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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: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.

Project description:BackgroundEconomic feasibility and sustainability of lignocellulosic ethanol production requires the development of robust microorganisms that can efficiently degrade and convert plant biomass to ethanol. The anaerobic thermophilic bacterium Clostridium thermocellum is a candidate microorganism as it is capable of hydrolyzing cellulose and fermenting the hydrolysis products to ethanol and other metabolites. C. thermocellum achieves efficient cellulose hydrolysis using multiprotein extracellular enzymatic complexes, termed cellulosomes.Methodology/principal findingsIn this study, we used quantitative proteomics (multidimensional LC-MS/MS and (15)N-metabolic labeling) to measure relative changes in levels of cellulosomal subunit proteins (per CipA scaffoldin basis) when C. thermocellum ATCC 27405 was grown on a variety of carbon sources [dilute-acid pretreated switchgrass, cellobiose, amorphous cellulose, crystalline cellulose (Avicel) and combinations of crystalline cellulose with pectin or xylan or both]. Cellulosome samples isolated from cultures grown on these carbon sources were compared to (15)N labeled cellulosome samples isolated from crystalline cellulose-grown cultures. In total from all samples, proteomic analysis identified 59 dockerin- and 8 cohesin-module containing components, including 16 previously undetected cellulosomal subunits. Many cellulosomal components showed differential protein abundance in the presence of non-cellulose substrates in the growth medium. Cellulosome samples from amorphous cellulose, cellobiose and pretreated switchgrass-grown cultures displayed the most distinct differences in composition as compared to cellulosome samples from crystalline cellulose-grown cultures. While Glycoside Hydrolase Family 9 enzymes showed increased levels in the presence of crystalline cellulose, and pretreated switchgrass, in particular, GH5 enzymes showed increased levels in response to the presence of cellulose in general, amorphous or crystalline.Conclusions/significanceOverall, the quantitative results suggest a coordinated substrate-specific regulation of cellulosomal subunit composition in C. thermocellum to better suit the organism's needs for growth under different conditions. To date, this study provides the most comprehensive comparison of cellulosomal compositional changes in C. thermocellum in response to different carbon sources. Such studies are vital to engineering a strain that is best suited to grow on specific substrates of interest and provide the building blocks for constructing designer cellulosomes with tailored enzyme composition for industrial ethanol production.

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

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