Three Surface Layer Homology Domains at the N Terminus of the Clostridium cellulovorans Major Cellulosomal Subunit EngE

The gene engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been isolated and sequenced. engE is comprised of an open reading frame (ORF) of 3,090 bp and encodes a protein of 1,030 amino acids with a molecular weight of 111,796. The amino acid sequence derived from engE revealed a structure consisting of catalytic and noncatalytic domains. The N-terminal-half region of EngE consisted of a signal peptide of 31 amino acid residues and three repeated surface layer homology (SLH) domains, which were highly conserved and homologous to an S-layer protein from the gram-negative bacterium Caulobacter crescentus. The C-terminal-half region, which is necessary for the enzymatic function of EngE and for binding of EngE to the scaffolding protein CbpA, consisted of a catalytic domain homologous to that of family 5 of the glycosyl hydrolases, a domain of unknown function, and a duplicated sequence (DS or dockerin) at its C terminus. engE is located downstream of an ORF, ORF1, that is homologous to the Bacillus subtilis phosphomethylpyrimidine kinase (pmk) gene. The unique presence of three SLH domains and a DS suggests that EngE is capable of binding both to CbpA to form a CbpA-EngE cellulosome complex and to the surface layer of C. cellulovorans.     

Structure Predictions and Interaction Studies Indicate Homology of Separase N-Terminal Regulatory Domains and Drosophila THR

The final resolution of sister chromatid cohesion during mitotic and meiotic divisions is mediated by activation of separase which cleaves a cohesin complex subunit. The structural basis of separase regulation is unknown. Separases from different eukaryotes share almost no sequence similarity, especially within the large N-terminal domain that precedes the protease domain except in Drosophila melanogaster. Moreover, sequence similarity among securin proteins, which associate as regulatory subunits with separase, is restricted to the signals that promote the mitotic degradation required for separase activation. Here, we address the surprising divergence of separase and securin sequences. The absence of an extended N-terminal separase domain in dipteran species is shown to be correlated with the expression of an extra regulatory subunit (THR). The interactions of THR with separase and securin in Drosophila melanogaster are analogous to those of the human N-terminal separase domain with its C-terminal domain and securin. Even heterologous interactions between Drosophila and human separase complex components occur in yeast two-hybrid experiments. Tertiary structure predictions reveal alpha-alpha superhelix folds in both THR and the N-terminal domains of non-dipteran separases. The compatibility of these folds with a wide range of primary sequences has likely allowed the rapid divergence of THR/N-terminal separase sequences and securins, which contact this region.     

Cellulosome and noncellulosomal cellulases of Clostridium cellulovorans

This paper reviews the properties of the cellulosome and noncellulosome cellulases produced by Clostridium cellulovorans, an anaerobic, mesophilic, spore-forming microorganism that produces copious amounts of cellulase. The three major subunits of the cellulosome, CbpA, exoglucanase S (ExgS), and P100, are described, as well as the properties of the functional domains of CbpA. The properties of two noncellulosomal endoglucanases, EngD and EngF, are compared. The functions of the cellulose-binding domain (CBD) of CbpA indicate its potential uses in biotechnology. Received: November 18, 1997 / Accepted: November 26, 1997     

Regulation of cellulose-inducible structures of Clostridium cellulovorans.

Scanning electron microscopy was used to detect ultrastructural protuberances on the cellulolytic anaerobe Clostridium cellulovorans. Numerous ultrastructural protuberances were observed on cellulose-grown cells, but few were detected on glucose-, fructose-, cellobiose-, or carboxymethylcellulose (CMC)-grown cells. Formation of these protuberances was detected within 2 h of incubation in cellulose medium, but 4 h incubation was required before numerous structures were observed on the cells. When a soluble carbohydrate or CMC was mixed with cellulose-grown cells, the ultrastructural protuberances could no longer be detected. In fact, no protuberances were observed within 5 min following the addition of glucose, cellobiose, or methylglucose to cellulose-grown cells. The presence of these protuberances corresponded with the binding of the Bandeiraea simplicifolia BSI-B4 isolectin to the cell. Cellulose-grown cells had a greater level of observable lectin binding than cellobiose-grown cells, and lectin binding was not detected on glucose- or fructose-grown cells. In addition, lectin binding ability was lost by cellulose-grown cells following the addition of glucose, fructose, or methylglucose to the cellulose medium. A cellulose-affinity protein fraction expressing cellulase activity was also detected in cell extracts of cellobiose- or cellulose-grown cultures. However, this protein fraction was not detected in extracts of glucose-grown cultures, and was rapidly lost (within 5 min) following the addition of glucose to cellulose-grown cultures. The ability of C. cellulovorans to adhere to cellulose was also affected by the energy substrate, but not in the same manner as the protuberance formation or the cellulase-containing protein fraction. Rather, cellobiose-, cellulose-, and CMC-grown cultures adhered to cellulose, but this adherence was not affected by addition of glucose to the medium. This is the first report that soluble carbohydrates caused the rapid loss of some cellulose-inducible systems of C. cellulovorans.     

Cohesin-dockerin interactions of cellulosomal subunits of Clostridium cellulovorans

The cellulosome of Clostridium cellulovorans consists of three major subunits: CbpA, EngE, and ExgS. The C. cellulovorans scaffolding protein (CbpA) contains nine hydrophobic repeated domains (cohesins) for the binding of enzymatic subunits. Cohesin domains are quite homologous, but there are some questions regarding their binding specificity because some of the domains have regions of low-level sequence similarity. Two cohesins which exhibit 60% sequence similarity were investigated for their ability to bind cellulosomal enzymes. Cohesin 1 (Coh1) was found to contain amino acid residues corresponding to amino acids 312 to 453 of CbpA, which contains a total of 1,848 amino acid residues. Coh6 was determined to contain amino acid residues corresponding to residues 1113 to 1254 of CbpA. By genetic construction, these two cohesins were each fused to MalE, producing MalE-Coh1 and MalE-Coh6. The abilities of two fusion proteins to bind to EngE, ExgS, and CbpA were compared. Although MalE-Coh6 could bind EngE and ExgS, little or no binding of the enzymatic subunits was observed with MalE-Coh1. Significantly, the abilities of the two fusion proteins to bind CbpA were similar. The binding of dockerin-containing enzymes to cohesin-containing proteins was suggested as a model for assembly of cellulosomes. In our examination of the role of dockerins, it was also shown that the binding of endoglucanase B (EngB) to CbpA was dependent on the presence of EngB's dockerin. These results suggest that different cohesins may function with differing efficiency and specificity, that cohesins may play some role in the formation of polycellulosomes through Coh-CbpA interactions, and that dockerins play an important role during the interaction of cellulosomal enzymes and cohesins present in CbpA.     

Cellulose promotes extracellular assembly of Clostridium cellulovorans cellulosomes.

Cellulosome synthesis by Clostridium cellulovorans was investigated by growing the cells in media containing different carbon sources. Supernatant from cells grown with cellobiose contained no cellulosomes and only the free forms of cellulosomal major subunits CbpA, P100, and P70 and the minor subunits with enzymatic activity. Supernatant from cells grown on pebble-milled cellulose and Avicel contained cellulosomes capable of degrading crystalline cellulose. Supernatants from cells grown with cellobiose, pebble-milled cellulose, and Avicel contained about the same amount of carboxymethyl cellulase activity. Although the supernatant from the medium containing cellobiose did not initially contain active cellulosomes, the addition of crystalline cellulose to the cell-free supernatant fraction converted the free major forms to cellulosomes with the ability to degrade crystalline cellulose. The binding of P100 and P70 to crystalline cellulose was dependent on their attachment to the endoglucanase-binding domains of CbpA. These data strongly indicate that crystalline cellulose promotes cellulosome assembly.     

Properties of cellulosomal family 9 cellulases from Clostridium cellulovorans

The cellulosomal family 9 cellulase genes engH, engK, engL, engM, and engY of Clostridium cellulovorans have been cloned and sequenced. We compared the enzyme activity of family 9 cellulosomal cellulases from C. cellulovorans and their derivatives. EngH has the highest activity toward soluble cellulose derivatives such as carboxymethylcellulose (CMC) as well as insoluble cellulose such as acid-swollen cellulose (ASC). EngK has high activity toward insoluble cellulose such as ASC and Avicel. The results of thin-layer chromatography showed that the cleavage products of family 9 cellulases were varied. These results indicated that family 9 endoglucanases possess different modes of attacking substrates and produce varied products. To investigate the functions of the carbohydrate-binding module (CBM) and the catalytic module, truncated derivatives of EngK, EngH, and EngY were constructed and characterized. EngHΔCBM and EngYΔCBM devoid of the CBM lost activity toward all substrates including CMC. EngKΔCBM and EngMΔCBM did not lose activity toward CMC but lost activity toward Avicel. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.     

Synergistic interaction of Clostridium cellulovorans cellulosomal cellulases and HbpA

Clostridium cellulovorans, an anaerobic bacterium, produces a small nonenzymatic protein called HbpA, which has a surface layer homology domain and a type I cohesin domain similar to those found in the cellulosomal scaffolding protein CbpA. In this study, we demonstrated that HbpA could bind to cell wall fragments from C. cellulovorans and insoluble polysaccharides and form a complex with cellulosomal cellulases endoglucanase B (EngB) and endoglucanase L (EngL). Synergistic degradative action of the cellulosomal cellulase and HbpA complexes was demonstrated on acid-swollen cellulose, Avicel, and corn fiber. We propose that HbpA functions to bind dockerin-containing cellulosomal enzymes to the cell surface and complements the activity of cellulosomes.     

Exoproteome Profiles of Clostridium cellulovorans Grown on Various Carbon Sources

The cellulosome is a complex of cellulosomal proteins bound to scaffolding proteins. This complex is considered the most efficient system for cellulose degradation. Clostridium cellulovorans, which is known to produce cellulosomes, changes the composition of its cellulosomes depending on the growth substrates. However, studies have investigated only cellulosomal proteins; profile changes in noncellulosomal proteins have rarely been examined. In this study, we performed a quantitative proteome analysis of the whole exoproteome of C. cellulovorans, including cellulosomal and noncellulosomal proteins, to illustrate how various substrates are efficiently degraded. C. cellulovorans was cultured with cellobiose, xylan, pectin, or phosphoric acid-swollen cellulose (PASC) as the sole carbon source. PASC was used as a cellulose substrate for more accurate quantitative analysis. Using an isobaric tag method and a liquid chromatography mass spectrometer equipped with a long monolithic silica capillary column, 639 proteins were identified and quantified in all 4 samples. Among these, 79 proteins were involved in saccharification, including 35 cellulosomal and 44 noncellulosomal proteins. We compared protein abundance by spectral count and found that cellulosomal proteins were more abundant than noncellulosomal proteins. Next, we focused on the fold change of the proteins depending on the growth substrates. Drastic changes were observed mainly among the noncellulosomal proteins. These results indicate that cellulosomal proteins were primarily produced to efficiently degrade any substrate and that noncellulosomal proteins were specifically produced to optimize the degradation of a particular substrate. This study highlights the importance of noncellulosomal proteins as well as cellulosomes for the efficient degradation of various substrates.     

Membrane Binding and Self-Association of the Epsin N-Terminal Homology Domain

Epsin possesses a conserved epsin N-terminal homology (ENTH) domain that acts as a phosphatidylinositol 4,5-bisphosphate‐lipid‐targeting and membrane‐curvature‐generating element. Upon binding phosphatidylinositol 4,5‐bisphosphate, the N-terminal helix (H₀) of the ENTH domain becomes structured and aids in the aggregation of ENTH domains, which results in extensive membrane remodeling. In this article, atomistic and coarse-grained (CG) molecular dynamics (MD) simulations are used to investigate the structure and the stability of ENTH domain aggregates on lipid bilayers. EPR experiments are also reported for systems composed of different ENTH-bound membrane morphologies, including membrane vesicles as well as preformed membrane tubules. The EPR data are used to help develop a molecular model of ENTH domain aggregates on preformed lipid tubules that are then studied by CG MD simulation. The combined computational and experimental approach suggests that ENTH domains exist predominantly as monomers on vesiculated structures, while ENTH domains self-associate into dimeric structures and even higher‐order oligomers on the membrane tubes. The results emphasize that the arrangement of ENTH domain aggregates depends strongly on whether the local membrane curvature is isotropic or anisotropic. The molecular mechanism of ENTH‐domain-induced membrane vesiculation and tubulation and the implications of the epsin's role in clathrin-mediated endocytosis resulting from the interplay between ENTH domain membrane binding and ENTH domain self-association are also discussed.     

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

An endoglucanase gene, engB, from Clostridium cellulovorans, previously cloned into pUC19, has been further characterized and its product investigated. The enzyme, EngB, encoded by the gene was secreted into the periplasmic space of Escherichia coli. The enzyme was active against carboxymethylcellulose, xylan and lichenan but not Avicel (crystalline cellulose). The sequenced gene showed an open reading frame of 1323 base pairs and coded for a protein with a molecular mass of 48.6 kDa. The mRNA contained a typical Gram-positive ribosome-binding site sequence GGAGG and a sequence coding for a putative signal peptide. There is high amino acid and base sequence homology between the N-terminal regions of EngB and another C. cellulovorans endoglucanase, EngD, but they differ significantly in their C-termini. Deletion analyses revealed that up to 32 amino acids of the N-terminus and 52 amino acids of the C-terminus were not required for catalytic activity. The conserved reiterated domains at the C-terminus of EngB were similar to those from endoglucanases from other cellulytic bacteria. According to our deletion analyses, this region is not needed for catalytic activity.     

Production of Minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800

Two genes encoding EngB endoglucanase and mini-CbpA1 scaffolding protein of Clostridium cellulovorans were constructed and coexpressed in Bacillus subtilis WB800. The resulting minicellulosomes were isolated by gel filtration chromatography and characterized. Biochemical and immunological evidence indicated that fraction II contained minicellulosomes consisting of mini-CbpA1 and EngB. The in vivo synthesis of minicellulosomes suggests that it will be possible in the future to insert into B. subtilis cellulosomal genes that will allow growth on cellulosic materials and the production of various designer cellulosomes with specific functions.     

Production of minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800

Two genes encoding EngB endoglucanase and mini-CbpA1 scaffolding protein of Clostridium cellulovorans were constructed and coexpressed in Bacillus subtilis WB800. The resulting minicellulosomes were isolated by gel filtration chromatography and characterized. Biochemical and immunological evidence indicated that fraction II contained minicellulosomes consisting of mini-CbpA1 and EngB. The in vivo synthesis of minicellulosomes suggests that it will be possible in the future to insert into B. subtilis cellulosomal genes that will allow growth on cellulosic materials and the production of various designer cellulosomes with specific functions.     

Genome Sequence of the Cellulosome-Producing Mesophilic Organism Clostridium cellulovorans 743B

Clostridium cellulovorans 743B was isolated from a wood chip pile and is an anaerobic and mesophilic spore-forming bacterium. This organism degrades native substrates in soft biomass such as corn fiber and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Here we report the genome sequence of C. cellulovorans 743B.The biotechnological potential of polysaccharolytic enzymes has resulted in the isolation and characterization of a large number of anaerobic, Gram-positive, spore-forming bacteria, the majority of which have been allocated to the genus Clostridium. Among clostridia, the cellulosomes produced by Clostridium species are particularly designed for efficient degradation of plant cell wall polysaccharides. The component parts of the multicomponent complex are integrated by virtue of a unique family of integrating modules, the cohesins and the dockerins, whose distribution and specificity dictate the overall cellulosome architecture (3). The cellulosome system in Clostridium cellulovorans 743B (ATCC 35296) has been studied extensively for the last 20 years and has resulted in providing basic information about mesophilic cellulosomes. This organism was isolated from a wood chip pile and is an anaerobic spore-forming bacterium whose optimal growth temperature is 37°C (9). It has the ability to utilize cellulose, xylan, pectin, cellobiose, glucose, fructose, galactose, and mannose as carbon sources for growth. Its fermentation products include H₂, CO₂, acetate, butyrate, formate, lactate, and ethanol. When it is grown in the presence of cellulose, electron micrographs have shown that large protuberances are present on its cell surface (4), while small or no protuberances are evident when cells are grown in the presence of glucose or cellobiose (5).We sequenced a total length of 101,749,598 bp and analyzed 381,514 reads by Genome Sequencer FLX 454./Roche sequencing (8) (GS-FLX version) to highly oversample the genome (20× coverage) and generated 123,892 paired-end sequence tags, to enable the assembly of all tags using the GS De Novo Assembler version 1.1.03.24 (Roche Diagnostics) and the Genome Analyzer II and sequencing kit 36-Cycle Run (Illumina). Finally, we assembled 30 scaffolds (sets of 601 ordered and oriented contigs; total length of 5,123,527 bp) to generate approximately 5.1 Mbp of nearly contiguous Clostridium botulinum E3 strain Alaska E43 (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_010723","term_id":"188587536","term_text":"NC_010723"}}NC_010723) complete genome sequence. We analyzed a number of predicted genes included in the C. cellulovorans genome using CRITICA (version 1.05b) (2) and Glimmer 2 (version 2.10) (6) to find regions in proteins with known functions. We annotated and classified according to Gene Ontology (GO) (1). In silico Molecular Cloning Genomic Edition ver. 3.0.26 software (In Silico Biology, Co., Ltd., Japan) was used for individual genomic analysis.The C. cellulovorans 743B (ATCC 35296) genome consists of 5,123,527 bp. A total of 4,220 polypeptide-encoding open reading frames (ORFs) were identified using CRITICA, while 4,297 ORFs were identified using Glimmer 2. The number of ORFs identical between CRITICA and Glimmer 2 was 2,773. Sixty-three tRNAs and 33 anticodons were also identified using tRNAscan-SE (7). In comparison of the genome sizes among cellulosomal clostridia such as Clostridium cellulolyticum H10 (4.07 Mbp) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP001348","term_id":"219997787","term_text":"CP001348"}}CP001348) and Clostridium thermocellum ATCC 27405 (3.84 Mbp) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP000568","term_id":"125712750","term_text":"CP000568"}}CP000568), the C. cellulovorans genome was over 1 Mbp larger than the other genomes. Moreover, the number of predicted genes (4,220 by CRITICA) in the C. cellulovorans genome was the largest among them. On the other hand, the G+C content in C. cellulovorans was 31.1%, similar to that (30.9%) in Clostridium acetobutylicum ATCC 824 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE001437","term_id":"25168256","term_text":"AE001437"}}AE001437), while the G+C contents in C. cellulolyticum and C. thermocellum were 37.7% and 39.0%, respectively.A protein BLAST search against the database of clusters of orthologous groups (COGs) of proteins indicated that 4,171 genes were annotated by 4,220 predicted coding sequences using CRITICA while 4,098 genes were identified by 4,297 predicted coding sequences using Glimmer 2. On the other hand, a protein BLAST search against the NCBI nr database indicated that 4,184 genes were annotated by 4,220 predicted coding sequences using CRITICA while 4,071 genes were identified by 4,297 predicted coding sequences using Glimmer 2. Interestingly, 57 cellulosomal genes were found in the C. cellulovorans genome and coded for not only carbohydrate-active enzymes but also lipases, peptidases, and proteinase inhibitors. Moreover, two novel genes encoding a scaffolding protein were found in the genome. Thus, by examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way in which natural selection molds functional DNA sequence evolution. Our analysis, coupled with the genome sequence data, will provide a road map for constructing enhanced cellulosome-producing Clostridium strains for industrial applications such as biofuel production.     

Regulation of expression of cellulosomal cellulase and hemicellulase genes in Clostridium cellulovorans

The regulation of expression of the genes encoding the cellulases and hemicellulases of Clostridium cellulovorans was studied at the mRNA level with cells grown under various culture conditions. A basic pattern of gene expression and of relative expression levels was obtained from cells grown in media containing poly-, di- or monomeric sugars. The cellulase (cbpA and engE) and hemicellulase (xynA) genes were coordinately expressed in medium containing cellobiose or cellulose. Growth in the presence of cellulose, xylan, and pectin gave rise to abundant expression of most genes (cbpA-exgS, engH, hbpA, manA, engM, engE, xynA, and/or pelA) studied. Moderate expression of cbpA, engH, manA, engE, and xynA was observed when cellobiose or fructose was used as the carbon source. Low levels of mRNA from cbpA, manA, engE, and xynA were observed with cells grown in lactose, mannose, and locust bean gum, and very little or no expression of cbpA, engH, manA, engE, and xynA was detected in glucose-, galactose-, maltose-, and sucrose-grown cells. The cbpA-exgS and engE genes were most frequently expressed under all conditions studied, whereas expression of xynA and pelA was more specifically induced at higher levels in xylan- or pectin-containing medium, respectively. Expression of the genes (cbpA, hbpA, manA, engM, and engE) was not observed in the presence of most soluble di- or monosaccharides such as glucose. These results support the hypotheses that there is coordinate expression of some cellulases and hemicellulases, that a catabolite repression type of mechanism regulates cellulase expression in rapidly growing cells, and that the presence of hemicelluloses has an effect on cellulose utilization by the cell.     

Role of TolR N-Terminal, Central, and C-Terminal Domains in Dimerization and Interaction with TolA and TolQ

The Tol-PAL system of Escherichia coli is a multiprotein system involved in maintaining the cell envelope integrity and is necessary for the import of some colicins and phage DNA into the bacterium. It is organized into two complexes, one near the outer membrane between TolB and PAL and one in the cytoplasmic membrane between TolA, TolQ, and TolR. In the cytoplasmic membrane, all of the Tol proteins have been shown to interact with each other. Cross-linking experiments have shown that the TolA transmembrane domain interacts with TolQ and TolR. Suppressor mutant analyses have localized the TolQ-TolA interaction to the first transmembrane domain of TolQ and have shown that the third transmembrane domain of TolQ interacts with the transmembrane domain of TolR. To get insights on the composition of the cytoplasmic membrane complex and its possible contacts with the outer membrane complex, we focused our attention on TolR. Cross-linking and immunoprecipitation experiments allowed the identification of Tol proteins interacting with TolR. The interactions of TolR with TolA and TolQ were confirmed, TolR was shown to dimerize, and the resulting dimer was shown to interact with TolQ. Deletion mutants of TolR were constructed, and they allowed us to determine the TolR domains involved in each interaction. The TolR transmembrane domain was shown to be involved in the TolA-TolR and TolQ-TolR interactions, while TolR central and C-terminal domains appeared to be involved in TolR dimerization. The role of the TolR C-terminal domain in the TolA-TolR interaction and its association with the membranes was also demonstrated. Furthermore, phenotypic studies clearly showed that the three TolR domains (N terminal, central, and C terminal) and the level of TolR production are important for colicin A import and for the maintenance of cell envelope integrity.