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Distinctive ligand-binding specificities of tandem PA14 biomass-sensory elements from Clostridium thermocellum and Clostridium clariflavum.

ABSTRACT: Cellulolytic clostridia use a highly efficient cellulosome system to degrade polysaccharides. To regulate genes encoding enzymes of the multi-enzyme cellulosome complex, certain clostridia contain alternative sigma I (σ^I ) factors that have cognate membrane-associated anti-σ^I factors (RsgIs) which act as polysaccharide sensors. In this work, we analyzed the structure-function relationship of the extracellular sensory elements of Clostridium (Ruminiclostridium) thermocellum and Clostridium clariflavum (RsgI3 and RsgI4, respectively). These elements were selected for comparison, as each comprised two tandem PA14-superfamily motifs. The X-ray structures of the PA14 modular dyads from the two bacterial species were determined, both of which showed a high degree of structural and sequence similarity, although their binding preferences differed. Bioinformatic approaches indicated that the DNA sequence of promoter of sigI/rsgI operons represents a strong signature, which helps to differentiate binding specificity of the structurally similar modules. The σ^I4 -dependent C. clariflavum promoter sequence correlates with binding of RsgI4_PA14 to xylan and was identified in genes encoding xylanases, whereas the σ^I3 -dependent C. thermocellum promoter sequence correlates with RsgI3_PA14 binding to pectin and regulates pectin degradation-related genes. Structural similarity between clostridial PA14 dyads to PA14-containing proteins in yeast helped identify another crucial signature element: the calcium-binding loop 2 (CBL2), which governs binding specificity. Variations in the five amino acids that constitute this loop distinguish the pectin vs xylan specificities. We propose that the first module (PA14^A ) is dominant in directing the binding to the ligand in both bacteria. The two X-ray structures of the different PA14 dyads represent the first reported structures of tandem PA14 modules.

SUBMITTER: Grinberg IR

PROVIDER: S-EPMC6852018 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe thermophilic anaerobic bacterium Clostridium thermocellum is a multifunctional ethanol producer, capable of both saccharification and fermentation, that is central to the consolidated bioprocessing (CBP) approach of converting lignocellulosic biomass to ethanol without external enzyme supplementation. Although CBP organisms have evolved efficient machinery for biomass deconstruction, achieving complete solubilization requires targeted approaches, such as pretreatment, to prepare recalcitrant biomass feedstocks for further biological digestion. Here, differences between how C. thermocellum and fungal cellulases respond to senescent switchgrass prepared by four different pretreatment techniques revealed relationships between biomass substrate composition and its digestion by the two biological approaches.ResultsAlamo switchgrass was pretreated using hydrothermal, dilute acid, dilute alkali, and co-solvent-enhanced lignocellulosic fractionation (CELF) pretreatments to produce solids with varying glucan, xylan, and lignin compositions. C. thermocellum achieved highest sugar release and metabolite production from de-lignified switchgrass prepared by CELF and dilute alkali pretreatments demonstrating greater resilience to the presence of hemicellulose sugars than fungal enzymes. 100% glucan solubilization and glucan plus xylan release from switchgrass were achieved using the CELF-CBP combination. Lower glucan solubilization and metabolite production by C. thermocellum was observed on solids prepared by dilute acid and hydrothermal pretreatments with higher xylan removal from switchgrass than lignin removal. Further, C. thermocellum (2% by volume inoculum) showed ~?48% glucan solubilization compared to <?10% through fungal enzymatic hydrolysis (15 and 65 mg protein/g glucan loadings) of unpretreated switchgrass indicating the effectiveness of C. thermocellum's cellulosome. Overall, C. thermocellum performed equivalent to 65 and better than 15 mg protein/g glucan fungal enzymatic hydrolysis on all substrates except CELF-pretreated substrates. CELF pretreatments of switchgrass produced solids that were highly digestible regardless of whether C. thermocellum or fungal enzymes were chosen.ConclusionsThe unparalleled comprehensive nature of this work with a comparison of four pretreatment and two biological digestion techniques provides a strong platform for future integration of pretreatment with CBP. Lignin removal had a more positive impact on biological digestion of switchgrass than xylan removal from the biomass. However, the impact of switchgrass structural properties, including cellulose, hemicellulose, and lignin characterization, would provide a better understanding of lignocellulose deconstruction.

Project description:UnlabelledClostridium clariflavum is an anaerobic, cellulosome-forming thermophile, containing in its genome genes for a large number of cellulosomal enzyme and a complex scaffoldin system. Previously, we described the major cohesin-dockerin interactions of the cellulosome components, and on this basis a model of diverse cellulosome assemblies was derived. In this work, we cultivated C. clariflavum on cellobiose-, microcrystalline cellulose-, and switchgrass-containing media and isolated cell-free cellulosome complexes from each culture. Gel filtration separation of the cellulosome samples revealed two major fractions, which were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to identify the key players of the cellulosome assemblies therein. From the 13 scaffoldins present in the C. clariflavum genome, 11 were identified, and a variety of enzymes from different glycoside hydrolase and carbohydrate esterase families were identified, including the glycoside hydrolase families GH48, GH9, GH5, GH30, GH11, and GH10. The expression level of the cellulosomal proteins varied as a function of the carbon source used for cultivation of the bacterium. In addition, the catalytic activity of each cellulosome was examined on different cellulosic substrates, xylan and switchgrass. The cellulosome isolated from the microcrystalline cellulose-containing medium was the most active of all the cellulosomes that were tested. The results suggest that the expression of the cellulosome proteins is regulated by the type of substrate in the growth medium. Moreover, both cell-free and cell-bound cellulosome complexes were produced which together may degrade the substrate in a synergistic manner. These observations are compatible with our previously published model of cellulosome assemblies in this bacterium.ImportanceBecause the reservoir of unsustainable fossil fuels, such as coal, petroleum, and natural gas, is overutilized and continues to contribute to environmental pollution and CO2 emission, the need for appropriate alternative energy sources becomes more crucial. Bioethanol produced from dedicated crops and cellulosic waste can provide a partial answer, yet a cost-effective production method must be developed. The cellulosome system of the anaerobic thermophile C. clariflavum comprises a large number of cellulolytic and hemicellulolytic enzymes, which self-assemble in a number of different cellulosome architectures for enhanced cellulosic biomass degradation. Identification of the major cellulosomal components expressed during growth of the bacterium and their influence on its catalytic capabilities provide insight into the performance of the remarkable cellulosome of this intriguing bacterium. The findings, together with the thermophilic characteristics of the proteins, render C. clariflavum of great interest for future use in industrial cellulose conversion processes.

Project description:The Gram-positive, anaerobic, cellulolytic, thermophile Clostridium (Ruminiclostridium) thermocellum secretes a multi-enzyme system called the cellulosome to solubilize plant cell wall polysaccharides. During the saccharolytic process, the enzymatic composition of the cellulosome is modulated according to the type of polysaccharide(s) present in the environment. C. thermocellum has a set of eight alternative RNA polymerase sigma (?) factors that are activated in response to extracellular polysaccharides and share sequence similarity to the Bacillus subtilis ?I factor. The aim of the present work was to demonstrate whether individual C. thermocellum ?I-like factors regulate specific cellulosomal genes, focusing on C. thermocellum ?I6 and ?I3 factors. To search for putative ?I6- and ?I3-dependent promoters, bioinformatic analysis of the upstream regions of the cellulosomal genes was performed. Because of the limited genetic tools available for C. thermocellum, the functionality of the predicted ?I6- and ?I3-dependent promoters was studied in B. subtilis as a heterologous host. This system enabled observation of the activation of 10 predicted ?I6-dependent promoters associated with the C. thermocellum genes: sigI6 (itself, Clo1313_2778), xyn11B (Clo1313_0522), xyn10D (Clo1313_0177), xyn10Z (Clo1313_2635), xyn10Y (Clo1313_1305), cel9V (Clo1313_0349), cseP (Clo1313_2188), sigI1 (Clo1313_2174), cipA (Clo1313_0627), and rsgI5 (Clo1313_0985). Additionally, we observed the activation of 4 predicted ?I3-dependent promoters associated with the C. thermocellum genes: sigI3 (itself, Clo1313_1911), pl11 (Clo1313_1983), ce12 (Clo1313_0693) and cipA. Our results suggest possible regulons of ?I6 and ?I3 in C. thermocellum, as well as the ?I6 and ?I3 promoter consensus sequences. The proposed -35 and -10 promoter consensus elements of ?I6 are CNNAAA and CGAA, respectively. Additionally, a less conserved CGA sequence next to the C in the -35 element and a highly conserved AT sequence three bases downstream of the -10 element were also identified as important nucleotides for promoter recognition. Regarding ?I3, the proposed -35 and -10 promoter consensus elements are CCCYYAAA and CGWA, respectively. The present study provides new clues for understanding these recently discovered alternative ?I factors.

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Distinctive ligand-binding specificities of tandem PA14 biomass-sensory elements from Clostridium thermocellum and Clostridium clariflavum.

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