In vivo immobilization of enzymatically active polypeptides on the cell surface of Staphylococcus carnosus

Many surface proteins of Gram-positive bacteria are covalently anchored to the cell wall by a ubiquitous mechanism, involving a specific, C-terminal sorting signal. To achieve cell-wall immobilization of a normally secreted enzyme in vivo, we constructed a hybrid protein consisting of Staphylococcus hyicus lipase and the C-terminal region of Staphylococcus aureus fibronectin binding protein B (FnBPB). This region comprised the authentic cell-wall-spanning region and cell-wall sorting signal of FnBPB. Expression of the hybrid protein in Staphylococcus carnosus resulted in efficient cell-wall anchoring of enzymatically active lipase. The cell-wall-immobilized lipase (approximately 10000 molecules per cell) retained more than 80% of the specific activity, compared to the C-terminally unmodified S. hyicus lipase secreted by S. carnosus cells. After releasing the hybrid protein from the cell wall by lysostaphin treatment, its specific activity was indistinguishable from that of the unmodified lipase. Thus, the C-terminal region of FnBPB per se was fully compatible with folding of the lipase to an active conformation. To study the influence of the distance between the cell-wall sorting signal and the C-terminus of the lipase on the activity of the immobilized lipase, the length of this spacer region was varied. Reduction of the spacer length gradually reduced the activity of the surface-immobilized lipase. On the other hand, elongation of this spacer did not stimulate the activity of the immobilized lipase, indicating that the spacer must exceed a critical length of approx. 90 amino acids to allow efficient folding of the enzyme, which probably can only be achieved outside the pep-tidoglycan web of the cell wall. When the lipase was replaced by another enzyme, the Escherichia coliβ-lactamase, the resulting hybrid was also efficiently anchored in an active conformation to the cell wall of S, carnosus. These results demonstrate that it is possible to immobilize normally soluble enzymes on the cell wall of S. carnosus - without radically altering their catalytic activity - by fusing them to a cell-wall-immobilization unit, consisting of a suitable cellwall-spanning region and a standard cell-wall sorting signal.     

Cleavage of cellulose by a CBM33 protein

Bacterial proteins categorized as family 33 carbohydrate-binding modules (CBM33) were recently shown to cleave crystalline chitin, using a mechanism that involves hydrolysis and oxidation. We show here that some members of the CBM33 family cleave crystalline cellulose as demonstrated by chromatographic and mass spectrometric analyses of soluble products released from Avicel or filter paper on incubation with CelS2, a CBM33-containing protein from Streptomyces coelicolor A3(2). These enzymes act synergistically with cellulases and may thus become important tools for efficient conversion of lignocellulosic biomass. Fungal proteins classified as glycoside hydrolase family 61 that are known to act synergistically with cellulases are likely to use a similar mechanism.     

Influence of avocado (Persea americana) Cx-cellulase on the structural features of avocado cellulose

Avocado (Persea americana Mill.) fruit produce copious quantities of the enzyme Cx-cellulase (EC 3.2.1.4) during ripening. The possibility that Cx-cellulase is able to disrupt cellulose microfibril oranization was investigated using molecular weight (M_r), x-ray diffraction, and ultrastructural analyses of cell walls from unripe avocado fruit incubated with the purified enzyme. Results indicate that Cx-cellulase causes a downshift in the M_r of unbranched cell-wall polymers in the M_r range of 10⁶–10⁷ Da. There is an increase in the proportion of crystalline cellulose, and cellulose fibrils appear to lose cohesiveness in response to enzyme activity. We propose that Cx-cellulase attacks avocado cellulose at accessible sites in the peripheral and integral noncrystalline regions of the microfibril, resulting in a loss of cohesiveness within the fibril structure and an alteration in the binding of associated cell-wall matrix polysaccharides. The initial loss of avocado mesocarp firmness during fruit ripening may be linked to the onset of Cx-cellulase activity.Abbreviations CMC carboxymethylcellulose - DMAC dimethylacetamide - DS developmental stage - M molecular weight - XG xyloglucan     

In-situ Raman microprobe studies of plant cell walls: Macromolecular organization and compositional variability in the secondary wall of Picea mariana (Mill.) B.S.P.

Native-state organization and distribution of cell-wall components in the secondary wall of woody tissue from P. mariana (Black Spruce) have been investigated using polarized Raman microspectroscopy. Evidence for orientation is detected through Raman intensity variations resulting from rotations of the exciting electric vector with respect to cell-wall geometry. Spectral features associated with cellulose and lignin were studied. The changes in cellulose bands indicate that the pyranose rings of the anhydroglucose repeat units are in planes perpendicular to the cross section, while methine C–H bonds are in planes parallel to the cross section. Changes in bands associated with lignin indicate that the aromatic rings of the phenyl-propane units are most often in the plane of the cell-wall surface. However, regions where lignin orientation departs from this pattern also occur. These results represent direct evidence of molecular organization with respect to cellular morphological features in woody tissue, and indicate that cell-wall components are more highly organized than had been recognized. Studies carried out in order to establish the usefulness and sensitivity of the Raman technique to differences of composition within the cell walls provide evidence of variations in the distribution of cellulose and lignin. Such compositional differences were more prominent between the walls of different cells than within a particular cell wall.     

Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei

Molecular dynamics simulations were carried out for a system consisting of the carbohydrate-binding module (CBM) of the cellulase CBH I from Trichoderma reesei (Hypocrea jecorina) in a concentrated solution of β-d-glucopyranose, to determine whether there is any tendency for the sugar molecules to bind to the CBM. In spite of the general tendency of glucose to behave as an osmolyte, a marked tendency for the sugar molecules to bind to the protein was observed. However, the glucose molecules tended to bind only to specific sites on the protein. As expected, the hydrophobic face of the sugar molecules, comprising the axial H1, H3, and H5 aliphatic protons, tended to adhere to the flat faces of the three tyrosine side chains on the planar binding surface of the CBM. However, a significant tendency to bind to a groove-like feature on the upper surface of the CBM was also observed. These results would not be inconsistent with a model of the mechanism for this globular domain in which the cellodextrin chain being removed from the surface of crystalline cellulose passes over the upper surface of the CBM, presumably then available for hydrolysis in the active site tunnel of this processive cellulase.     

Role of cell-wall biogenesis in the initiation of auxin-mediated growth in coleoptiles of Zea mays L.

The involvement of cell-wall polymer synthesis in auxin-mediated elongation of coleoptile segments from Zea mays L. was investigated with particular regard to the growth-limiting outer epidermis. There was no effect of indole acetic acid (IAA) on the incorporation of labeled glucose into the major polysaccharide wall fractions (cellulose, hemicellulose) within the first 2 h of IAA-induced growth. 2,6-Dichlorobenzonitrile inhibited cellulose synthesis strongly but had no effect on IAA-induced segment elongation even after a pretreatment period of 24 h, indicating that the growth response is independent of the apposition of new cellulose microfibrils at the epidermal cell wall. The incorporation of labeled leucine into total and cell-wall protein of the epidermis was promoted by IAA during the first 30 min of IAA-induced growth. Inhibition of IAA-induced growth by protein and RNA-synthesis inhibitors (cycloheximide, cordycepin) was accompanied by an inhibition of leucine incorporation into the epidermal cell wall during the first 30 min of induced growth but had no effect on the concomitant incorporation of monosaccharide precursors into the cellulose or hemicellulose fractions of this wall. It is concluded that at least one of the epidermal cell-wall proteins fulfills the criteria for a growth-limiting protein induced by IAA at the onset of the growth response. In contrast, the synthesis of the polysaccharide wall fractions cellulose and hemicellulose, as well as their transport and integration into the growing epidermal wall, appears to be independent of growth-limiting protein and these processes are therefore no part of the mechanism of growth control by IAA.Abbreviations CHI cycloheximide - COR cordycepin - DCB 2,6-dichlorobenzonitrile - GLP growth-limiting protein(s) - IAA indole-3-acetic acid     

Biochemical characterization of a glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii

The glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii (AkCel61) is a modular enzyme that consists of a catalytic domain and a carbohydrate-binding module belonging to family 1 (CBM1) that are connected by a Ser-Thr linker region longer than 100 amino acids. We expressed the recombinant AkCel61, wild-type enzyme (rAkCel61), and a truncated enzyme consisting of the catalytic domain (rAkCel61ΔCBM) in Pichia pastoris and analyzed their biochemical properties. Purified rAkCel61 and rAkCel61ΔCBM migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were demonstrated to have apparent molecular masses of 81,000 and 34,000 Da, respectively. After treatment with endoglycosidase H, both proteins showed an increase in mobility, thus, demonstrating estimated molecular masses of 78,000 and 28,000 Da, respectively. Mass spectrometry analysis revealed that rAkCel61 and rAkCel61ΔCBM expressed in P. pastoris are heterogeneous due to protein glycosylation. The rAkCel61 protein bound to crystalline cellulose but not to arabinoxylan. The rAkCel61 and rAkCel61ΔCBM proteins produced small amounts of oligosaccharides from soluble carboxymethylcellulose. They also exhibited a slight hydrolytic activity toward laminarin. However, they showed no detectable activity toward microcrystalline cellulose, arabinoxylan, and pectin. Both recombinant enzymes also showed no detectable activity toward p-nitrophenyl β-d-glucoside, p-nitrophenyl β-d-cellobioside, and p-nitrophenyl β-d-cellotrioside. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of cell-wall polymers during cotton fiber development

Although the fibers of cotton (Gossypium hirsutum L.) are single cells with a secondary wall composed primarily of cellulose, the cell-wall polymers of the fibers are technically difficult to characterize with respect to molecular weights. This limitation hinders understanding how the fiber wall composition changes during development, particularly with respect to genotypic variations, and how the molecular composition is related to physical properties. We analyzed cell-wall polymers from cotton fibers (cultivar, Texas Marker-1) at several developmental stages (8–60 days post-anthesis; DPA) by gel-permeation chromatography of components soluble in dimethyl acetamide and lithium chloride. This procedure solubilizes fiber cell-wall components directly without prior extraction or derivatization, processes that could lead to degradation of high-molecular-weight components. Cellwall polymers from fibers at primary cell-wall stages had lower molecular weights than the cellulose from fibers at the secondary wall stages; however, the high-molecularweight cellulose characteristic of mature cotton was detected as early as 8 DPA. High-molecular-weight material decreased during the period of 10–18 DPA with concomitant increase in lower-molecular-weight wall components, possibly indicating hydrolysis during the later stages of elongation.Abbreviations DMAC dimethyl acetamide - DP degree of polymerization - DPA days post anthesis - GPC gel-permeation chromatography - MW molecular weight - MWD molecular-weight distribution - TM-1 Texas Marker 1     

Characteristics and structural requirements of apical sorting of the rat growth hormone through the O-glycosylated stalk region of intestinal sucrase-isomaltase

The apical sorting of the small intestinal membrane glycoprotein sucrase-isomaltase (SI) depends on the presence of O-linked glycans and the transmembrane domain. Here, we investigate the role of O-glycans carried by the Ser/Thr-rich stalk region of SI as an apical sorting signal and evaluate the spatial requirements for an efficient recognition of this signal. Several hybrid proteins are generated comprising the unsorted and unglycosylated protein, the rat growth hormone (rGH), fused to either the transmembrane domain of SI (GH-SI(TM)), or the transmembrane and the stalk domains (GH-SI(SR/TM)). Both constructs are randomly distributed over the apical and basolateral membranes of MDCK cells indicating that neither the transmembrane domain nor the O-glycans are sufficient per se for an apical delivery. Only when a polyglycine spacer is inserted between the stalk region of SI and the luminal part of rGH in the GH-SI(Gly/SR/TM) fusion protein does efficient apical sorting of an O-glycosylated protein as well as a time-dependent association with detergent-insoluble lipid microdomains occur. Obviously, the polyglycine spacer facilitates the accessibility of the O-glycans in GH-SI(Gly/SR/TM) to a putative sorting receptor, whereas these glycans are inadequately recognized in GH-SI(SR/TM). We conclude that the O-glycans in the stalk region of SI act as an apical sorting signal within a sorting machinery that comprises at least a carbohydrate-binding protein and fulfills specific spatial requirements provided, for example by a polyglycine spacer in the context of rGH or the P-domain within the SI enzyme complex.     

Purification, characterization and modular organization of a cellulose-binding protein, CBP105, a processive β-1,4-endoglucanase from Cellulomonas flavigena

A cellulose-binding protein of 105 kDa (CBP105) from Cellulomonas flavigena was purified and its gene was cloned. CBP105 is a processive endoglucanase with maximum activity on carboxymethyl cellulose (CMC) at pH 7.5 and 60°C. Limited proteolysis suggested that CBP105 is composed of one catalytic domain (CD) and two carbohydrate-binding modules (CBM). The nucleotide sequence of the cbp105 gene (AY729806) indicates that CBP105 is a modular enzyme with a family 9 glycoside hydrolase CD linked to a family 3 CBM, two fibronectin III-like domains and a family 2 CBM. This structural organization may be responsible for CBP105 processive CMC degradation.     

Cellulosomal carbohydrate-binding module from Clostridium josui binds to crystalline and non-crystalline cellulose,and soluble polysaccharides

To understand the lignocellulose degradation activity of the Clostridium josui cellulosome, a carbohydrate-binding module of the scaffoldin CjCBM3 was characterized. CjCBM3 shows binding to crystalline cellulose, non-crystalline cellulose and soluble polysaccharides. The binding isotherm of CjCBM3 to acid-swollen cellulose is best fitted by the Langmuir two-site model, suggesting that there are two CjCBM3 binding sites on acid-swollen cellulose with different affinities. The second site shows lower affinity and larger binding capacity, suggesting that the cellulosome is directly targeted to the cellulose surface with high affinity, where larger amounts of the cellulosome bind to cellulose with low affinity.     

Exploring the Structure-Function Loop Adaptability of a (β/α)(8)-Barrel Enzyme through Loop Swapping and Hinge Variability

Cellulosomes are large extracellular multi-enzyme complexes that exhibit elevated activity on plant cell-wall polysaccharides. In the present study, the relationships between the conformational flexibility and efficacy of cellulosomes, and the inter-modules linkers of their scaffold protein were investigated. For this purpose, the length of the intrinsically disordered Ser/Thr-rich 50-residue linker connecting a Clostridium thermocellum and a Clostridium cellulolyticum cohesin in a hybrid scaffoldin (Scaf4) was changed by sequences ranging from 4 to 128 residues. The composition was also modified and new linkers composed of series of N, S or repeats of the EPPV motif were generated. Two model cellulases (Cel48F and Cel9G) appended with appropriate dockerins were subsequently bound to the engineered scaffoldins. All the resulting minicomplexes displayed the same activity on crystalline cellulose as the complex based on the initial Scaf4, and were found to be 2-fold more active than Cel48F and Cel9G bound to separate cohesins. Small-angle X-ray scattering assays of the engineered scaffoldins confirmed, however, that the size and the conformational flexibility of some of the new inter-cohesins linkers differed significantly from that of the initial 50 residue linker displayed by the parental Scaf4. Our data suggest that the synergy induced by proximity does not require a specific inter-cohesins sequence or distance. The present study reveals that complexation onto the hybrid scaffoldins modifies the type of soluble sugars released from crystalline cellulose by the selected cellulases, compared to the free enzyme system.     

The crystalline polymorphism of mannan in plant cell walls and after recrystallisation

Mannan-rich plant cell walls were mechanically disintegrated and chemically extracted in order to ascertain their morphology and structure by electron microscopy and electron diffraction. For Acetabularia crenulata and Codium fragile, the cell-wall fragments were found to consist of alkali-resistant fibrillar mannan II encrusted with alkali-soluble granular mannan I. In the case of ivory nuts (Phytelephas macrocarpa) there is, in addition, a microfibrillar cellulose component which was also identified. The mannan I—mannan II polymorphism was also obtained when various mannan fractions were recrystallized from solution. In these recrystallizations, the occurrence of one or the other polymorph was found to depend on several parameters: the molecular weight of the mannan, the temperature of crystallization and the polarity of the crystallization medium.Abbreviations DP degree of polymerization - EDTA ethylenediaminetetraacetic acid Affiliated with the Scientific and Medical University of Grenoble     

Use of a fluorescence labelled,carbohydrate-binding module from Phanerochaete chrysosporium Cel7D for studying wood cell wall ultrastructure

The -amino group of the carbohydrate-binding module (CBM) from Phanerochaete chrysosporium cellulase Cel7D was covalently labelled with fluorescein isothiocyanate. The fluorescein-labelled CBM was characterised regarding substrate binding, showing specificity only to cellulose and not to mannan and xylan. Conjugation of fluorescein isothiocyanate to CBM did not affect its binding to cellulose. The labelled CBM was successfully used as a probe for detecting cellulose in lignocellulose material such as never dried spruce and birch wood as well as pulp fibres.     

Calcium lonophore-induced stimulation of secretory activity in Chlamydomonas reinhardii

The cell-wall lysin in gametes from Chlamydomonas reinhardii which under normal mating conditions is activated by flagellar cell contact was found to be susceptible to stimulation by the antibiotic ionophore A 23187 provided that CA²⁺ was included in the medium. Ionophore-induced release of the cell-wall lysin did not deend on the mating type or the gametic state of the cells. Vegetative cells which normally do not exhibit any mating capacity reacted with cell-wall lysis like gametes stimulated by cell contact.Ionophore-dependent Ca²⁺-transfer across the cell membranes generated a signal for cell-wall lysis only in cells with intact flagella. Deflagellated cells did not respond to A 23187 before regeneration of the amputated organelles. Another indication for a possible role of flagella in Ca²⁺-mediated cell-wall lysis was obtained from a conditional flagellar-assembly mutant of C. reinhardii which had been isolated and described by Huang et al. (1977). Upon shift-up the mutant strain immediately became unresponsive to A 23187 and Ca²⁺ but regained susceptibility soon after being retransferred to permissive conditions (20°C).     

A second generation snp-derived Escherichia coli-Streptomyces shuttle expression vector that is generally transferable by conjugation

An Escherichia coli-Streptomyces shuttle vector (pJN100) was constructed, by inserting an origin of transfer (oriT), derived from the E. coli broad host range plasmid RK2, into pANT1202, a high-copy-number vector for gene expression in Streptomyces. The resulting conjugably transferable vector contains the pANT1202-derived SnpR (LysR-like protein) activated snpA promoter that drives strong heterologous expression of proteins. We initially demonstrated that plasmid pJN100 was transferred with high frequency (10(-5-7) exconjugants per recipient) into several Streptomyces strains that were refractory to transformation by other means. Plasmid pJN100 was also shown to be stable in E. coli and Streptomyces. We confirmed functional protein expression by using a pJN100 derivative to complement a mutant of Streptomyces griseus with a disrupted chromosomal copy of the gene nonM, a gene encoding an essential reductase in the nonactin biosynthesis gene cluster. High levels of protein expression were confirmed using Western blotting to assess the production of the serine esterase NonR, an enzyme responsible for nonactin resistance in the nonactin producer S. griseus.     

Biochemical and genetic characterization of ChiA,the major enzyme component for the solubilization of chitin by<Emphasis Type="Italic"> Cellulomonas uda</Emphasis>

Cellulomonas uda efficiently solubilized chitinous substrates with a simple chitinase system composed of an endochitinase, designated ChiA, which hydrolyzed insoluble substrates into long-chain chitooligosaccharides, and an as yet uncharacterized exochitinase activity. ChiA, isolated from culture supernatant fluids, was found to be a glycosylated endochitinase with an apparent molecular mass of approximately 70 kDa and pI of 8.5. The gene encoding ChiA was cloned in Escherichia coli and sequenced, revealing an open reading frame of 1,716 bp encoding a 571-amino-acid protein with a predicted molecular mass of 59.2 kDa. The region upstream of chiA included a conserved –35 hexamer flanked by two direct repeats analogous to those found in many Streptomyces chitinase promoters, and thought to function as binding sequences for regulatory proteins. Analysis of the deduced amino acid sequence showed a modular protein consisting of a signal peptide at its N terminus, a family 2 carbohydrate-binding module (CBM2) that was closely related to the substrate-binding domains of glycosyl hydrolases from distantly related bacteria, and a family 18 glycosyl hydrolase catalytic module related to Streptomyces chitinases. In contrast to the fibronectin type III domains of Streptomyces chitinases, the linker region between modules in ChiA consisted of a long proline- and threonine-rich module, thought to contribute to the glycosylation and flexibility of the mature protein.Abbreviations CBM Carbohydrate-binding module - P-T Proline- and threonine-rich domain - Fn3 Type III repetitive sequences of fibronectin domain - PKD Polycystic kidney disease I domain     

Fusion of carbohydrate binding modules from <Emphasis Type="Italic">Thermotoga neapolitana</Emphasis> with a family 10 xylanase from <Emphasis Type="Italic">Bacillus halodurans</Emphasis> S7

Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains two carbohydrate-binding modules (CBM) which belong to family 22. These CBMs bind xylan but not to cellulose. The gene encoding the mature peptide of these CBMs was fused with an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The (His)₆ tagged hybrid protein was purified by immobilized metal affinity chromatography and characterized. Xylan binding by the chimeric protein was influenced by NaCl concentration and pH of the binding medium. Binding increased with increasing salt concentration up to 200 mM. Higher extent of binding was observed under acidic conditions. The fusion of the CBM structures enhanced the hydrolytic efficiency of the xylanase against insoluble xylan, but decreased the stability of the enzyme. The optimum temperature and pH for the activity of the xylanase did not change.     

Characterization of inhibitors of cellulose synthesis in cotton fibers

Several compounds were tested for their ability to inhibit the in-vivo synthesis of cellulose and other cell-wall polysaccharides in fibers of cotton (Gossypium hirsutum L.) developing on in-vitro cultured ovules. Inhibitory effects were measured by the ability of the compounds to inhibit the incorporation of radioactivity from [U-¹⁴C]glucose into these cell-wall polymers. Of the compounds surveyed, 2,6-dichlorobenzonitrile (DCB) was the most effective and specific one for its effects on cellulose synthesis when compared to its effect on the synthesis of other cell-wall components. At 10 M DCB caused 80% inhibition of cellulose synthesis, and the effect was reversed upon removal of the DCB, with recovery to 90% of the control rate. Two analogs of DCB, 2-chloro-6-fluorobenzonitrile and 2,6-dichlorobenzene carbothiamide, were as specific and nearly as effective as DCB with respect to their effects on cellulose synthesis. Coumarin, generally regarded as an inhibitor of cellulose synthesis in other plant systems, was effective in cotton fibers in millimolar concentrations and, like DCB, was relatively specific with regard to its effect on cellulose synthesis. DCB and coumarin inhibited the synthesis of both primary and secondary wall cellulose. Bacitracin, an inhibitor of the cycling of phosphorylated polyprenols involved in cell-wall synthesis in bacteria, and ethylenediaminetetracetic acid (EDTA) and ethyleneglycol-bis-(-amino-ethylether)-N,N-tetracetic acid (EGTA), chelators of civalent cations, were also effective, although only at relatively high concentrations, in inhibiting incorporation of radioactivity into cellulose.Abbreviations DCB 2,6-dichlorobenzonitrite - CFB 2-chloro-6-fluorobenzonitrile - EDTA ethylenediaminetetracetic acid - EGTA ethyleneglycol-bis-(-amino-ethylether)-N,N-tetracetic acid