Intracellular removal of acetyl, feruloyl and p-coumaroyl decorations on arabinoxylo-oligosaccharides imported from lignocellulosic biomass degradation by Ruminiclostridium cellulolyticum.

BACKGROUND: Xylans are polysaccharides that are naturally abundant in agricultural by-products, such as cereal brans and straws. Microbial degradation of arabinoxylan is facilitated by extracellular esterases that remove acetyl, feruloyl, and p-coumaroyl decorations. The bacterium Ruminiclostridium cellulolyticum possesses the Xua (xylan utilization associated) system, which is responsible for importing and intracellularly degrading arabinoxylodextrins. This system includes an arabinoxylodextrins importer, four intracellular glycosyl hydrolases, and two intracellular esterases, XuaH and XuaJ which are encoded at the end of the gene cluster. RESULTS: Genetic studies demonstrate that the genes xuaH and xuaJ are part of the xua operon, which covers xuaABCDD'EFGHIJ. This operon forms a functional unit regulated by the two-component system XuaSR. The esterases encoded at the end of the cluster have been further characterized: XuaJ is an acetyl esterase active on model substrates, while XuaH is a xylan feruloyl- and p-coumaryl-esterase. This latter is active on oligosaccharides derived from wheat bran and wheat straw. Modelling studies indicate that XuaH has the potential to interact with arabinoxylobiose acylated with mono- or diferulate. The intracellular esterases XuaH and XuaJ are believed to allow the cell to fully utilize the complex acylated arabinoxylo-dextrins imported into the cytoplasm during growth on wheat bran or straw. CONCLUSIONS: This study reports for the first time that a cytosolic feruloyl esterase is part of an intracellular arabinoxylo-dextrin import and degradation system, completing its cytosolic enzymatic arsenal. This system represents a new pathway for processing highly-decorated arabinoxylo-dextrins, which could provide a competitive advantage to the cell and may have interesting biotechnological applications.

Modulation of bacterial multicellularity via spatio-specific polysaccharide secretion.

The development of multicellularity is a key evolutionary transition allowing for differentiation of physiological functions across a cell population that confers survival benefits; among unicellular bacteria, this can lead to complex developmental behaviors and the formation of higher-order community structures. Herein, we demonstrate that in the social δ-proteobacterium Myxococcus xanthus, the secretion of a novel biosurfactant polysaccharide (BPS) is spatially modulated within communities, mediating swarm migration as well as the formation of multicellular swarm biofilms and fruiting bodies. BPS is a type IV pilus (T4P)-inhibited acidic polymer built of randomly acetylated ß-linked tetrasaccharide repeats. Both BPS and exopolysaccharide (EPS) are produced by dedicated Wzx/Wzy-dependent polysaccharide-assembly pathways distinct from that responsible for spore-coat assembly. While EPS is preferentially produced at the lower-density swarm periphery, BPS production is favored in the higher-density swarm interior; this is consistent with the former being known to stimulate T4P retraction needed for community expansion and a function for the latter in promoting initial cell dispersal. Together, these data reveal the central role of secreted polysaccharides in the intricate behaviors coordinating bacterial multicellularity.

A Novel Two-Component System, XygS/XygR, Positively Regulates Xyloglucan Degradation, Import, and Catabolism in Ruminiclostridium cellulolyticum.

Cellulolytic microorganisms play a key role in the global carbon cycle by decomposing structurally diverse plant biopolymers from dead plant matter. These microorganisms, in particular anaerobes such as Ruminiclostridium cellulolyticum that are capable of degrading and catabolizing several different polysaccharides, require a fine-tuned regulation of the biosynthesis of their polysaccharide-degrading enzymes. In this study, we present a bacterial regulatory system involved in the regulation of genes enabling the metabolism of the ubiquitous plant polysaccharide xyloglucan. The characterization of R. cellulolyticum knockout mutants suggests that the response regulator XygR and its cognate histidine kinase XygS are essential for growth on xyloglucan. Using in vitro and in vivo analyses, we show that XygR binds to the intergenic region and activates the expression of two polycistronic transcriptional units encoding an ABC transporter dedicated to the uptake of xyloglucan oligosaccharides and the two-component system itself together with three intracellular glycoside hydrolases responsible for the sequential intracellular degradation of the imported oligosaccharides into mono- and disaccharides. Interestingly, XygR also upregulates the expression of a distant gene coding for the most active extracellular cellulosomal xyloglucanase of R. cellulolyticum by binding to the upstream intergenic region.IMPORTANCERuminiclostridium cellulolyticum is a Gram-positive, mesophilic, anaerobic, cellulolytic, and hemicellulolytic bacterium. The last property qualifies this species as a model species for the study of hemicellulose degradation, import of degradation products, and overall regulation of these phenomena. In this study, we focus on the regulation of xyloglucan dextrin import and intracellular degradation and show that the two components of the two-component regulation system XygSR are essential for growth on xyloglucan and that the response regulator XygR regulates the transcription of genes involved in the extracellular degradation of the polysaccharide, the import of degradation products, and their intracellular degradation.

Cell-surface exposure of a hybrid 3-cohesin scaffoldin allowing the functionalization of Escherichia coli envelope.

Cellulosomes are large plant cell wall degrading complexes secreted by some anaerobic bacteria. They are typically composed of a major scaffolding protein containing multiple receptors called cohesins, which tightly anchor a small complementary module termed dockerin harbored by the cellulosomal enzymes. In the present study, we have successfully cell surface exposed in Escherichia coli a hybrid scaffoldin, Scaf6, fused to the curli protein CsgA, the latter is known to polymerize at the surface of E. coli to form extracellular fibers under stressful environmental conditions. The C-terminal part of the chimera encompasses the hybrid scaffoldin composed of three cohesins from different bacterial origins and a carbohydrate-binding module targeting insoluble cellulose. Using three cellulases hosting the complementary dockerin modules and labeled with different fluorophores, we have shown that the hybrid scaffoldin merged to CsgA is massively exposed at the cell surface of E. coli and that each cohesin module is fully operational. Altogether these data open a new route for a series of biotechnological applications exploiting the cell-surface exposure of CsgA-Scaf6 in various industrial sectors such as vaccines, biocatalysts or bioremediation, simply by grafting the small dockerin module to the desired proteins before incubation with the engineered E. coli.

ABC Transporters Required for Hexose Uptake by Clostridium phytofermentans.

The mechanisms by which bacteria uptake solutes across the cell membrane broadly impact their cellular energetics. Here, we use functional genomic, genetic, and biophysical approaches to reveal how Clostridium (Lachnoclostridium) phytofermentans, a model bacterium that ferments lignocellulosic biomass, uptakes plant hexoses using highly specific, nonredundant ATP-binding cassette (ABC) transporters. We analyze the transcription patterns of its 173 annotated sugar transporter genes to find those upregulated on specific carbon sources. Inactivation of these genes reveals that individual ABC transporters are required for uptake of hexoses and hexo-oligosaccharides and that distinct ABC transporters are used for oligosaccharides versus their constituent monomers. The thermodynamics of sugar binding shows that substrate specificity of these transporters is encoded by the extracellular solute-binding subunit. As sugars are not phosphorylated during ABC transport, we identify intracellular hexokinases based on in vitro activities. These mechanisms used by Clostridia to uptake plant hexoses are key to understanding soil and intestinal microbiomes and to engineer strains for industrial transformation of lignocellulose.IMPORTANCE Plant-fermenting Clostridia are anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine. Clostridia degrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organism Clostridium phytofermentans We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.

Characterization of all family-9 glycoside hydrolases synthesized by the cellulosome-producing bacterium Clostridium cellulolyticum.

The genome of Clostridium cellulolyticum encodes 13 GH9 enzymes that display seven distinct domain organizations. All but one contain a dockerin module and were formerly detected in the cellulosomes, but only three of them were previously studied (Cel9E, Cel9G, and Cel9M). In this study, the 10 uncharacterized GH9 enzymes were overproduced in Escherichia coli and purified, and their activity pattern was investigated in the free state or in cellulosome chimeras with key cellulosomal cellulases. The newly purified GH9 enzymes, including those that share similar organization, all exhibited distinct activity patterns, various binding capacities on cellulosic substrates, and different synergies with pivotal cellulases in mini-cellulosomes. Furthermore, one enzyme (Cel9X) was characterized as the first genuine endoxyloglucanase belonging to this family, with no activity on soluble and insoluble celluloses. Another GH9 enzyme (Cel9V), whose sequence is 78% identical to the cellulosomal cellulase Cel9E, was found inactive in the free and complexed states on all tested substrates. The sole noncellulosomal GH9 (Cel9W) is a cellulase displaying a broad substrate specificity, whose engineered form bearing a dockerin can act synergistically in minicomplexes. Finally, incorporation of all GH9 cellulases in trivalent cellulosome chimera containing Cel48F and Cel9G generated a mixture of heterogeneous mini-cellulosomes that exhibit more activity on crystalline cellulose than the best homogeneous tri-functional complex. Altogether, our data emphasize the importance of GH9 diversity in bacterial cellulosomes, confirm that Cel9G is the most synergistic GH9 with the major endoprocessive cellulase Cel48F, but also identify Cel9U as an important cellulosomal component during cellulose depolymerization.

Recombinant Bacillus subtilis that grows on untreated plant biomass.

Lignocellulosic biomass is a promising feedstock to produce biofuels and other valuable biocommodities. A major obstacle to its commercialization is the high cost of degrading biomass into fermentable sugars, which is typically achieved using cellulolytic enzymes from Trichoderma reesei. Here, we explore the use of microbes to break down biomass. Bacillus subtilis was engineered to display a multicellulase-containing minicellulosome. The complex contains a miniscaffoldin protein that is covalently attached to the cell wall and three noncovalently associated cellulase enzymes derived from Clostridium cellulolyticum (Cel48F, Cel9E, and Cel5A). The minicellulosome spontaneously assembles, thus increasing the practicality of the cells. The recombinant bacteria are highly cellulolytic and grew in minimal medium containing industrially relevant forms of biomass as the primary nutrient source (corn stover, hatched straw, and switch grass). Notably, growth did not require dilute acid pretreatment of the biomass and the cells achieved densities approaching those of cells cultured with glucose. An analysis of the sugars released from acid-pretreated corn stover indicates that the cells have stable cellulolytic activity that enables them to break down 62.3% ± 2.6% of the biomass. When supplemented with beta-glucosidase, the cells liberated 21% and 33% of the total available glucose and xylose in the biomass, respectively. As the cells display only three types of enzymes, increasing the number of displayed enzymes should lead to even more potent cellulolytic microbes. This work has important implications for the efficient conversion of lignocellulose to value-added biocommodities.

Role of the Solute-Binding Protein CuaD in the Signaling and Regulating Pathway of Cellobiose and Cellulose Utilization in Ruminiclostridium cellulolyticum.

In Ruminiclostridium cellulolyticum, cellobiose is imported by the CuaABC ATP-binding cassette transporter containing the solute-binding protein (SBP) CuaA and is further degraded in the cytosol by the cellobiose phosphorylase CbpA. The genes encoding these proteins have been shown to be essential for cellobiose and cellulose utilization. Here, we show that a second SBP (CuaD), whose gene is adjacent to two genes encoding a putative two-component regulation system (CuaSR), forms a three-component system with CuaS and CuaR. Studies of mutant and recombinant strains of R. cellulolyticum have indicated that cuaD is important for the growth of strains on cellobiose and cellulose. Furthermore, the results of our RT-qPCR experiments suggest that both the three (CuaDSR)- and the two (CuaSR)-component systems are able to perceive the cellobiose signal. However, the strain producing the three-component system is more efficient in its cellobiose and cellulose utilization. As CuaD binds to CuaS, we propose an in-silico model of the complex made up of two extracellular domains of CuaS and two of CuaD. CuaD allows microorganisms to detect very low concentrations of cellobiose due to its high affinity and specificity for this disaccharide, and together with CuaSR, it triggers the expression of the cuaABC-cbpA genes involved in cellodextrins uptake.

Selfish uptake versus extracellular arabinoxylan degradation in the primary degrader Ruminiclostridium cellulolyticum, a new string to its bow.

BACKGROUND: Primary degraders of polysaccharides play a key role in anaerobic biotopes, where plant cell wall accumulates, providing extracellular enzymes to release fermentable carbohydrates to fuel themselves and other non-degrader species. Ruminiclostridium cellulolyticum is a model primary degrader growing amongst others on arabinoxylan. It produces large multi-enzymatic complexes called cellulosomes, which efficiently deconstruct arabinoxylan into fermentable monosaccharides. RESULTS: Complete extracellular arabinoxylan degradation was long thought to be required to fuel the bacterium during this plant cell wall deconstruction stage. We discovered and characterized a second system of "arabinoxylan" degradation in R. cellulolyticum, which challenged this paradigm. This "selfish" system is composed of an ABC transporter dedicated to the import of large and possibly acetylated arabinoxylodextrins, and a set of four glycoside hydrolases and two esterases. These enzymes show complementary action modes on arabinoxylo-dextrins. Two α-L-arabinofuranosidases target the diverse arabinosyl side chains, and two exo-xylanases target the xylo-oligosaccharides backbone either at the reducing or the non-reducing end. Together, with the help of two different esterases removing acetyl decorations, they achieve the depolymerization of arabinoxylo-dextrins in arabinose, xylose and xylobiose. The in vivo study showed that this new system is strongly beneficial for the fitness of the bacterium when grown on arabinoxylan, leading to the conclusion that a part of arabinoxylan degradation is achieved in the cytosol, even if monosaccharides are efficiently provided by the cellulosomes in the extracellular space. These results shed new light on the strategies used by anaerobic primary degrader bacteria to metabolize highly decorated arabinoxylan in competitive environments. CONCLUSION: The primary degrader model Ruminiclostridium cellulolyticum has developed a "selfish" strategy consisting of importing into the bacterium, large arabinoxylan-dextrin fractions released from a partial extracellular deconstruction of arabinoxylan, thus complementing its efficient extracellular arabinoxylan degradation system. Genetic studies suggest that this system is important to support fitness and survival in a competitive biotope. These results provide a better understanding of arabinoxylan catabolism in the primary degrader, with biotechnological application for synthetic microbial community engineering for the production of commodity chemicals from lignocellulosic biomass.

Multiple detection of both attractants and repellents by the dCache-chemoreceptor SO_1056 of Shewanella oneidensis.

Chemoreceptors are usually transmembrane proteins dedicated to the detection of compound gradients or signals in the surroundings of a bacterium. After detection, they modulate the activation of CheA-CheY, the core of the chemotactic pathway, to allow cells to move upwards or downwards depending on whether the signal is an attractant or a repellent, respectively. Environmental bacteria such as Shewanella oneidensis harbour dozens of chemoreceptors or MCPs (methyl-accepting chemotaxis proteins). A recent study revealed that MCP SO_1056 of S. oneidensis binds chromate. Here, we show that this MCP also detects an additional attractant (l-malate) and two repellents (nickel and cobalt). The experiments were performed in vivo by the agarose-in-plug technique after overproducing MCP SO_1056 and in vitro, when possible, by submitting the purified ligand-binding domain (LBD) of SO_1056 to a thermal shift assay (TSA) coupled to isothermal titration calorimetry (ITC). ITC assays revealed a KD of 3.4 µm for l-malate and of 47.7 µm for nickel. We conclude that MCP SO_1056 binds attractants and repellents of unrelated composition. The LBD of SO_1056 belongs to the double Cache_1 family and is highly homologous to PctA, a chemoreceptor from Pseudomonas aeruginosa that detects several amino acids. Therefore, LBDs of the same family can bind diverse compounds, confirming that experimental approaches are required to define accurate LBD-binding molecules or signals.

The issue of secretion in heterologous expression of Clostridium cellulolyticum cellulase-encoding genes in Clostridium acetobutylicum ATCC 824.

The genes encoding the cellulases Cel5A, Cel8C, Cel9E, Cel48F, Cel9G, and Cel9M from Clostridium cellulolyticum were cloned in the C. acetobutylicum expression vector pSOS952 under the control of a Gram-positive constitutive promoter. The DNA encoding the native leader peptide of the heterologous cellulases was maintained. The transformation of the solventogenic bacterium with the corresponding vectors generated clones in the cases of Cel5A, Cel8C, and Cel9M. Analyses of the recombinant strains indicated that the three cellulases are secreted in an active form to the medium. A large fraction of the secreted cellulases, however, lost the C-terminal dockerin module. In contrast, with the plasmids pSOS952-cel9E, pSOS952-cel48F, and pSOS952-cel9G no colonies were obtained, suggesting that the expression of these genes has an inhibitory effect on growth. The deletion of the DNA encoding the leader peptide of Cel48F in pSOS952-cel48F, however, generated strains of C. acetobutylicum in which mature Cel48F accumulates in the cytoplasm. Thus, the growth inhibition observed when the wild-type cel48F gene is expressed seems related to the secretion of the cellulase. The weakening of the promoter, the coexpression of miniscaffoldin-encoding genes, or the replacement of the native signal sequence of Cel48F by that of secreted heterologous or endogenous proteins failed to generate strains secreting Cel48F. Taken together, our data suggest that a specific chaperone(s) involved in the secretion of the key family 48 cellulase, and probably Cel9G and Cel9E, is missing or insufficiently synthesized in C. acetobutylicum.

Scaffoldin modules serving as "cargo" domains to promote the secretion of heterologous cellulosomal cellulases by Clostridium acetobutylicum.

The secretion of large heterologous cellulases by Clostridium acetobutylicum was formerly shown to be deleterious. To circumvent this issue, various scaffoldins' modules were grafted at their N termini. Family 3a cellulose binding module combined with an X2 module(s) was found to trigger the secretion of Clostridium cellulolyticum cellulases by the solventogenic bacterium.

Assembly of minicellulosomes on the surface of Bacillus subtilis.

To cost-efficiently produce biofuels, new methods are needed to convert lignocellulosic biomass into fermentable sugars. One promising approach is to degrade biomass using cellulosomes, which are surface-displayed multicellulase-containing complexes present in cellulolytic Clostridium and Ruminococcus species. In this study we created cellulolytic strains of Bacillus subtilis that display one or more cellulase enzymes. Proteins containing the appropriate cell wall sorting signal are covalently anchored to the peptidoglycan by coexpressing them with the Bacillus anthracis sortase A (SrtA) transpeptidase. This approach was used to covalently attach the Cel8A endoglucanase from Clostridium thermocellum to the cell wall. In addition, a Cel8A-dockerin fusion protein was anchored on the surface of B. subtilis via noncovalent interactions with a cell wall-attached cohesin module. We also demonstrate that it is possible to assemble multienzyme complexes on the cell surface. A three-enzyme-containing minicellulosome was displayed on the cell surface; it consisted of a cell wall-attached scaffoldin protein noncovalently bound to three cellulase-dockerin fusion proteins that were produced in Escherichia coli. B. subtilis has a robust genetic system and is currently used in a wide range of industrial processes. Thus, grafting larger, more elaborate minicellulosomes onto the surface of B. subtilis may yield cellulolytic bacteria with increased potency that can be used to degrade biomass.

Engineering of a new Escherichia coli strain efficiently metabolizing cellobiose with promising perspectives for plant biomass-based application design.

The necessity to decrease our fossil energy dependence requests bioprocesses based on biomass degradation. Cellobiose is the main product released by cellulases when acting on the major plant cell wall polysaccharide constituent, the cellulose. Escherichia coli, one of the most common model organisms for the academy and the industry, is unable to metabolize this disaccharide. In this context, the remodeling of E. coli to catabolize cellobiose should thus constitute an important progress for the design of such applications. Here, we developed a robust E. coli strain able to metabolize cellobiose by integration of a small set of modifications in its genome. Contrary to previous studies that use adaptative evolution to achieve some growth on this sugar by reactivating E. coli cryptic operons coding for cellobiose metabolism, we identified easily insertable modifications impacting the cellobiose import (expression of a gene coding a truncated variant of the maltoporin LamB, modification of the expression of lacY encoding the lactose permease) and its intracellular degradation (genomic insertion of a gene encoding either a cytosolic ß-glucosidase or a cellobiose phosphorylase). Taken together, our results provide an easily transferable set of mutations that confers to E. coli an efficient growth phenotype on cellobiose (doubling time of 2.2 â€‹h in aerobiosis) without any prior adaptation.

Supported lysozyme for improved antimicrobial surface protection.

Surface protection against biofilms is still an open challenge. Current strategies rely on coatings that are meant to guarantee antiadhesive or antimicrobial effects. While it seems difficult to ensure antiadhesion in complex media and against all the adhesive arsenal of microbes, strategies based on antimicrobials lack from sustainable functionalization methodologies to allow the perfect efficiency of the grafted molecules. Here we used the high affinity ligand-receptor interaction between biotin and streptavidin to functionalize surfaces with lysozyme, an enzyme that degrades the bacterial peptidoglycan cell wall. Biotinylated lysozyme was grafted on surfaces coated with streptavidin receptors. Using atomic force microscopy (AFM)-based single molecule force spectroscopy, we showed that grafting through ligand-receptor interaction allows the correct orientation of the enzyme on the substrate for enhanced activity towards the microbial target. The antibacterial efficiency was tested against Micrococcus luteus and revealed that surface protection was improved when lysozyme was grafted through the ligand-receptor interaction. These results suggest that bio-molecular interactions are promising for a sustainable grafting of antimicrobial agents on surfaces.

Handling Several Sugars at a Time: a Case Study of Xyloglucan Utilization by Ruminiclostridium cellulolyticum.

Xyloglucan utilization by Ruminiclostridium cellulolyticum was formerly shown to imply the uptake of large xylogluco-oligosaccharides, followed by cytosolic depolymerization into glucose, galactose, xylose, and cellobiose. This raises the question of how the anaerobic bacterium manages the simultaneous presence of multiple sugars. Using genetic and biochemical approaches targeting the corresponding metabolic pathways, we observed that, surprisingly, all sugars are catabolized, collectively, but glucose consumption is prioritized. Most selected enzymes display unusual features, especially the GTP-dependent hexokinase of glycolysis, which appeared reversible and crucial for xyloglucan utilization. In contrast, mutant strains lacking either galactokinase, cellobiose-phosphorylase, or xylulokinase still catabolize xyloglucan but display variably altered growth. Furthermore, the xylogluco-oligosaccharide depolymerization process appeared connected to the downstream pathways through an intricate network of competitive and noncompetitive inhibitions. Altogether, our data indicate that xyloglucan utilization by R. cellulolyticum relies on an energy-saving central carbon metabolism deviating from current bacterial models, which efficiently prevents carbon overflow. IMPORTANCE The study of the decomposition of recalcitrant plant biomass is of great interest as the limiting step of terrestrial carbon cycle and to produce plant-derived valuable chemicals and energy. While extracellular cellulose degradation and catabolism have been studied in detail, few publications describe the complete metabolism of hemicelluloses and, to date, the published models are limited to the extracellular degradation and sequential entry of simple sugars. Here, we describe how the model anaerobic bacterium Ruminiclostridium cellulolyticum deals with the synchronous intracellular release of glucose, galactose, xylose, and cellobiose upon cytosolic depolymerization of imported xyloglucan oligosaccharides. The described novel metabolic strategy involves the simultaneous activity of different metabolic pathways coupled to a network of inhibitions controlling the carbon flux and is distinct from the ubiquitously observed sequential uptake and metabolism of carbohydrates known as the diauxic shift. Our results highlight the diversity of cellular responses related to a complex environment.

Modulation of cellulosome composition in Clostridium cellulolyticum: adaptation to the polysaccharide environment revealed by proteomic and carbohydrate-active enzyme analyses.

Clostridium cellulolyticum is a model mesophilic anaerobic bacterium that efficiently degrades plant cell walls. The recent genome release offers the opportunity to analyse its complete degradation system. A total of 148 putative carbohydrate-active enzymes were identified, and their modular structures and activities were predicted. Among them, 62 dockerin-containing proteins bear catalytic modules from numerous carbohydrate-active enzymes' families and whose diversity reflects the chemical and structural complexity of the plant carbohydrate. The composition of the cellulosomes produced by C. cellulolyticum upon growth on different substrates (cellulose, xylan, and wheat straw) was investigated by LC MS/MS. The majority of the proteins encoded by the cip-cel operon, essential for cellulose degradation, were detected in all cellulosome preparations. In the presence of wheat straw, the natural and most complex of the substrates studied, additional proteins predicted to be involved in hemicellulose degradation were produced. A 32-kb gene cluster encodes the majority of these proteins, all harbouring carbohydrate-binding module 6 or carbohydrate-binding module 22 xylan-binding modules along dockerins. This newly identified xyl-doc gene cluster, specialised in hemicellulose degradation, comes in addition of the cip-cel operon for plant cell wall degradation. Hydrolysis efficiencies determined on the different substrates corroborates the finding that cellulosome composition is adapted to the growth substrate.

Uridine diphosphate N-acetylglucosamine orchestrates the interaction of GlmR with either YvcJ or GlmS in Bacillus subtilis.

In bacteria, glucosamine-6-phosphate (GlcN6P) synthase, GlmS, is an enzyme required for the synthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a precursor of peptidoglycan. In Bacillus subtilis, an UDP-GlcNAc binding protein, GlmR (formerly YvcK), essential for growth on non-glycolytic carbon sources, has been proposed to stimulate GlmS activity; this activation could be antagonized by UDP-GlcNAc. Using purified proteins, we demonstrate that GlmR directly stimulates GlmS activity and the presence of UDP-GlcNAc (at concentrations above 0.1 mM) prevents this regulation. We also showed that YvcJ, whose gene is associated with yvcK (glmR), interacts with GlmR in an UDP-GlcNAc dependent manner. Strains producing GlmR variants unable to interact with YvcJ show decreased transformation efficiency similar to that of a yvcJ null mutant. We therefore propose that, depending on the intracellular concentration of UDP-GlcNAc, GlmR interacts with either YvcJ or GlmS. When UDP-GlcNAc concentration is high, this UDP-sugar binds to YvcJ and to GlmR, blocking the stimulation of GlmS activity and driving the interaction between GlmR and YvcJ to probably regulate the cellular role of the latter. When the UDP-GlcNAc level is low, GlmR does not interact with YvcJ and thus does not regulate its cellular role but interacts with GlmS to stimulate its activity.

Catalytic subunit exchanges in the cellulosomes produced by Ruminiclostridium cellulolyticum suggest unexpected dynamics and adaptability of their enzymatic composition.

Cellulosomes are complex nanomachines produced by cellulolytic anaerobic bacteria such as Ruminiclostridium cellulolyticum (formerly known as Clostridium cellulolyticum). Cellulosomes are composed of a scaffoldin protein displaying several cohesin modules on which enzymatic components can bind to through their dockerin module. Although cellulosomes have been studied for decades, very little is known about the dynamics of complex assembly. We have investigated the ability of some dockerin-bearing enzymes to chase the catalytic subunits already bound onto a miniscaffoldin displaying a single cohesin. The stability of the preassembled enzyme-scaffoldin complex appears to depend on the nature of the dockerin, and we have identified a key position in the dockerin sequence that is involved in the stability of the complex with the cohesin. Depending on the residue occupying this position, the dockerin can establish with the cohesin partner either a nearly irreversible or a reversible interaction, independently of the catalytic domain associated with the dockerin. Site-directed mutagenesis of this residue can convert a dockerin able to form a highly stable complex with the miniscaffoldin into a reversible complex forming one and vice versa. We also show that refunctionalization can occur with natural purified cellulosomes. Altogether, our results shed light on the dynamics of cellulosomes, especially their capacity to be remodeled even after their assembly is 'achieved', suggesting an unforeseen adaptability of their enzymatic composition over time.