Cellulose-binding Protein Research Articles

Secretory protein Rv1987, a ‘probable chitinase’ from Mycobacterium tuberculosis is a novel chitin and cellulose binding protein lacking enzymatic function

Bacterial chitinases serve to hydrolyse chitin as food source or as defence mechanism. Given that chitin is not produced by mammals, it is intriguing that Mycobacterium tuberculosis, an exclusively human pathogen harbours Rv1987, a probable chitinase and secretes it. Interestingly genes annotated as chitinases are widely distributed among Mycobacterium tuberculosis complex species, clinical isolates and other human pathogens M. abscessus and M. ulcerans. However, Mycobacterial chitinases are not characterized and hence the functions remain unknown. In the present study, we show that Rv1987 is a chitin and cellulose binding protein lacking enzymatic activity in contrary to its current annotation. Further, we show Rv1987 has moon lighting functions in M. tuberculosis pathobiology signifying roles of bacterial cellulose binding clusters in infections.

Addition of Various Cellulosic Components to Bacterial Nanocellulose: A Comparison of Surface Qualities and Crystalline Properties.

Bacterial nanocellulose (BNC) is a biocompatible material with a lot of potential. To make BNC commercially feasible, improvements in its production and surface qualities must be made. Here, we investigated the in situ fermentation and generation of BNC by addition of different cellulosic substrates such as Avicel and carboxymethylcellulose (CMC) and using Komagataeibacter sp. SFCB22-18. The addition of cellulosic substrates improved BNC production by a maximum of about 5 times and slightly modified its structural properties. The morphological and structural properties of BNC were investigated by using Fourier transform-infrared spectroscopy (FT-IR), scanning electron microscopy and X-ray diffraction. Furthermore, a type-A cellulose-binding protein derived from Clostridium thermocellum, CtCBD3, was used in a novel biological analytic approach to measure the surface crystallinity of the BNC. Because Avicel and CMC may adhere to microfibrils during BNC synthesis or crystallization, cellulose-binding protein could be a useful tool for identifying the crystalline properties of BNC with high sensitivity.

A sensitive paper-based lateral flow immunoassay platform using engineered cellulose-binding protein linker fused with antibody-binding domains

An advanced paper-based LFIA using engineered cellulose-binding protein linker fused with antibody-binding domains was developed to resolve problems associated with conventional LFIA strip, including the limitation of quantitative analysis and low sensitivity. CBP31-BC linker was prepared by genetically fusing the cellulose-binding modules of family 3 (CBM3) and family 1 (CBM1) with antibody-binding B and C domains of Protein A. Cellulose-binding analysis showed that the addition of two CBMs led to high cellulose-binding capacity in CBP31-BC. Transmission electron microscopy and quartz crystal microbalance analyses demonstrated that the cellulose-binding ability of CBP31-BC enabled the BC domain to be efficiently exposed, allowing about 6-fold higher antibody-binding efficiency (∼32.5 %) than that (∼5.5 %) of sole BC. To evaluate the feasibility of the CBP31-BC linker-employed LFIA platform, an immunoassay of prostate-specific antigen (PSA) was performed as a model reaction. The CBP31-BC-employed paper-based LFIA detected PSA at levels as low as 0.25 ng/mL in 20 min, which is about 10-fold more sensitive to conventional LFIAs that use simple physical adsorption. CBP31-BC-employed LFIA also exhibited a lower detection range of 0.25–2.5 ng/mL when compared to conventional LFIAs. These results might be due to efficient oriented immobilization of the capture antibody by CBP31-BC linker on cellulose membrane. Thus, our developed CBP31-BC linker-employed paper-based LFIA platform holds great promise for the detection of target markers due to significantly enhanced sensitivity.

In Silico Characterization of Meloidogyne Genus Nematode Cellulose Binding Proteins

Abstract Root-knot nematodes are a group of endoparasites species that induce the formation of giant cells in the hosts, by which they guarantee their feeding and development. Meloidogyne species infect over 2000 plant species, and are highly destructive, causing damage to many crops around the world. M. enterolobii is considered the most aggressive species in tropical regions, such as Africa and South America. Phytonematodes are able to penetrate and migrate within plant tissues, establishing a sophisticated interaction with their hosts through parasitism factors, which include a series of cell wall degradation enzymes and plant cell modification. Among the parasitism factors documented in the M. enterolobii species, cellulose binding protein (CBP), a nematode excretion protein that appears to be associated with the breakdown of cellulose present in the plant cell wall. In silico analysis can be of great importance for the identification, structural and functional characterization of genomic sequences, besides making possible the prediction of structures and functions of proteins. The present work characterized 12 sequences of the CBP protein of nematodes of the genus Meloidogyne present in genomic databases. The results showed that all CBP sequences had signal peptide and that, after their removal, they had an isoelectric point that characterized them as unstable in an acid medium. The values of the average hydrophilicity demonstrated the hydrophilic character of the analyzed sequences. Phylogenetic analyzes were also consistent with the taxonomic classification of the nematode species of this study. Five motifs were identified, which are present in all sequences analyzed. These results may provide theoretical grounds for future studies of plant resistance to nematode infection.

Thermodynamics of adsorption on nanocellulose surfaces

Understanding the thermodynamic interactions of cellulose nanomaterials with their environment is important to understand the forces behind their self-organization and co-organisation with other compounds, and to be able to use self-assembly to form new functional multicomponent materials. This review analyzes published studies that determined the thermodynamic parameters of the surface interactions of and adsorption of various compounds (proteins, polymers, and small molecules/ions) onto cellulose nanomaterials. We compiled the data reported and performed a meta-analysis for better comparison and to find trends in the published data. We first introduce the methods employed and describe the adsorption isotherm models typically used to describe the adsorption thermodynamics on nanocellulose surfaces. We then discuss and analyze the published results for the interaction of the various compounds with nanocellulose surfaces. The systems that have been reported on most were adsorption of natural binding proteins and various pollutants from water, such as heavy metal ions, dyes, and drugs. Interactions between cellulose surfaces and the cellulose binding module were generally both enthalpy- and entropy-driven, where the negative binding enthalpy indicates the formation of specific interactions between peptides and the carbohydrate backbone. On the other hand, interactions with charged molecules were mostly endothermic and purely entropy-driven, indicating that the adsorption on nanocellulose surfaces can be described as an interaction between opposite charges, where the entropy increase that arises from the release of surface-structured water molecules and counterions from the electronic double layer supplies the major contribution to the free energy of adsorption. We performed a meta-analysis on all published data, and found a linear relationship between ∆H and ∆S with the slope equal to the reference temperature, irrespective of whether the interacting compound is a specific cellulose binding protein, a non-specific binding protein, a polymer, or a small molecule/ion. This indicates that the process of adsorption is the same for all compounds and takes place with a constant change in Gibbs free energy of interaction, ∆G, where a change in interaction enthalpy is offset by change in entropy change upon binding and vice versa.

The Functional Characterization of Podosphaera xanthii Candidate Effector Genes Reveals Novel Target Functions for Fungal Pathogenicity.

Podosphaera xanthii is the main causal agent of powdery mildew disease in cucurbits. In a previous study, we determined that P. xanthii expresses approximately 50 Podosphaera effector candidates (PECs), identified based on the presence of a predicted signal peptide and the absence of functional annotation. In this work, we used host-induced gene silencing (HIGS), employing Agrobacterium tumefaciens as a vector for the delivery of the silencing constructs (ATM-HIGS), to identify genes involved in early plant-pathogen interaction. The analysis of seven selected PEC-encoding genes showed that six of them, PEC007, PEC009, PEC019, PEC032, PEC034, and PEC054, are required for P. xanthii pathogenesis, as revealed by reduced fungal growth and increased production of hydrogen peroxide by host cells. In addition, protein models and protein-ligand predictions allowed us to identify putative functions for these candidates. The biochemical activities of PEC019, PEC032, and PEC054 were elucidated using their corresponding proteins expressed in Escherichia coli. These proteins were confirmed as phospholipid-binding protein, α-mannosidase, and cellulose-binding protein. Further, BLAST searches showed that these three effectors are widely distributed in phytopathogenic fungi. These results suggest novel targets for fungal effectors, such as host-cell plasma membrane, host-cell glycosylation, and damage-associated molecular pattern-triggered immunity.

Ethylenediamine pretreatment of corn stover facilitates high gravity fermentation with low enzyme loading

This work investigated the effect of ethylenediamine pretreatment on reducing enzyme loading in high gravity fermentation. At optimal conditions of ethylenediamine pretreatment, 85.5% lignin was removed. Enzyme adsorption analysis using a fluorescent cellulose-binding protein showed 35.2% increase of productive adsorption of enzymes to ethylenediamine pretreated biomass, which was caused by high delignification and dramatically increased surface roughness and porosity. In SScF at 15% glucan loading, up to 82.2 g/L ethanol was achieved with a relatively low enzyme loading of 3.6 FPU/g dry matter. It suggested that the remarkably high digestibility of EDA pretreated corn stover could effectively reduce the enzyme loading in the high gravity fermentation of cellulosic ethanol.

Visualizing cellulase adsorption and quantitatively determining cellulose accessibility with an updated fungal cellulose-binding module-based fluorescent probe protein

BackgroundCellulose accessibility to cellulases (CAC) is a direct factor determining the enzymatic digestibility of lignocellulosic cellulose. Improving CAC by pretreatment is a prerequisite step for the efficient release of fermentable sugars from biomass cell wall. However, conventional methods to study the porosimetry of solid materials showed some limitations to be used for investigating CAC. In this work, an updated novel fusion protein comprising a fungal cellulose-binding module (CBM) from Cel7A cellobiohydrolase I (CBH I) of Trichoderma reesei QM6 and a di-green fluorescent protein (GFP2) was constructed for quantitative determination of CAC.ResultsThe obtained probe protein had similar molecular size (e.g., weight) with that of Cel7A and could give detectable signal for quantitative analysis. Several construction strategies were compared with regard to the site of His-tag and order of CBM and GFP2 modules in the protein sequence, in order to achieve good expression quantity and usability of the probe protein. His6–CBM–GFP2 has been identified as the best probe protein for investigating the effects of structural features of cellulosic substrates on cellulose accessibility. Substrate samples with different contents of xylan, lignin, and degree of substitution of cellulose –OH by formyl group were obtained, respectively, by mild H2SO4 pre-hydrolysis, NaClO2 selective delignification, and treatment of filter paper cellulose with concentrated formic acid. The determined CAC was in a wide range of 0.6–20.4 m2/g depending on the contents of hemicelluloses, lignin, and formyl group as well as cellulose degree of crystallization.ConclusionsThe obtained fusion probe protein could be used as a versatile tool to quantitatively investigate the impacts of biomass structural features on CAC and hydrolyzability of cellulose substrates, as well as nonproductive adsorption of cellulase enzymes on lignin.

Steered molecular dynamics simulations reveal the role of Ca2+ in regulating mechanostability of cellulose-binding proteins.

The conversion of cellulosic biomass into biofuels requires degradation of the biomass into fermentable sugars. The most efficient natural cellulase system for carrying out this conversion is an extracellular multi-enzymatic complex named the cellulosome. In addition to temperature and pH stability, mechanical stability is important for functioning of cellulosome domains, and experimental techniques such as Single Molecule Force Spectroscopy (SMFS) have been used to measure the mechanical strength of several cellulosomal proteins. Molecular dynamics computer simulations provide complementary atomic-resolution quantitative maps of domain mechanical stability for identification of experimental leads for protein stabilization. In this study, we used multi-scale steered molecular dynamics computer simulations, benchmarked against new SMFS measurements, to measure the intermolecular contacts that confer high mechanical stability to a family 3 Carbohydrate Binding Module protein (CBM3) derived from the archetypal Clostridium thermocellum cellulosome. Our data predicts that electrostatic interactions in the calcium binding pocket modulate the mechanostability of the cellulose-binding module, which provides an additional design rule for the rational re-engineering of designer cellulosomes for biotechnology. Our data offers new molecular insights into the origins of mechanostability in cellulose binding domains and gives leads for synthesis of more robust cellulose-binding protein modules. On the other hand, simulations predict that insertion of a flexible strand can promote alternative unfolding pathways and dramatically reduce the mechanostability of the carbohydrate binding module, which gives routes to rational design of tailormade fingerprint complexes for force spectroscopy experiments.

The elution of certain protein affinity tags with millimolar concentrations of diclofenac

Diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid) is a sparingly soluble, nonsteroidal anti-inflammatory drug therapeutically acting at low micromolar concentrations. In pH range from 8 to 11, its aqueous solubility can be increased up to 200 times by the presence of counter ions such as sodium. Our protein interaction studies revealed that a millimolar concentration of sodium diclofenac is able to elute glutathione S-transferase (GST), cellulose binding protein (CBD), and maltose binding protein (MBP) but not histidine-tagged or PDZ-tagged proteins from their affinity resins. The elution efficiency of diclofenac is comparable with the eluting agents normally used at similar concentrations. Native gel electrophoresis of sodium diclofenac-treated proteins showed that the interaction is non-covalent and non-denaturing. These results suggest that sodium diclofenac, in addition to its pharmaceutical applications, can also be exploited as a lead for the development of new proteomics reagents.

Expression, purification, crystallization and preliminary X-ray analysis of CttA, a putative cellulose-binding protein from Ruminococcus flavefaciens.

A number of anaerobic microorganisms produce multi-modular, multi-enzyme complexes termed cellulosomes. These extracellular macromolecular nanomachines are designed for the efficient degradation of plant cell-wall carbohydrates to smaller sugars that are subsequently used as a source of carbon and energy. Cellulolytic strains from the rumens of mammals, such as Ruminococcus flavefaciens, have been shown to have one of the most complex cellulosomal systems known. Cellulosome assembly requires the binding of dockerin modules located in cellulosomal enzymes to cohesin modules located in a macromolecular scaffolding protein. Over 220 genes encoding dockerin-containing proteins have been identified in the R. flavefaciens genome. The dockerin-containing enzymes can be incorporated into the primary scaffoldin (ScaA), which in turn can bind to adaptor scaffoldins (ScaB or ScaC) and subsequently to anchoring scaffoldin (ScaE), thereby attaching the whole complex to the cell surface. However, unlike other cellulosomes such as that from Clostridium thermocellum, the Ruminococcus species lack a specific carbohydrate-binding module (CBM) on ScaA which recruits the entire complex onto the surface of the substrate. Instead, a cellulose-binding protein, CttA, comprising two putative tandem novel carbohydrate-binding modules and a C-terminal X-dockerin module, which can bind to the cohesin of ScaE, may mediate the attachment of bacterial cells to cellulose. Here, the expression, purification and crystallization of the carbohydrate-binding modular part of the CttA from R. flavefaciens are described. X-ray data have been collected to resolutions of 3.23 and to 1.61 Å in space groups P3(1)21 or P3(2)21 and P2(1), respectively. The structure was phased using bound iodide from the crystallization buffer by SAD experiments.

Hydrolytic effects of scaffolding proteins CbpB and CbpC on crystalline cellulose mediated by the major cellulolytic complex from Clostridium cellulovorans

The role of the scaffolding proteins, cellulose binding protein B and C (CbpB and CbpC, respectively) were identified in cellulolytic complex (cellulosome) of Clostridium cellulovorans for efficient degradation of cellulose. Recombinant CbpB and CbpC directly anchored to the cell surface of C. cellulovorans. In addition, CbpB and CbpC showed increased hydrolytic activity on crystalline cellulose incubated with exoglucanase S (ExgS) and endoglucanase Z (EngZ) compared with the activity of free enzymes. Moreover, the results showed synergistic effects of crystalline cellulose hydrolytic activity (1.8- to 2.2-fold) when CbpB and CbpC complex with ExgS and EngZ are incubated with cellulolytic complex containing mini-CbpA. The results suggest C. cellulovorans critically uses CbpB and CbpC, which can directly anchor cells for the hydrolysis of cellulosic material with the major cellulosome complex.

Duplication of Genes in an ATP-binding Cassette Transport System Increases Dynamic Range While Maintaining Ligand Specificity

Many bacteria exist in a state of feast or famine where high nutrient availability leads to periods of growth followed by nutrient scarcity and growth stagnation. To adapt to the constantly changing nutrient flux, metabolite acquisition systems must be able to function over a broad range. This, however, creates difficulties as nutrient concentrations vary over many orders of magnitude, requiring metabolite acquisition systems to simultaneously balance ligand specificity and the dynamic range in which a response to a metabolite is elicited. Here we present how a gene duplication of a periplasmic binding protein in a mannose ATP-binding cassette transport system potentially resolves this dilemma through gene functionalization. Determination of ligand binding affinities and specificities of the gene duplicates with fluorescence and circular dichroism demonstrates that although the binding specificity is maintained the Kd values for the same ligand differ over three orders of magnitude. These results suggest that this metabolite acquisition system can transport ligand at both low and high environmental concentrations while preventing saturation with related and less preferentially metabolized compounds. The x-ray crystal structures of the β-mannose-bound proteins help clarify the structural basis of gene functionalization and reveal that affinity and specificity are potentially encoded in different regions of the binding site. These studies suggest a possible functional role and adaptive advantage for the presence of two periplasmic-binding proteins in ATP-binding cassette transport systems and a way bacteria can adapt to varying nutrient flux through functionalization of gene duplicates.

A new family of β-helix proteins with similarities to the polysaccharide lyases.

Microorganisms that degrade biomass produce diverse assortments of carbohydrate-active enzymes and binding modules. Despite tremendous advances in the genomic sequencing of these organisms, many genes do not have an ascribed function owing to low sequence identity to genes that have been annotated. Consequently, biochemical and structural characterization of genes with unknown function is required to complement the rapidly growing pool of genomic sequencing data. A protein with previously unknown function (Cthe_2159) was recently isolated in a genome-wide screen using phage display to identify cellulose-binding protein domains from the biomass-degrading bacterium Clostridium thermocellum. Here, the crystal structure of Cthe_2159 is presented and it is shown that it is a unique right-handed parallel β-helix protein. Despite very low sequence identity to known β-helix or carbohydrate-active proteins, Cthe_2159 displays structural features that are very similar to those of polysaccharide lyase (PL) families 1, 3, 6 and 9. Cthe_2159 is conserved across bacteria and some archaea and is a member of the domain of unknown function family DUF4353. This suggests that Cthe_2159 is the first representative of a previously unknown family of cellulose and/or acid-sugar binding β-helix proteins that share structural similarities with PLs. Importantly, these results demonstrate how functional annotation by biochemical and structural analysis remains a critical tool in the characterization of new gene products.

Solid state NMR chemical shift assignment and conformational analysis of a cellulose binding protein facilitated by optimized glycerol enrichment.

Magic-angle spinning solid-state NMR has been applied to study CBM3b-Cbh9A (CBM3b), a cellulose binding module protein belonging to family 3b. It is a 146-residue protein having a unique nine-stranded β-sandwich fold, in which 35% of the structure is in a β-sheet conformation and the remainder of the protein is composed of loops and unstructured regions. Yet, the protein can be crystalized and it forms elongated needles. Close to complete chemical shift assignment of the protein was obtained by combining two- and three-dimensional experiments using a fully labeled sample and a glycerol-labeled sample. The use of an optimized protocol for glycerol-based sparse labeling reduces sample preparation costs and facilitates the assignment of the large number of aromatic signals in this protein. Conformational analysis shows good correlation between the NMR-predicted secondary structure and the reported X-ray crystal structure, in particular in the structured regions. Residues which show high B-factor values are situated mainly in unstructured regions, and are missing in our spectra indicating conformational flexibility rather than heterogeneity. Interestingly, long-range contacts, which could be clearly detected for tyrosine residues, could not be observed for aromatic phenylalanine residues pointing into the hydrophobic core, suggesting possible high ring mobility. These studies will allow us to further investigate the cellulose-bound form of CBM proteins.

Characterization of a potential Listeria monocytogenes virulence factor associated with attachment to fresh produce.

A study to determine the attachment of L. monocytogenes serotype 4b strain F2365 on vegetables and fruits was conducted. In an initial study, we screened 32 genes encoding surface proteins and lipases of the strain to find highly expressed genes on lettuce leaves. The results showed that transcription levels of LMOf2365_0413, LMOf2365_0498, LMOf2365_0859, LMOf2365_2052, and LMOf2365_2812 were significantly upregulated on lettuce leaves. In silico analysis showed that LMOf2365_0859 contains a putative cellulose binding domain. Thus, we hypothesized that this gene may be involved in an attachment to vegetables, and named it lcp (gene encoding Listeria cellulose binding protein [LCP]). lcp mutant (Δlcp) and lcp complement (F2365::pMAD::cat::lcp) strains were generated by homologous recombination. The abilities of a wild-type (WT) strain, the Δlcp strain, and the complemented strain to attach to lettuce leaves were evaluated, which indicated that the attachment of the Δlcp strain to lettuce was significantly less than that of the WT and the complemented strains. Similar results were observed for baby spinach and cantaloupe. Fluorescence microscopy and field emission scanning microscopy analysis further supported these findings. The binding of L. monocytogenes to cellulose was determined using cellulose acetate-coated plates. The results showed that a binding ability of the Δlcp strain was significantly lower than that of the wild type. Combined, these results strongly suggest that LCP plays an important role in an attachment to vegetables and fruits.

Atypical Cohesin-Dockerin Complex Responsible for Cell Surface Attachment of Cellulosomal Components: BINDING FIDELITY, PROMISCUITY, AND STRUCTURAL BUTTRESSES

The rumen bacterium Ruminococcus flavefaciens produces a highly organized multienzyme cellulosome complex that plays a key role in the degradation of plant cell wall polysaccharides, notably cellulose. The R. flavefaciens cellulosomal system is anchored to the bacterial cell wall through a relatively small ScaE scaffoldin subunit, which bears a single type IIIe cohesin responsible for the attachment of two major dockerin-containing scaffoldin proteins, ScaB and the cellulose-binding protein CttA. Although ScaB recruits the catalytic machinery onto the complex, CttA mediates attachment of the bacterial substrate via its two putative carbohydrate-binding modules. In an effort to understand the structural basis for assembly and cell surface attachment of the cellulosome in R. flavefaciens, we determined the crystal structure of the high affinity complex (Kd = 20.83 nM) between the cohesin module of ScaE (CohE) and its cognate X-dockerin (XDoc) modular dyad from CttA at 1.97-Å resolution. The structure reveals an atypical calcium-binding loop containing a 13-residue insert. The results further pinpoint two charged specificity-related residues on the surface of the cohesin module that are responsible for specific versus promiscuous cross-strain binding of the dockerin module. In addition, a combined functional role for the three enigmatic dockerin inserts was established whereby these extraneous segments serve as structural buttresses that reinforce the stalklike conformation of the X-module, thus segregating its tethered complement of cellulosomal components from the cell surface. The novel structure of the RfCohE-XDoc complex sheds light on divergent dockerin structure and function and provides insight into the specificity features of the type IIIe cohesin-dockerin interaction.

A Cellulolytic and Xylanolytic Enzyme Complex from an Alkalothermoanaerobacterium, Tepidimicrobium xylanilyticum BT14

A cellulolytic and xylanolytic enzyme complex-producing alkalothermoanaerobacterium strain, Tepidimicrobium xylanilyticum BT14 is described. The cell was Gram-positive, rod-shaped and endospore-forming. Based on 16S rRNA gene analysis and various lines of biochemical and physiological properties, the strain BT14 was a new member of the genus Tepidimicrobium. The strain BT14 cells had ability to bind to Avicel, xylan and corn hull. The pH and temperature optima for growth were 9.0 and 60 degrees C, respectively. The strain BT14 was able to use a variety of carbon sources. When the bacterium was grown on corn hulls under an anaerobic condition, a cellulolytic and xylanolytic enzyme complex was produced. Crude enzyme containing cellulase and xylanase of the strain BT14 was active in broad ranges of pH and temperature. The optimum conditions for cellulase and xylanase activities were pH 8.0 and 9.0 at 60 degrees C, respectively. The crude enzyme had ability to bind to Avicel and xylan. The analysis of native-PAGE and native-zymograms indicated the cellulose-binding protein showing both cellulase and xylanase activities while SDS-PAGE zymograms showed 4 bands of cellulases and 5 bands of xylanases. Evidence of cohesin-like amino acid sequence seemed to indicate that the protein complex shared direct relationship to the cellulosome of Clostridium thermocellum. The crude enzyme from the strain BT14 showed effective degradation of plant biomass. When grown on corn hulls at pH 9.0 and 60 degrees C under anaerobic conditions, the strain BT14 produced ethanol and acetate as the main fermentation products.

Paradoxical conservation of a set of three cellulose-targeting genes in Mycobacterium tuberculosis complex organisms

The genome of the tuberculosis agent Mycobacterium tuberculosis encodes a putative cellulose-binding protein (CBD2), one candidate cellulase (Cel12), and one fully active cellulase (Cel6). This observation is puzzling, because cellulose is a major component of plant cell walls, whereas M. tuberculosis is a human pathogen without known contact with plants. In order to investigate the biological role of such cellulose-targeting genes in M. tuberculosis we report here the search for and transcription analysis of this set of genes in the genus Mycobacterium. An in silico search for cellulose-targeting orthologues found that only 2.5 % of the sequenced bacterial genomes encode the Cel6, Cel12 and CBD2 gene set simultaneously, including those of the M. tuberculosis complex (MTC) members. PCR amplification and sequencing further demonstrated the presence of these three genes in five non-sequenced MTC bacteria. Among mycobacteria, the combination of Cel6, Cel12 and CBD2 was unique to MTC members, with the exception of Mycobacterium bovis BCG Pasteur, which lacked CBD2. RT-PCR in M. tuberculosis H37Rv indicated that the three cellulose-targeting genes were transcribed into mRNA. The present work shows that MTC organisms are the sole mycobacteria among very few organisms to encode the three cellulose-targeting genes CBD2, Cel6 and Cel12. Our data point toward a unique, yet unknown, relationship with non-plant cellulose-producing hosts such as amoebae.

Structural Analysis of Semi-specific Oligosaccharide Recognition by a Cellulose-binding Protein of Thermotoga maritima Reveals Adaptations for Functional Diversification of the Oligopeptide Periplasmic Binding Protein Fold

Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instances, PBPs bind their cognate ligands with exquisite specificity, distinguishing, for example, between sugar epimers or structurally similar anions. By contrast, oligopeptide-binding proteins bind their ligands through interactions with the peptide backbone but do not distinguish between different side chains. The extremophile Thermotoga maritima possesses a remarkable array of carbohydrate-processing metabolic systems, including the hydrolysis of cellulosic polymers. Here, we present the crystal structure of a T. maritima cellobiose-binding protein (tm0031) that is homologous to oligopeptide-binding proteins. T. maritima cellobiose-binding protein binds a variety of lengths of beta(1-->4)-linked glucose oligomers, ranging from two rings (cellobiose) to five (cellopentaose). The structure reveals that binding is semi-specific. The disaccharide at the nonreducing end binds specifically; the other rings are located in a large solvent-filled groove, where the reducing end makes several contacts with the protein, thereby imposing an upper limit of the oligosaccharides that are recognized. Semi-specific recognition, in which a molecular class rather than individual species is selected, provides an efficient solution for the uptake of complex mixtures.