scholarly journals Synergistic Interaction of Clostridium cellulovorans Cellulosomal Cellulases and HbpA

2007 ◽  
Vol 189 (20) ◽  
pp. 7190-7194 ◽  
Author(s):  
Satoshi Matsuoka ◽  
Hideaki Yukawa ◽  
Masayuki Inui ◽  
Roy H. Doi
Keyword(s):  
Cell Wall ◽  
Surface Layer ◽  
Cell Surface ◽  
Anaerobic Bacterium ◽  
Synergistic Interaction ◽  
Corn Fiber ◽  
Scaffolding Protein ◽  
Type I ◽  
Clostridium Cellulovorans ◽  
Insoluble Polysaccharides

ABSTRACT 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.

scholarly journals Cell-Surface-Anchoring Role of N-Terminal Surface Layer Homology Domains of Clostridium cellulovorans EngE

2002 ◽  
Vol 184 (4) ◽  
pp. 884-888 ◽  
Author(s):  
Akihiko Kosugi ◽  
Koichiro Murashima ◽  
Yutaka Tamaru ◽  
Roy H. Doi
Keyword(s):  
Cell Wall ◽  
Surface Layer ◽  
Cell Surface ◽  
Catalytic Domain ◽  
Glycosyl Hydrolase ◽  
Sodium Dodecyl ◽  
Scaffolding Protein ◽  
Clostridium Cellulovorans ◽  
Surface Anchoring

ABSTRACT engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been cloned and sequenced (Y. Tamaru and R. H. Doi, J. Bacteriol. 181:3270-3276, 1999). The N-terminal-half region of EngE possesses three repeated surface layer homology (SLH) domains, which are homologous to those of some bacterial S-layer proteins. Also, the C-terminal-half region consists of a catalytic domain of glycosyl hydrolase family 5 and a duplicated sequence (dockerin) for binding EngE to scaffolding protein CbpA. Our hypothesis is that the SLH domains serve in the role of anchoring to the cell surface. This model was investigated by using recombinant EngEs (rEngE) with and without SLH domains that were synthesized in Escherichia coli and cell wall preparations from C. cellulovorans. When rEngE and SLH polypeptides of EngE were incubated with cell wall fragments prepared by sodium dodecyl sulfate treatment, these proteins bound strongly to the cell wall. However, rEngEs without SLH domains lost their ability to bind to cell walls. When rEngE was incubated with mini-CbpA, consisting of two cohesin domains, and cell wall fragments, the mini-CbpA was able to bind to the cell wall with rEngE. However, the binding of mini-CbpA was dramatically inhibited by addition of a chelating reagent, such as EDTA, which prevents cohesin-dockerin interactions. These results suggest not only that the SLH domains of EngE can bind to the cell surface but also that EngE plays an anchoring role for cellulosomes through the interaction of its dockerin domain with a CbpA cohesin.

scholarly journals Hydrophilic Domains of Scaffolding Protein CbpA Promote Glycosyl Hydrolase Activity and Localization of Cellulosomes to the Cell Surface of Clostridium cellulovorans

2004 ◽  
Vol 186 (19) ◽  
pp. 6351-6359 ◽  
Author(s):  
Akihiko Kosugi ◽  
Yoshihiko Amano ◽  
Koichiro Murashima ◽  
Roy H. Doi
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Cell Walls ◽  
Cellulose Degradation ◽  
Glycosyl Hydrolase ◽  
Scaffolding Protein ◽  
Crystalline Cellulose ◽  
Minor Role ◽  
Primary Role ◽  
Clostridium Cellulovorans

ABSTRACT CbpA, the scaffolding protein of Clostridium cellulovorans cellulosomes, possesses one family 3 cellulose binding domain, nine cohesin domains, and four hydrophilic domains (HLDs). Among the three types of domains, the function of the HLDs is still unknown. We proposed previously that the HLDs of CbpA play a role in attaching the cellulosome to the cell surface, since they showed some homology to the surface layer homology domains of EngE. Several recombinant proteins with HLDs (rHLDs) and recombinant EngE (rEngE) were examined to determine their binding to the C. cellulovorans cell wall fraction. Tandemly linked rHLDs showed higher affinity for the cell wall than individual rHLDs showed. EngE was shown to have a higher affinity for cell walls than rHLDs have. C. cellulovorans native cellulosomes were found to have higher affinity for cell walls than rHLDs have. When immunoblot analysis was carried out with the native cellulosome fraction bound to cell wall fragments, the presence of EngE was also confirmed, suggesting that the mechanism anchoring CbpA to the C. cellulovorans cell surface was mediated through EngE and that the HLDs play a secondary role in the attachment of the cellulosome to the cell surface. During a study of the role of HLDs on cellulose degradation, the mini-cellulosome complexes with HLDs degraded cellulose more efficiently than complexes without HLDs degraded cellulose. The rHLDs also showed binding affinity for crystalline cellulose and carboxymethyl cellulose. These results suggest that the CbpA HLDs play a major role and a minor role in C. cellulovorans cellulosomes. The primary role increases cellulose degradation activity by binding the cellulosome complex to the cellulose substrate; secondarily, HLDs aid the binding of the CbpA/cellulosome to the C. cellulovorans cell surface.

scholarly journals A Large Gene Cluster for the Clostridium cellulovorans Cellulosome

2000 ◽  
Vol 182 (20) ◽  
pp. 5906-5910 ◽  
Author(s):  
Yutaka Tamaru ◽  
Shuichi Karita ◽  
Atef Ibrahim ◽  
Helen Chan ◽  
Roy H. Doi
Keyword(s):  
Surface Layer ◽  
Dna Sequencing ◽  
Gene Cluster ◽  
Common Ancestor ◽  
Catalytic Domain ◽  
Gene Clusters ◽  
Scaffolding Protein ◽  
Clostridium Cellulovorans ◽  
Hydrophobic Protein ◽  
Large Gene

ABSTRACT A large gene cluster for the Clostridium cellulovoranscellulosome has been cloned and sequenced upstream and downstream of the cbpA and exgS genes (C.-C. Liu and R. H. Doi, Gene 211:39–47, 1998). Gene walking revealed that theengL gene cluster (Y. Tamaru and R. H. Doi, J. Bacteriol. 182:244–247, 2000) was located downstream of thecbpA-exgS genes. Further DNA sequencing revealed that this cluster contains the genes for the scaffolding protein CbpA, the exoglucanase ExgS, several endoglucanases of family 9, the mannanase ManA, and the hydrophobic protein HbpA containing a surface layer homology domain and a hydrophobic (or cohesin) domain. The sequence of the clustered genes iscbpA-exgS-engH-engK-hbpA-engL-manA-engM-engN and is about 22 kb in length. The engN gene did not have a complete catalytic domain, indicating that engN is a truncated gene. This large gene cluster is flanked at the 5′ end by a putative noncellulosomal operon consisting of nifV-orf1-sigX-regAand at the 3′ end by noncellulosomal genes with homology to transposase (trp) and malate permease (mle). Since gene clusters for the cellulosome are also found in C. cellulolyticum and C. josui, they seem to be typical of mesophilic clostridia, indicating that the large gene clusters may arise from a common ancestor with some evolutionary modifications.

scholarly journals Determination of Subunit Composition of Clostridium cellulovorans Cellulosomes That Degrade Plant Cell Walls

2002 ◽  
Vol 68 (4) ◽  
pp. 1610-1615 ◽  
Author(s):  
Koichiro Murashima ◽  
Akihiko Kosugi ◽  
Roy H. Doi
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Cell Walls ◽  
Plant Cell Wall ◽  
Scaffolding Protein ◽  
Plant Cell Walls ◽  
Higher Plant ◽  
Clostridium Cellulovorans ◽  
Plant Cell Wall Degradation

ABSTRACT Clostridium cellulovorans produces a cellulase enzyme complex (cellulosome). In this study, we isolated two plant cell wall-degrading cellulosomal fractions from culture supernatant of C. cellulovorans and determined their subunit compositions and enzymatic activities. One of the cellulosomal fractions showed fourfold-higher plant cell wall-degrading activity than the other. Both cellulosomal fractions contained the same nine subunits (the scaffolding protein CbpA, endoglucanases EngE and EngK, cellobiohydrolase ExgS, xylanase XynA, mannanase ManA, and three unknown proteins), although the relative amounts of the subunits differed. Since only cellobiose was released from plant cell walls by the cellulosomal fractions, cellobiohydrolases were considered to be key enzymes for plant cell wall degradation.

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

1999 ◽  
Vol 181 (10) ◽  
pp. 3270-3276 ◽  
Author(s):  
Yutaka Tamaru ◽  
Roy H. Doi
Keyword(s):  
Surface Layer ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Caulobacter Crescentus ◽  
Scaffolding Protein ◽  
Glycosyl Hydrolases ◽  
Clostridium Cellulovorans ◽  
Reading Frame ◽  
Enzymatic Function ◽  
C Terminus

ABSTRACT The gene engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovoranscellulosome, 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 theBacillus 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.

scholarly journals Degradation of Corn Fiber by Clostridium cellulovorans Cellulases and Hemicellulases and Contribution of Scaffolding Protein CbpA

2005 ◽  
Vol 71 (7) ◽  
pp. 3504-3511 ◽  
Author(s):  
Roger Koukiekolo ◽  
Hee-Yeon Cho ◽  
Akihiko Kosugi ◽  
Masayuki Inui ◽  
Hideaki Yukawa ◽  
...  
Keyword(s):  
Synergistic Effects ◽  
Extracellular Enzyme ◽  
Corn Fiber ◽  
Scaffolding Protein ◽  
Clostridium Cellulovorans ◽  
Synergistic Interactions ◽  
Hemicellulose Degradation ◽  
Enzyme Subunits ◽  
Rate Limiting ◽  
Potential Rate

ABSTRACT Clostridium cellulovorans, an anaerobic bacterium, degrades native substrates efficiently by producing an extracellular enzyme complex called the cellulosome. All cellulosomal enzyme subunits contain dockerin domains that can bind to hydrophobic domains termed cohesins which are repeated nine times in CbpA, the nonenzymatic scaffolding protein of C. cellulovorans cellulosomes. In this study, the synergistic interactions of cellulases (endoglucanase E, EngE; endoglucanase L, EngL) and hemicellulases (arabinofuranosidase A, ArfA; xylanase A, XynA) were determined on the degradation of corn fiber, a natural substrate containing mainly xylan, arabinan, and cellulose. The degradation by XynA and ArfA of cellulose/arabinoxylan was greater than that of corn fiber and resulted in 2.6-fold and 1.4-fold increases in synergy, respectively. Synergistic effects were observed in increments in both simultaneous and sequential reactions with ArfA and XynA. These synergistic enzymes appear to represent potential rate-limiting enzymes for efficient hemicellulose degradation. When mini-cellulosomes were constructed from the cellulosomal enzymes (XynA and EngL) and mini-CbpA with cohesins 1 and 2 (mini-CbpA1&2) and mini-CbpA with cohesins 5 and 6 (mini-CbpA5&6), higher activity was observed than that for the corresponding enzymes alone. Based on the degradation of different types of celluloses and hemicelluloses, the interaction between cellulosomal enzymes (XynA and EngL) and mini-CbpA displayed a diversity that suggests that dockerin-cohesin interaction from C. cellulovorans may be more selective than random.

scholarly journals Unique Contribution of the Cell Wall-Binding Endoglucanase G to the Cellulolytic Complex in Clostridium cellulovorans

2013 ◽  
Vol 79 (19) ◽  
pp. 5942-5948 ◽  
Author(s):  
Sang Duck Jeon ◽  
Ji Eun Lee ◽  
Su Jung Kim ◽  
Sung Hyun Park ◽  
Gi-Wook Choi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Glycosyl Hydrolase ◽  
Surface Display ◽  
Scaffolding Protein ◽  
Crystalline Cellulose ◽  
Unique Contribution ◽  
Surface Plasmon Resonance Analysis ◽  
Clostridium Cellulovorans ◽  
Content Type ◽  
Hydrolysis Of

ABSTRACTThe cellulosomes produced byClostridium cellulovoransare organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain inC. cellulovorans. The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio = 3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30- to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces ofC. cellulovoransusing its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that inC. cellulovorans, EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.

scholarly journals Regulation of Expression of Cellulosomes and Noncellulosomal (Hemi)Cellulolytic Enzymes in Clostridium cellulovorans during Growth on Different Carbon Sources

2004 ◽  
Vol 186 (13) ◽  
pp. 4218-4227 ◽  
Author(s):  
Sung Ok Han ◽  
Hee-Yeon Cho ◽  
Hideaki Yukawa ◽  
Masayuki Inui ◽  
Roy H. Doi
Keyword(s):  
Cell Wall ◽  
Carbon Source ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Carbon Sources ◽  
Corn Fiber ◽  
Cellulolytic Enzymes ◽  
Synergistic Activity ◽  
Clostridium Cellulovorans ◽  
Carbon Substrates

ABSTRACT Cellulosomes and noncellulosomal (hemi)cellulolytic enzymes are produced by Clostridium cellulovorans to degrade plant cell walls. To understand their synergistic relationship, changes in mRNA and protein expression in cellulosomes and noncellulosomal (hemi)cellulolytic enzymes (hereafter called noncellulosomal enzymes) of cultures grown on cellobiose, cellulose, pectin, xylan, and corn fiber or mixtures thereof were examined. Cellulase expression, favored particularly by the presence of Avicel, was found with all substrates. Comparison of cellulosome and noncellulosomal enzymes showed that expression profiles were strongly affected by the carbon source. High xylanase or pectate lyase expression was observed when C. cellulovorans was grown on xylan or pectin, respectively. Mixed carbon substrates (cellulose-pectin-xylan mixture or corn fiber) induced a wider variety of enzymes than a single carbon source, such as cellobiose, pectin, or xylan. Cellulosomal proteome profiles were more affected by the carbon source than the noncellulosomal enzymes. Transcription and protein analyses revealed that cellulosomes and noncellulosomal enzymes were expressed simultaneously on mixed carbon sources, but their degree of inducibility varied when the substrate was either cellulose or cellobiose. Cellulosomes and noncellulosomal enzymes had synergistic activity on various carbon substrates. These results indicated that expression of plant cell wall-degrading enzymes is highly influenced by the available carbon source and that synergy between cellulosomes and noncellulosomal enzymes contribute to plant cell wall degradation.

Influence of Calcium Ions on the Plant Cell Surface: Membrane Fusions and Conformational Changes

1974 ◽  
Vol 32 ◽  
pp. 154-155
Author(s):  
D. James Morré ◽  
Charles E. Bracker ◽  
William J. VanDerWoude
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Conformational Changes ◽  
Calcium Ions ◽  
Surface Membrane ◽  
Carboxyl Groups ◽  
Cross Linking ◽  
Ultrastructural Evidence ◽  
Glycine Max L ◽  
Wall Extensibility

Calcium ions in the concentration range 5-100 mM inhibit auxin-induced cell elongation and wall extensibility of plant stems. Inhibition of wall extensibility requires that the tissue be living; growth inhibition cannot be explained on the basis of cross-linking of carboxyl groups of cell wall uronides by calcium ions. In this study, ultrastructural evidence was sought for an interaction of calcium ions with some component other than the wall at the cell surface of soybean (Glycine max (L.) Merr.) hypocotyls.

scholarly journals Direct Interaction of Polar Scaffolding Protein Wag31 with Nucleoid-Associated Protein Rv3852 Regulates Its Polar Localization

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1558
Author(s):  
Rajni Garg ◽  
Chinmay Anand ◽  
Sohini Ganguly ◽  
Sandhya Rao ◽  
Rinkee Verma ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Envelope ◽  
Transmembrane Helix ◽  
Ectopic Expression ◽  
Direct Interaction ◽  
Fine Tuning ◽  
Scaffolding Protein ◽  
Physical Interaction ◽  
Cell Wall Synthesis ◽  
Interaction Approach

Rv3852 is a unique nucleoid-associated protein (NAP) found exclusively in Mycobacterium tuberculosis (Mtb) and closely related species. Although annotated as H-NS, we showed previously that it is very different from H-NS in its properties and is distinct from other NAPs, anchoring to cell membrane by virtue of possessing a C-terminal transmembrane helix. Here, we investigated the role of Rv3852 in Mtb in organizing architecture or synthesis machinery of cell wall by protein–protein interaction approach. We demonstrated a direct physical interaction of Rv3852 with Wag31, an important cell shape and cell wall integrity determinant essential in Mtb. Wag31 localizes to the cell poles and possibly acts as a scaffold for cell wall synthesis proteins, resulting in polar cell growth in Mtb. Ectopic expression of Rv3852 in M. smegmatis resulted in its interaction with Wag31 orthologue DivIVAMsm. Binding of the NAP to Wag31 appears to be necessary for fine-tuning Wag31 localization to the cell poles, enabling complex cell wall synthesis in Mtb. In Rv3852 knockout background, Wag31 is mislocalized resulting in disturbed nascent peptidoglycan synthesis, suggesting that the NAP acts as a driver for localization of Wag31 to the cell poles. While this novel association between these two proteins presents one of the mechanisms to structure the elaborate multi-layered cell envelope of Mtb, it also exemplifies a new function for a NAP in mycobacteria.

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