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.

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.

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.

The xyl-doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α-l-arabinofuranosidases with complementary modes of action.

BACKGROUND: The α-l-arabinofuranosidases (α-l-ABFs) are exoenzymes involved in the hydrolysis of α-l-arabinosyl linkages in plant cell wall polysaccharides. They play a crucial role in the degradation of arabinoxylan and arabinan and they are used in many biotechnological applications. Analysis of the genome of R. cellulolyticum showed that putative cellulosomal α-l-ABFs are exclusively encoded by the xyl-doc gene cluster, a large 32-kb gene cluster. Indeed, among the 14 Xyl-Doc enzymes encoded by this gene cluster, 6 are predicted to be α-l-ABFs belonging to the CAZyme families GH43 and GH62. RESULTS: The biochemical characterization of these six Xyl-Doc enzymes revealed that four of them are α-l-ABFs. GH4316-1229 (RcAbf43A) which belongs to the subfamily 16 of the GH43, encoded by the gene at locus Ccel_1229, has a low specific activity on natural substrates and can cleave off arabinose decorations located at arabinoxylan chain extremities. GH4310-1233 (RcAbf43Ad2,3), the product of the gene at locus Ccel_1233, belonging to subfamily 10 of the GH43, can convert the double arabinose decorations present on arabinoxylan into single O2- or O3-linked decorations with high velocity (k cat = 16.6 ± 0.6 s-1). This enzyme acts in synergy with GH62-1234 (RcAbf62Am2,3), the product of the gene at locus Ccel_1234, a GH62 α-l-ABF which hydrolyzes α-(1 → 3) or α-(1 → 2)-arabinosyl linkages present on polysaccharides and arabinoxylooligosaccharides monodecorated. Finally, a bifunctional enzyme, GH62-CE6-1240 (RcAbf62Bm2,3Axe6), encoded by the gene at locus Ccel_1240, which contains a GH62-α-l-ABF module and a carbohydrate esterase (CE6) module, catalyzes deacylation of plant cell wall polymers and cleavage of arabinosyl mono-substitutions. These enzymes are also active on arabinan, a component of the type I rhamnogalacturonan, showing their involvement in pectin degradation. CONCLUSION: Arabinofuranosyl decorations on arabinoxylan and pectin strongly inhibit the action of xylan-degrading enzymes and pectinases. α-l-ABFs encoded by the xyl-doc gene cluster of R. cellulolyticum can remove all the decorations present in the backbone of arabinoxylan and arabinan, act synergistically, and, thus, play a crucial role in the degradation of plant cell wall polysaccharides.

Turning a potent family-9 free cellulase into an operational cellulosomal component and vice versa.

Ruminiclostridium cellulolyticum and Lachnoclostridium phytofermentans are cellulolytic clostridia either producing extracellular multienzymatic complexes termed cellulosomes or secreting free cellulases respectively. In the free state, the cellulase Cel9A secreted by L. phytofermentans is much more active on crystalline cellulose than any cellulosomal family-9 enzyme produced by R. cellulolyticum. Nevertheless, the incorporation of Cel9A in vitro in hybrid cellulosomes was formerly shown to generate artificial complexes with altered activity, whereas its incorporation in vivo in native R. cellulolyticum cellulosomes resulted in a strain displaying a weakened cellulolytic phenotype. In this study, we investigated why Cel9A is so potent in the free state but functions poorly as a cellulosomal component, in contrast to the most similar enzyme synthesized by R. cellulolyticum, Cel9G, weakly active in the free state but whose activity on crystalline cellulose is drastically increased in cellulosomes. We show that the removal of the C-terminal moiety of Cel9A encompassing the two X2 modules and the family-3b carbohydrate binding module (CBM3b), reduces its activity on crystalline cellulose. Grafting a dockerin module further diminishes the activity, but this truncated cellulosomal form of Cel9A displays important synergies in hybrid cellulosomes with the pivotal family-48 cellulosomal enzyme of R. cellulolyticum. The exact inverse approach was applied to the cellulosomal Cel9G. Grafting the two X2 modules and the CBM3b of Cel9A to Cel9G strongly increases its activity on crystalline cellulose, to reach Cel9A activity levels. Altogether these data emphasize the specific features required to generate an efficient free or cellulosomal family-9 cellulase.

A seven-gene cluster in Ruminiclostridium cellulolyticum is essential for signalization, uptake and catabolism of the degradation products of cellulose hydrolysis.

BACKGROUND: Like a number of anaerobic and cellulolytic Gram-positive bacteria, the model microorganism Ruminiclostridium cellulolyticum produces extracellular multi-enzymatic complexes called cellulosomes, which efficiently degrade the crystalline cellulose. Action of the complexes on cellulose releases cellobiose and longer cellodextrins but to date, little is known about the transport and utilization of the produced cellodextrins in the bacterium. A better understanding of the uptake systems and fermentation of sugars derived from cellulose could have a major impact in the field of biofuels production. RESULTS: We characterized a putative ABC transporter devoted to cellodextrins uptake, and a cellobiose phosphorylase (CbpA) in R. cellulolyticum. The genes encoding the components of the ABC transporter (a binding protein CuaA and two integral membrane proteins) and CbpA are expressed as a polycistronic transcriptional unit induced in the presence of cellobiose. Upstream, another polycistronic transcriptional unit encodes a two-component system (sensor and regulator), and a second binding protein CuaD, and is constitutively expressed. The products might form a three-component system inducing the expression of cuaABC and cbpA since we showed that CuaR is able to recognize the region upstream of cuaA. Biochemical analysis showed that CbpA is a strict cellobiose phosphorylase inactive on longer cellodextrins; CuaA binds to all cellodextrins (G2-G5) tested, whereas CuaD is specific to cellobiose and presents a higher affinity to this sugar. This results are in agreement with their function in transport and signalization, respectively. Characterization of a cuaD mutant, and its derivatives, indicated that the ABC transporter and CbpA are essential for growth on cellobiose and cellulose. CONCLUSIONS: For the first time in a Gram-positive strain, we identified a three-component system and a conjugated ABC transporter/cellobiose phosphorylase system which was shown to be essential for the growth of the model cellulolytic bacterium R. cellulolyticum on cellobiose and cellulose. This efficient and energy-saving system of transport and phosphorolysis appears to be the major cellobiose utilization pathway in R. cellulolyticum, and seems well adapted to cellulolytic life-style strain. It represents a new way to enable engineered strains to utilize cellodextrins for the production of biofuels or chemicals of interest from cellulose.

Mechanisms involved in xyloglucan catabolism by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum.

Xyloglucan, a ubiquitous highly branched plant polysaccharide, was found to be rapidly degraded and metabolized by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum. Our study shows that at least four cellulosomal enzymes displaying either endo- or exoxyloglucanase activities, achieve the extracellular degradation of xyloglucan into 4-glucosyl backbone xyloglucan oligosaccharides. The released oligosaccharides (composed of up to 9 monosaccharides) are subsequently imported by a highly specific ATP-binding cassette transporter (ABC-transporter), the expression of the corresponding genes being strongly induced by xyloglucan. This polysaccharide also triggers the synthesis of cytoplasmic ß-galactosidase, α-xylosidase, and ß-glucosidase that act sequentially to convert the imported oligosaccharides into galactose, xylose, glucose and unexpectedly cellobiose. Thus R. cellulolyticum has developed an energy-saving strategy to metabolize this hemicellulosic polysaccharide that relies on the action of the extracellular cellulosomes, a highly specialized ABC-transporter, and cytoplasmic enzymes acting in a specific order. This strategy appears to be widespread among cellulosome-producing mesophilic bacteria which display highly similar gene clusters encoding the cytosolic enzymes and the ABC-transporter.

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.

A two-component system (XydS/R) controls the expression of genes encoding CBM6-containing proteins in response to straw in Clostridium cellulolyticum.

The composition of the cellulosomes (multi enzymatic complexes involved in the degradation of plant cell wall polysaccharides) produced by Clostridium cellulolyticum differs according to the growth substrate. In particular, the expression of a cluster of 14 hemicellulase-encoding genes (called xyl-doc) seems to be induced by the presence of straw and not of cellulose. Genes encoding a putative two-component regulation system (XydS/R) were found upstream of xyl-doc. First evidence for the involvement of the response regulator, XydR, part of this two-component system, in the expression of xyl-doc genes was given by the analysis of the cellulosomes produced by a regulator overproducing strain when grown on cellulose. Nano-LC MS/MS analysis allowed the detection of the products of all xyl-doc genes and of the product of the gene at locus Ccel_1656 predicted to bear a carbohydrate binding domain targeting hemicellulose. RT-PCR experiments further demonstrated that the regulation occurs at the transcriptional level and that all xyl-doc genes are transcriptionally linked. mRNA quantification in a regulator knock-out strain and in its complemented derivative confirmed the involvement of the regulator in the expression of xyl-doc genes and of the gene at locus Ccel_1656 in response to straw. Electrophoretic mobility shift assays using the purified regulator further demonstrated that the regulator binds to DNA regions located upstream of the first gene of the xyl-doc gene cluster and upstream of the gene at locus Ccel_1656.

Random mutagenesis of Clostridium cellulolyticum by using a Tn1545 derivative.

Further understanding of the plant cell wall degradation system of Clostridium cellulolyticum and the possibility of metabolic engineering in this species highlight the need for a means of random mutagenesis. Here, we report the construction of a Tn1545-derived delivery tool which allows monocopy random insertion within the genome.

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.

Transcriptional regulation of the Clostridium cellulolyticum cip-cel operon: a complex mechanism involving a catabolite-responsive element.

The cip-cel cluster of genes plays an important role in the catabolism of the substrate cellulose by Clostridium cellulolyticum. It encodes several key components of the cellulosomes, including the scaffolding protein CipC and the major cellulase Cel48F. All the genes of this cluster display linked transcription, focusing attention on the promoter upstream from the first gene, cipC. We analyzed the regulation of the cipC promoter using a transcriptional fusion approach. A single promoter is located between nucleotides -671 and -643 with respect to the ATG start codon, and the large mRNA leader sequence is processed at position -194. A catabolite-responsive element (CRE) 414 nucleotides downstream from the transcriptional start site has been shown to be involved in regulating this operon by a carbon catabolite repression mechanism. This CRE is thought to bind a CcpA-like regulator complexed with a P-Ser-Crh-like protein. Sequences surrounding the promoter sequence may also be involved in direct (sequence-dependent DNA curvature) or indirect (unknown regulator binding) regulation.

Enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum explored by two-dimensional analysis: identification of seven genes encoding new dockerin-containing proteins.

The enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum grown on crystalline cellulose as a sole carbon and energy source was explored by two-dimensional electrophoresis. The cellulolytic system of C. cellulolyticum is composed of at least 30 dockerin-containing proteins (designated cellulosomal proteins) and 30 noncellulosomal components. Most of the known cellulosomal proteins, including CipC, Cel48F, Cel8C, Cel9G, Cel9E, Man5K, Cel9M, and Cel5A, were identified by using two-dimensional Western blot analysis with specific antibodies, whereas Cel5N, Cel9J, and Cel44O were identified by using N-terminal sequencing. Unknown enzymes having carboxymethyl cellulase or xylanase activities were detected by zymogram analysis of two-dimensional gels. Some of these enzymes were identified by N-terminal sequencing as homologs of proteins listed in the NCBI database. Using Trap-Dock PCR and DNA walking, seven genes encoding new dockerin-containing proteins were cloned and sequenced. Some of these genes are clustered. Enzymes encoded by these genes belong to glycoside hydrolase families GH2, GH9, GH10, GH26, GH27, and GH59. Except for members of family GH9, which contains only cellulases, the new modular glycoside hydrolases discovered in this work could be involved in the degradation of different hemicellulosic substrates, such as xylan or galactomannan.

ISCce1 and ISCce2, two novel insertion sequences in Clostridium cellulolyticum.

Two new insertion sequences, ISCce1 and ISCce2, were found to be inserted into the cipC gene of spontaneous mutants of Clostridium cellulolyticum. In these insertional mutants, the cipC gene was disrupted either by ISCce1 alone or by both ISCce1 and ISCce2. ISCce1 is 1,292 bp long and has one open reading frame. The open reading frame encodes a putative 348-amino-acid protein with significant levels of identity with putative proteins having unknown functions and with some transposases belonging to the IS481 and IS3 families. Imperfect 23-bp inverted repeats were found near the extremities of ISCce1. ISCce2 is 1,359 bp long, carries one open reading frame, and has imperfect 35-bp inverted repeats at its termini. The open reading frame encodes a putative 398-amino-acid protein. This protein shows significant levels of identity with transposases belonging to the IS256 family. Upon transposition, both ISCce1 and ISCce2 generate 8-bp direct repeats of the target sequence, but no consensus sequences could be identified at either insertion site. ISCce1 is copied at least 20 times in the genome, as assessed by Southern blot analysis. ISCce2 was found to be mostly inserted into ISCce1. In addition, as neither of the elements was detected in seven other Clostridium species, we concluded that they may be specific to the C. cellulolyticum strain used.

Construction and physiological studies of hydrogenase depleted mutants of Desulfovibrio fructosovorans.

Desulfovibrio fructosovorans possesses two periplasmic hydrogenases (a nickel-iron and an iron hydrogenase) and a cytoplasmic NADP-dependent hydrogenase. The hydAB genes encoding the periplasmic iron hydrogenase were replaced, in the wild-type strain as well as in single mutants depleted of one of the other two hydrogenases, by the acc1 gene encoding resistance to gentamycin. Molecular characterization and remaining activity measurements of the resulting single and double mutants were performed. All mutated strains exhibited similar growth when H(2) was the electron donor but they grew differently on fructose, lactate or pyruvate as electron donors. Our results indicate that the loss of one enzyme might be compensated by another even though hydrogenases have different localization in the cells.

Evidence for a fourth hydrogenase in Desulfovibrio fructosovorans.

A strain devoid of the three hydrogenases characterized for Desulfovibrio fructosovorans was constructed using marker exchange mutagenesis. As expected, the H(2)-dependent methyl viologen reduction activity of the strain was null, but physiological studies showed no striking differences between the mutated and wild-type strains. The H(+)-D(2) exchange activity measured in the mutated strain indicates the presence of a fourth hydrogenase in D. fructosovorans.