Clostridium thermocellum cellulosomal genes are regulated by extracytoplasmic polysaccharides via alternative sigma factors.
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SUBMITTER: Nataf Y
PROVIDER: S-EPMC2972930 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
| S-EPMC4711584 | biostudies-literature
Project description:Background:Enzymatic conversion of lignocellulosic biomass into soluble sugars is a major bottleneck in the plant biomass utilization. Several anaerobic organisms cope these issues via multiple-enzyme complex system so called 'cellulosome'. Hence, we proposed a "biomimic operon" concept for making an artificial cellulosome which can be used as a promising tool for the expression of cellulosomal enzymes in Bacillus subtilis. Results:According to the proteomic analysis of Clostridium thermocellum ATCC27405 induced by Avicel or cellobiose, we selected eight highly expressed cellulosomal genes including a scaffoldin protein gene (cipA), a cell-surface anchor gene (sdbA), two exoglucanase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ). Arranging these eight genes in two different orders, we constructed two different polycistronic operons using the ordered gene assembly in Bacillus method. This is the first study to express the whole CipA along with cellulolytic enzymes in B. subtilis. Each operon was successfully expressed in B. subtilis RM125, and the protein complex assembly, cellulose-binding ability, thermostability, and cellulolytic activity were demonstrated. The operon with a higher xylanase activity showed greater saccharification on complex cellulosic substrates such as Napier grass than the other operon. Conclusions:In this study, a strategy for constructing an efficient cellulosome system was developed and two different artificial cellulosomal operons were constructed. Both operons could efficiently express the cellulosomal enzymes and exhibited cellulose saccharification. This strategy can be applied to different industries with cellulose-containing materials, such as papermaking, biofuel, agricultural compost, mushroom cultivation, and waste processing industries.
| S-EPMC5991470 | biostudies-literature
Project description:The nucleotide sequence of the Clostridium thermocellum F7 cbhA gene, coding for the cellobiohydrolase CbhA, has been determined. An open reading frame encoding a protein of 1,230 amino acids was identified. Removal of a putative signal peptide yields a mature protein of 1,203 amino acids with a molecular weight of 135,139. Sequence analysis of CbhA reveals a multidomain structure of unusual complexity consisting of an N-terminal cellulose binding domain (CBD) homologous to CBD family IV, an immunoglobulin-like beta-barrel domain, a catalytic domain homologous to cellulase family E1, a duplicated domain similar to fibronectin type III (Fn3) modules, a CBD homologous to family III, a highly acidic linker region, and a C-terminal dockerin domain. The cellulosomal localization of CbhA was confirmed by Western blot analysis employing polyclonal antibodies raised against a truncated enzymatically active version of CbhA. CbhA was identified as cellulosomal subunit S3 by partial amino acid sequence analysis. Comparison of the multidomain structures indicates striking similarities between CbhA and a group of cellulases from actinomycetes. Average linkage cluster analysis suggests a coevolution of the N-terminal CBD and the catalytic domain and its spread by horizontal gene transfer among gram-positive cellulolytic bacteria.
| S-EPMC107808 | biostudies-literature
Project description:The high cellulose digestion capability of the anaerobic thermophilic bacterium, Clostridium thermocellum, can be attributed to its efficient glycoside hydrolase system, consisting of both a free-enzyme system and a cellulosomal system, wherein carbohydrate active enzymes (CAZymes) are organized by primary and secondary scaffoldin proteins into large protein complexes bound to the bacterial cell wall. Using an integrated series of experiments encompassing polysaccharide depolymerization assays, direct biochemical analyses and transcriptomics, we show herein that, in addition to cell-bound cellulosomes and “free” enzymes, C. thermocellum naturally employs a system of cell-free cellulosomes that are not designed to be tethered to the cell and can diffuse away to attack polysaccharide substrates at some distance from the cell. By characterizing the cells and the secretomes of a series of mutants in which genes for either individual scaffoldins or combinations thereof are deleted, we demonstrate that the cellulosome is far more important in cellulose degradation than are the free enzymes, and that the primary scaffoldin CipA is much more important for the action of the cellulosomes than is the collective contribution of the secondary scaffoldins. Extensive transcriptomics analyses confirm the above results and extend the study to the effects of scaffoldin deletions on the organism as a whole.
2015-02-28 | GSE63883 | GEO
Project description:BackgroundClostridium thermocellum is a thermophilic anaerobic bacterium that degrades cellulose by using a highly effective cellulosome, a macromolecular complex consisting of multiple cellulose degrading enzymes organized and attached to the cell surface by non-catalytic scaffoldins. However, due largely to lack of efficient methods for genetic manipulation of C. thermocellum, it is still unclear how the different scaffoldins and their functional modules contribute to cellulose hydrolysis.ResultsWe constructed C. thermocellum mutants with the primary scaffoldin CipA (cellulosome-integrating protein A) truncated at different positions or lacking four different secondary scaffoldins by using a newly developed thermotargetron system, and we analyzed cellulose hydrolysis, cellulosome formation, and cellulose binding of the mutants. A CipA truncation that deletes six type I cohesin modules, which bind cellulolytic enzymes, decreased cellulose hydrolysis rates by 46%, and slightly longer truncations that also delete the carbohydrate binding module decreased rates by 89 to 92%, indicating strong cellulosome-substrate synergy. By contrast, a small CipA truncation that deletes only the C-terminal type II dockerin (XDocII) module detached cellulosomes from the cells, but decreased cellulose hydrolysis rates by only 9%, suggesting a relatively small contribution of cellulosome-cell synergy. Disruptants lacking any of four different secondary scaffoldins (OlpB, 7CohII, Orf2p, or SdbA) showed moderately decreased cellulose hydrolysis rates, suggesting additive contributions. Surprisingly, the CipA-ΔXDocII mutant, which lacks cell-associated polycellulosomes, adheres to cellulose almost as strongly as wild-type cells, revealing an alternate, previously unknown cellulose-binding mechanism.ConclusionsOur results emphasize the important role of cellulosome-substrate synergy in cellulose degradation, demonstrate a contribution of secondary scaffoldins, and suggest a previously unknown, non-cellulosomal system for binding insoluble cellulose. Our findings provide new insights into cellulosome function and impact genetic engineering of microorganisms to enhance bioconversions of cellulose substrates.
| S-EPMC4045903 | biostudies-literature
Project description:The anaerobic, thermophilic cellulolytic bacterium Clostridium thermocellum is known for its elaborate cellulosome complex, but it also produces a separate free cellulase system. Among the free enzymes, the noncellulosomal enzyme Cel9I is a processive endoglucanase whose sequence and architecture are very similar to those of the cellulosomal enzyme Cel9R; likewise, the noncellulosomal exoglucanase Cel48Y is analogous to the principal cellulosomal enzyme Cel48S. In this study we used the designer cellulosome approach to examine the interplay of prominent cellulosomal and noncellulosomal cellulases from C. thermocellum. Toward this end, we converted the cellulosomal enzymes to noncellulosomal chimeras by swapping the dockerin module of the cellulosomal enzymes with a carbohydrate-binding module from the free enzyme analogues and vice versa. This enabled us to study the importance of the targeting effect of the free enzymes due to their carbohydrate-binding module and the proximity effect for cellulases on the designer cellulosome. C. thermocellum is the only cellulosome-producing bacterium known to express two different glycoside hydrolase family 48 enzymes and thus the only bacterial system that can currently be used for such studies. The different activities with crystalline cellulose were examined, and the results demonstrated that the individual chimeric cellulases were essentially equivalent to the corresponding wild-type analogues. The wild-type cellulases displayed a synergism of about 1.5-fold; the cellulosomal pair acted synergistically when they were converted into free enzymes, whereas the free enzymes acted synergistically mainly in the wild-type state. The targeting effect was found to be the major factor responsible for the elevated activity observed for these specific enzyme combinations, whereas the proximity effect appeared to play a negligible role.
| S-EPMC2869131 | biostudies-literature
Project description:Bacterial genomes are being sequenced at an exponentially increasing rate, but our inability to decipher their transcriptional wiring limits our ability to derive new biology from these sequences. De novo determination of regulatory interactions requires accurate prediction of regulators' DNA binding and precise determination of biologically significant binding sites. Here we address these challenges by solving the DNA-specificity code of extracytoplasmic function sigma factors (ECF σs), a major family of bacterial regulators, and determining their putative regulons. We generated an aligned collection of ECF σs and their promoters by leveraging the autoregulatory nature of ECF σs as a means of promoter discovery and analyzed it to identify and characterize the conserved amino acid-nucleotide interactions that determine promoter specificity. This enabled de novo prediction of ECF σ specificity, which we combined with a statistically rigorous phylogenetic footprinting pipeline based on precomputed orthologs to predict the direct targets of ∼67% of ECF σs. This global survey indicated that some ECF σs are conserved global regulators controlling many genes throughout the genome, which are important under many conditions, while others are local regulators, controlling a few closely linked genes in response to specific stimuli in select species. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.
| S-EPMC7776599 | biostudies-literature
Project description:Bacteria must sense alterations in their environment and respond with changes in function and/or structure in order to cope. Extracytoplasmic function sigma factors (ECF ?s) modulate transcription in response to cellular and environmental signals. The symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti carries genes for 11 ECF-like ?s (RpoE1 to -E10 and FecI). We hypothesized that some of these play a role in mediating the interaction between the bacterium and its plant symbiotic partner. The bacterium senses changes in its immediate environment as it establishes contact with the plant root, initiates invasion of the plant as the root nodule is formed, traverses several root cell layers, and enters plant cortical cells via endocytosis. We used genetics, transcriptomics, and functionality to characterize the entire S. meliloti cohort of ECF ?s. We discovered new targets for individual ?s, confirmed others by overexpressing individual ECF ?s, and identified or confirmed putative promoter motifs for nine of them. We constructed precise deletions of each ECF ? gene and its demonstrated or putative anti-? gene and also a strain in which all 11 ECF ? and anti-? genes were deleted. This all-ECF ? deletion strain showed no major defects in free-living growth, in Biolog Phenotype MicroArray assays, or in response to multiple stresses. None of the ECF ?s were required for symbiosis on the host plants Medicago sativa and Medicago truncatula: the strain deleted for all ECF ? and anti-? genes was symbiotically normal.IMPORTANCE Fixed (reduced) soil nitrogen plays a critical role in soil fertility and successful food growth. Much soil fertility relies on symbiotic nitrogen fixation: the bacterial partner infects the host plant roots and reduces atmospheric dinitrogen in exchange for host metabolic fuel, a process that involves complex interactions between the partners mediated by changes in gene expression in each partner. Here we test the roles of a family of 11 extracytoplasmic function (ECF) gene regulatory proteins (sigma factors [?s]) that interact with RNA polymerase to determine if they play a significant role in establishing a nitrogen-fixing symbiosis or in responding to various stresses, including cell envelope stress. We discovered that symbiotic nitrogen fixation occurs even when all 11 of these regulatory genes are deleted, that most ECF sigma factors control accessory functions, and that none of the ECF sigma factors are required to survive envelope stress.
| S-EPMC6180224 | biostudies-literature
Project description:The ability of bacterial core RNA polymerase (RNAP) to interact with different σ factors, thereby forming a variety of holoenzymes with different specificities, represents a powerful tool to coordinately reprogram gene expression. Extracytoplasmic function σ factors (ECFs), which are the largest and most diverse family of alternative σ factors, frequently participate in stress responses. The classification of ECFs in 157 different groups according to their phylogenetic relationships and genomic context has revealed their diversity. Here, we have clustered 55 ECF groups with experimentally studied representatives into two broad classes of stress responses. The remaining 102 groups still lack any mechanistic or functional insight, representing a myriad of systems yet to explore. In this work, we review the main features of ECFs and discuss the different mechanisms controlling their production and activity, and how they lead to a functional stress response. Finally, we focus in more detail on two well-characterized ECFs, for which the mechanisms to detect and respond to stress are complex and completely different: Escherichia coli RpoE, which is the best characterized ECF and whose structural and functional studies have provided key insights into the transcription initiation by ECF-RNAP holoenzymes, and the ECF15-type EcfG, the master regulator of the general stress response in Alphaproteobacteria.
| S-EPMC8103513 | biostudies-literature
Project description:Extracytoplasmic function σ factors that are stress inducible are often sequestered in an inactive complex with a membrane-associated anti-σ factor. Mycobacterium tuberculosis membrane-associated anti-σ factors have a small, stable RNA gene A (ssrA)-like degron for targeted proteolysis. Interaction between the unfoldase, ClpX, and a substrate with an accessible degron initiates energy-dependent proteolysis. Four anti-σ factors with a mutation in the degron provided a set of natural substrates to evaluate the influence of the degron on degradation strength in ClpX-substrate processivity. We note that a point mutation in the degron (X-Ala-Ala) leads to an order-of-magnitude difference in the dwell time of the substrate on ClpX. Differences in ClpX/anti-σ interactions were correlated with changes in unfoldase activities. Green fluorescent protein (GFP) chimeras or polypeptides with a length identical to that of the anti-σ factor degron also demonstrate degron-dependent variation in ClpX activities. We show that degron-dependent ClpX activity leads to differences in anti-σ degradation, thereby regulating the release of free σ from the σ/anti-σ complex. M. tuberculosis ClpX activity thus influences changes in gene expression by modulating the cellular abundance of ECF σ factors.IMPORTANCE The ability of Mycobacterium tuberculosis to quickly adapt to changing environmental stimuli occurs by maintaining protein homeostasis. Extracytoplasmic function (ECF) σ factors play a significant role in coordinating the transcription profile to changes in environmental conditions. Release of the σ factor from the anti-σ is governed by the ClpXP2P1 assembly. M. tuberculosis ECF anti-σ factors have an ssrA-like degron for targeted degradation. A point mutation in the degron leads to differences in ClpX-mediated proteolysis and affects the cellular abundance of ECF σ factors. ClpX activity thus synchronizes changes in gene expression with environmental stimuli affecting M. tuberculosis physiology.
| S-EPMC6398269 | biostudies-literature