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

ABSTRACT: 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.IMPORTANCE Ruminiclostridium 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.

SUBMITTER: Kampik C

PROVIDER: S-EPMC7531976 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:BackgroundIn anaerobic cellulolytic micro-organisms, cellulolysis results in the action of several cellulases gathered in extracellular multi-enzyme complexes called cellulosomes. Their action releases cellobiose and longer cellodextrins which are imported and further degraded in the cytosol to fuel the cells. In Ruminiclostridium cellulolyticum, an anaerobic and cellulolytic mesophilic bacteria, three cellodextrin phosphorylases named CdpA, CdpB, and CdpC, were identified in addition to the cellobiose phosphorylase (CbpA) previously characterized. The present study aimed at characterizing them, exploring their implication during growth on cellulose to better understand the life-style of cellulolytic bacteria on such substrate.ResultsThe three cellodextrin phosphorylases from R. cellulolyticum displayed marked different enzymatic characteristics. They are specific for cellodextrins of different lengths and present different k cat values. CdpC is the most active enzyme before CdpA, and CdpB is weakly active. Modeling studies revealed that a mutation of a conserved histidine residue in the phosphate ion-binding pocket in CdpB and CdpC might explain their activity-level differences. The genes encoding these enzymes are scattered over the chromosome of R. cellulolyticum and only the expression of the gene encoding the cellobiose phosphorylase and the gene cdpA is induced during cellulose growth. Characterization of four independent mutants constructed in R. cellulolyticum for each of the cellobiose and cellodextrin phosphorylases encoding genes indicated that only the cellobiose phosphorylase is essential for growth on cellulose.ConclusionsUnexpectedly, the cellobiose phosphorylase but not the cellodextrin phosphorylases is essential for the growth of the model bacterium on cellulose. This suggests that the bacterium adopts a "short" dextrin strategy to grow on cellulose, even though the use of long cellodextrins might be more energy-saving. Our results suggest marked differences in the cellulose catabolism developed among cellulolytic bacteria, which is a result that might impact the design of future engineered strains for biomass-to-biofuel conversion.

Project description:BackgroundAnaerobic, mesophilic, and cellulolytic Ruminiclostridium cellulolyticum produces an efficient cellulolytic extracellular complex named cellulosome, which consist of a non-catalytic multi-functional integrating subunit, organizing the various catalytic subunits into the complex. Main components of cellulosome were encoded by the cip-cel operon in R. cellulolyticum, and their stoichiometry is controlled by the mechanism of selective RNA processing and stabilization, which allows to confer each processed RNA portion from the cip-cel mRNA on different fates due to their stability and resolve the potential contradiction between the equimolar stoichiometry of transcripts with a within a transcription unit and the non-equimolar stoichiometry of subunits.ResultsIn this work, RNA processing events were found to occur at six intergenic regions (IRs) harboring stem-loop structures in cip-cel operon. These stem-loops not only stabilize processed transcripts at their both ends, but also act as cleavage signals specifically recognized by endoribonucleases. We further demonstrated that cleavage sites were often located downstream or 3' end of their associated stem-loops that could be classified into two types, with distinct GC-rich stems being required for RNA cleavage. However, the cleavage site in IR4 was found to be located upstream of the stem-loop, as determined by the bottom AT-pair region of this stem-loop, together with its upstream structure. Thus, our findings reveal the structural requirements for processing of cip-cel transcripts, which can be potentially used to control the stoichiometry of gene expression in an operon.ConclusionsOur findings reveal that stem-loop structures acting as RNA cleavage signals not only can be recognized by endoribonucleases and determine the location of cleavage sites but also determine the stoichiometry of their flanking processed transcripts by controlling stability in cip-cel operon. These features represent a complexed regulation of cellulosome in the post-transcriptional level, which can be exploited for designing synthetic elements to control gene expression.

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

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