isothermal titration calorimetry at 35 degrees C.

Cloning and characterization of transcription of the xylAB operon in Thermoanaerobacter ethanolicus.

The genes encoding xylose isomerase (xylA) and xylulose kinase (xylB) from the thermophilic anaerobe Thermoanaerobacter ethanolicus were found to constitute an operon with the transcription initiation site 169 nucleotides upstream from the previously assigned (K. Dekker, H. Yamagata, K. Sakaguchi, and S. Udaka, Agric. Biol. Chem. 55:221-227, 1991) promoter region. The bicistronic xylAB mRNA was processed by cleavage within the 5'-terminal portion of the XylB-coding sequence. Transcription of xylAB was induced in the presence of xylose, and, unlike in all other xylose-utilizing bacteria studied, was not repressed by glucose. The existence of putative xyl operator sequences suggested that xylose utilization is controlled by a repressor-operator mechanism. The T. ethanolicus xylB gene coded for a 500-amino-acid-residue protein with a deduced amino acid sequence highly homologous to those of other XylBs. This is the first report of an xylB nucleotide sequence and an xyLAB operon from a thermophilic anaerobic bacterium.