Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated.

Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its role in the structure and function of this important enzyme in biomass deconstruction.

Structural basis for the De-N-acetylation of Poly-ß-1,6-N-acetyl-D-glucosamine in Gram-positive bacteria.

Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In staphylococci, partial de-N-acetylation of the exopolysaccharide poly-ß-1,6-N-acetyl-d-glucosamine (PNAG) by the extracellular protein IcaB is required for biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of IcaB from Ammonifex degensii (IcaBAd) has been determined to 1.7 Å resolution. The structure of IcaBAd reveals a (ß/α)7 barrel common to the family four carbohydrate esterases (CE4s) with the canonical motifs circularly permuted. The metal dependence of IcaBAd is similar to most CE4s showing the maximum rates of de-N-acetylation with Ni(2+), Co(2+), and Zn(2+). From docking studies with ß-1,6-GlcNAc oligomers and structural comparison to PgaB from Escherichia coli, the Gram-negative homologue of IcaB, we identify Arg-45, Tyr-67, and Trp-180 as key residues for PNAG binding during catalysis. The absence of these residues in PgaB provides a rationale for the requirement of a C-terminal domain for efficient deacetylation of PNAG in Gram-negative species. Mutational analysis of conserved active site residues suggests that IcaB uses an altered catalytic mechanism in comparison to other characterized CE4 members. Furthermore, we identified a conserved surface-exposed hydrophobic loop found only in Gram-positive homologues of IcaB. Our data suggest that this loop is required for membrane association and likely anchors IcaB to the membrane during polysaccharide biosynthesis. The work presented herein will help guide the design of IcaB inhibitors to combat biofilm formation by staphylococci.

Cloning, expression and characterization of a novel thermophilic polygalacturonase from Caldicellulosiruptor bescii DSM 6725.

We cloned the gene ACM61449 from anaerobic, thermophilic Caldicellulosiruptor bescii, and expressed it in Escherichia coli origami (DE3). After purification through thermal treatment and Ni-NTA agarose column extraction, we characterized the properties of the recombinant protein (CbPelA). The optimal temperature and pH of the protein were 72 °C and 5.2, respectively. CbPelA demonstrated high thermal-stability, with a half-life of 14 h at 70 °C. CbPelA also showed very high activity for polygalacturonic acid (PGA), and released monogalacturonic acid as its sole product. The Vmax and Km of CbPelA were 384.6 U·mg⁻¹ and 0.31 mg·mL⁻¹, respectively. CbPelA was also able to hydrolyze methylated pectin (48% and 10% relative activity on 20%-34% and 85% methylated pectin, respectively). The high thermo-activity and methylated pectin hydrolization activity of CbPelA suggest that it has potential applications in the food and textile industry.

Biochemical and structural insights into xylan utilization by the thermophilic bacterium Caldanaerobius polysaccharolyticus.

Hemicellulose is the next most abundant plant cell wall component after cellulose. The abundance of hemicellulose such as xylan suggests that their hydrolysis and conversion to biofuels can improve the economics of bioenergy production. In an effort to understand xylan hydrolysis at high temperatures, we sequenced the genome of the thermophilic bacterium Caldanaerobius polysaccharolyticus. Analysis of the partial genome sequence revealed a gene cluster that contained both hydrolytic enzymes and also enzymes key to the pentose-phosphate pathway. The hydrolytic enzymes in the gene cluster were demonstrated to convert products from a large endoxylanase (Xyn10A) predicted to anchor to the surface of the bacterium. We further use structural and calorimetric studies to demonstrate that the end products of Xyn10A hydrolysis of xylan are recognized and bound by XBP1, a putative solute-binding protein, likely for transport into the cell. The XBP1 protein showed preference for xylo-oligosaccharides as follows: xylotriose > xylobiose > xylotetraose. To elucidate the structural basis for the oligosaccharide preference, we solved the co-crystal structure of XBP1 complexed with xylotriose to a 1.8-Å resolution. Analysis of the biochemical data in the context of the co-crystal structure reveals the molecular underpinnings of oligosaccharide length specificity.

Effect of C-terminal truncation on enzyme properties of recombinant amylopullulanase from Thermoanaerobacter pseudoethanolicus.

The smallest and enzymatically active molecule, TetApuQ818, was localized within the C-terminal Q818 amino acid residue after serial C-terminal truncation analysis of the recombinant amylopullulanase molecule (TetApuM955) from Thermoanaerobacter pseudoethanolicus. Kinetic analyses indicated that the overall catalytic efficiency, k (cat)/K (m), of TetApuQ818 was 8-32% decreased for the pullulan and the soluble starch substrate, respectively. Changes to the substrate affinity, K (m), and the turnover rate, k (cat), were decreased significantly in both enzymatic activities of TetApuQ818. TetApuQ818 exhibited less thermostability than TetApuM955 when the temperature was raised above 85°C, but it had similar substrate-binding ability and hydrolysis products toward various substrates as TetApuM955 did. Both enzymes showed similar spectroscopies of fluorescence and circular dichroism, suggesting the active folding conformation was maintained after this C-terminal Q818 deletion. This study suggested that the binding ability of insoluble starch by TetApuM955 did not rely on the putative C-terminal carbohydrate binding module family 20 (CBM20) and two FnIII regions of TetApu, though the integrity of the AamyC module of TetApuQ818 was required for the enzyme activity.

Substrate-driven gene expression in Roseburia inulinivorans: importance of inducible enzymes in the utilization of inulin and starch.

Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene-expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide/inulin utilization genes induced during growth on inulin included one encoding a ß-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, whereas a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis showed that the ß-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked sugar phosphotransferase transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the use of inulin. The R. inulinivorans ß-fructofuranosidase was overexpressed in Escherichia coli and shown to hydrolyze fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosaccharides. Genes induced on starch included the major extracellular α-amylase and two distinct α-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles.

Immobilization of Thermoanaerobium brockii alcohol dehydrogenase on SBA-15.

The activity of Thermoanaerobium brockii alcohol dehydrogenase (TBADH) adsorbed on mesoporous silica SBA-15 was compared with that of the free enzyme in water and in biphasic system (water phase up to 50% v/v water). TBADH was active at a water concentration ≥10% v/v. In the reduction reaction of sulcatone to sulcatol carried out in biphasic systems, the yield obtained with SBA-15-adsorbed TBADH was up to 5.5-fold higher than that with the free enzyme, which suggests a higher stability of the immobilized enzyme toward the organic solvent. The nature of the organic solvent substantially influenced the degree of conversion that, for example, was 7.4% in toluene and 31.6% in petroleum ether.

Isolation and characterization of a multienzyme complex (cellulosome) of the Paenibacillus curdlanolyticus B-6 grown on Avicel under aerobic conditions.

A multienzyme complex, cellulosome, of the facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6 was produced on microcrystalline cellulose (Avicel) under aerobic conditions. During growth on Avicel, the bacterial cells were found to be capable of adhesion to Avicel by scanning electron microscopic (SEM) analysis. The multienzyme complex of P. curdlanolyticus B-6 was isolated from the crude enzyme preparation by gel filtration chromatography on Sephacryl S-300 and affinity purification on cellulose. The isolated multienzyme complex was able to bind to both Avicel and insoluble xylan and consists of cellulolytic and xylanolytic enzymes such as avicelase, carboxymethyl cellulase (CMCase), cellobiohydrolase, beta-glucosidase, xylanase, beta-xylosidase and alpha-l-arabinofuranosidase. The molecular mass of the complex was estimated to be 1600 kDa. It composed of at least 12 proteins on SDS-PAGE and 10 CMCases and 11 xylanases on zymograms. The isolated multienzyme complex could degrade the raw lignocellulosic substances effectively.

Cloning, sequencing, and expression of the gene encoding a multidomain endo-beta-1,4-xylanase from Paenibacillus curdlanolyticus B-6, and characterization of the recombinant enzyme.

The nucleotide sequence of the Paenibacillus curdlanolyticus B-6 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,828 nucleotides encoding a protein of 1,276 amino acids with a predicted molecular mass of 142,726 Da. Sequence analysis indicated that Xyn10A is a multidomain enzyme comprising nine domains in the following order: three family 22 carbohydrate-binding modules (CBMs), a family 10 catalytic domain of glycosyl hydrolases (xylanase), a family 9 CBM, a glycine-rich region, and three surface layer homology (SLH) domains. Xyn10A was purified from a recombinant Escherichia coli by a single step of affinity purification on cellulose. It could effectively hydrolyze agricultural wastes and pure insoluble xylans, especially low substituted insoluble xylan. The hydrolysis products were a series of short-chain xylooligosaccharides, indicating that the purified enzyme was an endo-beta-1,4-xylanase. Xyn10A bound to various insoluble polysaccharides including Avicel, alpha-cellulose, insoluble birchwood and oat spelt xylans, chitin, and starches, and the cell wall fragments of P. curdlanolyticus B-6, indicating that both the CBM and the SLH domains are fully functioning in the Xyn10A. Removal of the CBMs from Xyn10A strongly reduced the ability of plant cell wall hydrolysis. These results suggested that the CBMs of Xyn10A play an important role in the hydrolysis of plant cell walls.

Paenibacillus thailandensis sp. nov. and Paenibacillus nanensis sp. nov., xylanase-producing bacteria isolated from soil.

Two strains of xylanase-producing bacteria, S3-4A(T) and MX2-3(T), isolated from soils in Thailand, were characterized on the basis of their phenotypic and chemotaxonomic characteristics, DNA-DNA relatedness and 16S rRNA gene sequences. The novel strains were Gram-positive, facultatively anaerobic, spore-forming, rod-shaped bacteria. They contained meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan. The DNA G+C contents of strains S3-4A(T) and MX2-3(T) were 52.7 and 52.9 mol%, respectively. The major isoprenoid quinone was MK-7. The dominant cellular fatty acids were anteiso-C(15 : 0) and iso-C(16 : 0). Phylogenetic analyses using 16S rRNA gene sequences showed that both novel strains were affiliated to the genus Paenibacillus. Strains S3-4A(T) and MX2-3(T) were closely related to Paenibacillus agaridevorans DSM 1355(T) with 97 % and 97.3 % gene sequence similarities, respectively. The DNA-DNA relatedness between strains S3-4A(T), MX2-3(T) and P. agaridevorans DSM 1355(T) was low (6.0-30.3 %). The novel strains could be clearly distinguished from P. agaridevorans DSM 1355(T) by physiological and biochemical characteristics. Therefore, these two strains represent novel species of the genus Paenibacillus, for which the names Paenibacillus thailandensis sp. nov. (type strain S3-4A(T)=KCTC 13043(T)=PCU 275(T)=TISTR 1827(T)) and Paenibacillus nanensis sp. nov. (type strain MX2-3(T)=KCTC 13044(T)=PCU 276(T)=TISTR 1828(T)) are proposed.

Paenibacillus siamensis sp. nov., Paenibacillus septentrionalis sp. nov. and Paenibacillus montaniterrae sp. nov., xylanase-producing bacteria from Thai soils.

Three strains of xylanase-producing bacteria, S5-3(T), X13-1(T) and MXC2-2(T), isolated from soils in Thailand, were characterized taxonomically based on their phenotypic and chemotaxonomic characteristics and 16S rRNA gene sequence comparisons. They were Gram-positive, facultatively anaerobic, spore-forming, rod-shaped bacteria. They contained meso-diaminopimelic acid in the cell-wall peptidoglycan. The DNA G+C contents were respectively 45.8, 47.3 and 48.8 mol%. The predominant isoprenoid quinone was MK-7, and anteiso-C(15 : 0) and C(16 : 0) were the dominant cellular fatty acids. Phylogenetic analyses based on 16S rRNA gene sequence comparison showed that they were affiliated to the genus Paenibacillus. Strains S5-3(T), X13-1(T) and MXC2-2(T) were closely related to Paenibacillus granivorans A30(T) and Paenibacillus agaridevorans DSM 1355(T) (94.7-98.6 % 16S rRNA gene sequence similarity). The similarity between the three novel strains ranged from 96.3 to 98.4 %. DNA-DNA relatedness, DNA G+C contents and some phenotypic characteristics could clearly distinguish the strains from each other and from related Paenibacillus species. Therefore, strains S5-3(T), X13-1(T) and MXC2-2(T) represent novel species of the genus Paenibacillus, for which the names Paenibacillus siamensis sp. nov. (type strain S5-3(T) =KCTC 13038(T) =PCU 279(T) =TISTR 1831(T)), Paenibacillus septentrionalis sp. nov. (type strain X13-1(T) =KCTC 13039(T) =PCU 280(T) =TISTR 1830(T)) and Paenibacillus montaniterrae sp. nov. (type strain MXC2-2(T) =KCTC 13036(T) =PCU 281(T) =TISTR 1836(T)) are proposed.

Effects of organic solvents on immobilized lipase in pectin microspheres.

Lipase from Brevibacillus agri 52 was found stable up to 90% diethylenglycol (DEG), glycerol (GLY), and 1,2 propanediol (1,2 PRO) at 37 degrees C for 1 h and the stability was reduced only approximately 20% after 12 h incubation, but in 40% dimethylsulfoxide (DMSO), lipase activity was stable only for 1 h. Inhibition of the biocatalysts with dimethylformamide (DMF) was detected at 20% solvent concentration. In water immiscible systems, the stability of lipase in n-hexane, n-tetradecane and n-heptane resembles the water activity, but in the presence of isobutanol, 1-hexanol, and butylbutirate, the stability was significantly reduced. Lipase 52 precipitates in the presence of 50% acetone or ethanol/water mixtures, but enzymatic activity was partially recovered by adding 20% GLY, DEG, 1,2 PRO, or DMSO to the reaction mixture. Furthermore, by increasing DEG in 70% DMF/DEG mixtures, the lipase activity was protected. Encapsulation of lipase in pectin gels cross-linked with calcium ions brings three to four times more enzymatic activity in 70% water miscible organic solvents compared to aqueous systems.

Cyclodextrin production and genetic characterization of cyclodextrin glucanotranferase of Paenibacillus graminis.

Paenibacillus graminis strains were described recently as cyclodextrin (CD) producers. Cyclodextrins are produced by cyclodextrin glucanotransferase (CGTase) which has not been characterized in P. graminis. Similar amounts of alpha- and beta-CDs were produced by P. graminis (MC22.13) and P. macerans (LMD24.10(T)). Primers were designed to sequence the gene encoding CGTase from P. graminis. A phylogenetic tree was constructed and P. graminis CGTase protein showed to be closer (79.4% protein identity) to P. macerans |P31835|. Hybridization studies suggested that the gene encoding CGTase is located in different positions in the genomes of P. macerans and P. graminis.

Functional and structural features of the oxyanion hole in a thermophilic esterase from Alicyclobacillus acidocaldarius.

Recent mutagenic and molecular modelling studies suggested a role for glycine 84 in the putative oxyanion loop of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius. A 114 times decrease of the esterase catalytic activity of the G84S mutant was observed, without changes in the thermal stability. The recently solved three-dimensional (3D) structure of EST2 in complex with a HEPES molecule permitted to demonstrate that G84 (together with G83 and A156) is involved in the stabilization of the oxyanion through a hydrogen bond from its main chain NH group. The structural data in this case did not allowed us to rationalize the effect of the mutation, since this hydrogen bond was predicted to be unaltered in the mutant. Since the mutation could shed light on the role of the oxyanion loop in the HSL family, experiments to elucidate at the mechanistic level the reasons of the observed drop in k (cat) were devised. In this work, the kinetic and structural features of the G84S mutant were investigated in more detail. The optimal temperature and pH for the activity of the mutated enzyme were found significantly changed (T = 65 degrees C and pH = 5.75). The catalytic constants K (M) and V(max) were found considerably altered in the mutant, with ninefold increased K (M) and 14-fold decreased V(max), at pH 5.75. At pH 7.1, the decrease in k (cat) was much more dramatic. The measurement of kinetic constants for some steps of the reaction mechanism and the resolution of the mutant 3D structure provided evidences that the observed effects were partly due to the steric hindrance of the S84-OH group towards the ester substrate and partly to its interference with the nucleophilic attack of a water molecule on the second tetrahedral intermediate.

Heterologous expression of a gene encoding a thermostable beta-galactosidase from Alicyclobacillus acidocaldarius.

A genomic DNA library screen yielded the nucleotide sequence of a 12 kb fragment containing a gene (2067 bp) coding a thermostable beta-galactosidase from Alicyclobacillus acidocaldarius ATCC 27009. The beta-galactosidase gene was expressed in Pichia pastoris, and up to 90 mg recombinant beta-galactosidase/l accumulated in shake flask cultures. Using o-nitrophenyl-beta-D: -galactopyranoside as a substrate, the optimum pH and temperature of the purified recombinant beta-galactosidase were 5.8-6.0 and 70 degrees C, respectively. The enzyme retained 90% of its activity when heated at 70 degrees C for 30 min. Approximately 48% of lactose in milk was hydrolyzed following treatment with the recombinant enzyme over 60 min at 65 degrees C.

Na+-pyrophosphatase: a novel primary sodium pump.

Membrane-bound pyrophosphatase (PPase) is commonly believed to couple pyrophosphate (PPi) hydrolysis to H+ transport across the membrane. Here, we demonstrate that two newly isolated bacterial membrane PPases from the mesophile Methanosarcina mazei (Mm-PPase) and the moderate thermophile Moorella thermoacetica and a previously described PPase from the hyperthermophilic bacterium Thermotoga maritima catalyze Na+ rather than H+ transport into Escherichia coli inner membrane vesicles (IMV). When assayed in uncoupled IMV, the three PPases exhibit an absolute requirement for Na+ but display the highest hydrolyzing activity in the presence of both Na+ and K+. Steady-state kinetic analysis of PPi hydrolysis by Mm-PPase revealed two Na+ binding sites. One of these sites can also bind K+, resulting in a 10-fold increase in the affinity of the other site for Na+ and a 2-fold increase in maximal velocity. PPi-driven 22Na+ transport into IMV containing Mm-PPase was unaffected by the protonophore carbonyl cyanide m-chlorophenylhydrazone, inhibited by the Na+ ionophore monensin, and activated by the K+ ionophore valinomycin. The Na+ transport was accompanied by the generation of a positive inside membrane potential as reported by Oxonol VI. These findings define Na+-dependent PPases as electrogenic Na+ pumps. Phylogenetic analysis suggests that ancient gene duplication preceded the split of Na+- and H+-PPases.

Isolation and identification of an endophytic strain EJS-3 producing novel fibrinolytic enzymes.

An endophytic strain EJS-3, which produces a novel fibrinolytic enzyme, was screened from root tissue of Stemona japonica (Blume) Miq, a chinese traditional medicine. This strain was identified as Paenibacillus polymyxa (DQ120522) by morphological, physiological, and biochemical tests and 16S rRNA gene sequence analysis. Two serine-type fibrinolytic enzymes with a relative molecular weight about 118 and 49 kDa, respectively, which are larger than known fibrinolytic enzymes, were found by the SDS-fibrin zymogram or by fibrin-inhibitor zymography gels. No work on P. polymyxa-producing fibrinolytic enzymes has been reported.

Role of the N-terminal region for the conformational stability of esterase 2 from Alicyclobacillus acidocaldarius.

In order to clarify the role played by the N-terminal region for the conformational stability of the thermophilic esterase 2 (EST2) from Alicyclobacillus acidocaldarius, two mutant forms have been investigated: a variant obtained by deleting the first 35 residues at the N-terminus (EST2-36del), and a variant obtained by mutating Lys102 to Gln (K102Q) to perturb the N-terminus by destroying the salt bridge E43-K102. The temperature- and denaturant-induced unfolding of EST2 and the two mutant forms have been studied by means of circular dichroism (CD), differential scanning calorimetry (DSC) and fluorescence measurements. In line with its thermophilic origin, the denaturation temperature of EST2 is high: T(d)=91 degrees C and 86 degrees C if detected by recording the CD signal at 222 nm and 290 nm, respectively. This difference suggests that the thermal denaturation process, even though reversible, is more complex than a two-state Nright arrow over left arrowD transition. The non-two-state behaviour is more pronounced in the case of the two mutant forms. The complex DSC profiles of EST2 and both mutant forms have been analysed by means of a deconvolution procedure. The thermodynamic parameters characterizing the two transitions obtained in the case of EST2 are: T(d,1)=81 degrees C, Delta(d)H(1)=440 kJ mol(-1), Delta(d)C(p,1)=7 kJ K(-1)mol(-1), T(d,2)=86 degrees C, Delta(d)H(2)=710 kJ mol(-1), and Delta(d)C(p,2)=9 kJ K(-1)mol(-1). The first transition occurs at lower temperatures in the two mutant forms, whereas the second transition is always centred at 86 degrees C. The results indicate that EST2 possesses two structural domains whose coupling is tight in the wild-type protein, but markedly weakens in the two mutant forms as a consequence of the perturbations in the N-terminal region.

New type of kdp region with a split sensor-kinase kdpD gene located within two divergent kdp operons from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius.

The kdp region from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius consists of two divergent operons: kdpZFABCN, which is tenfold induced at low K+ concentrations and encodes the K+-translocating P-type ATPase KdpZFABC as well as KdpN, a novel covalent homo-dimer of the cytoplasmic N-terminal part from sensor kinase KdpD; and secondly, the constitutively expressed kdpHE operon, encoding the remainder of KdpD and the response regulator KdpE.

Xyn11A, a multidomain multicatalytic enzyme from Pseudobutyrivibrio xylanivorans Mz5T.

The rumen bacterium Pseudobutyrivibrio xylanivorans Mz5T has a potent xylanolytic enzyme system. A small native peptide (approximately 30-kDa, designated Xyn11A) from the bacterium was first isolated and characterized by Edman degradation. The gene coding for Xyn11A was identified using PCR amplification with consensus primers. It was then fully sequenced to reveal an open reading frame of 1809 bp. The predicted N-terminal domain exhibited xylanolytic activity and was classed to the family 11 of glycosyl hydrolases; it is followed by a region with homology to a family 6 cellulose binding module. The C-terminal domain codes for a putative NodB-like polysaccharide deacetylase which is predicted to be an acetyl esterase implicated in debranching activity in the xylan backbone. As similar domain organization was also found in several other xylanases from a diverse range of bacteria, a common ancestor of such a xylanase is considered to be present and spread, possibly by horizontal gene transfer, to other microorganisms from different ecological niches.