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 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 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 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 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.

Isolation and partial characterization of a complementary protein from the mycoparasite Piptocephalis virginiana that specifically binds to two glycoproteins at the host cell surface

1997 ◽  
Vol 43 (7) ◽  
pp. 625-632 ◽  
Author(s):  
M. S. Manocha ◽  
D. Xiong ◽  
V. Govindsamy
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Western Blot ◽  
Host Cell ◽  
Immunofluorescence Microscopy ◽  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Primary Antibody ◽  
Rabbit Igg ◽  
Host Cell Surface

Immunofluorescence microscopy was used to detect in the mycoparasite Piptocephalis virginiana the presence of a complementary glycoprotein that binds specifically to the host cell surface glycoproteins b and c, reported earlier from our laboratory. Germinated spores of P. virginiana treated with cell wall extract of the host Mortierella pusilla, primary antibody prepared against cell wall glycoproteins b and c, and fluorescein isothiocyanate (FITC) – goat anti-rabbit IgG conjugate showed fluorescence. Immunobinding analysis identified from the mycoparasite a protein of 100 kDa that binds with the host glycoproteins b and c, separately as well as collectively. Its purification was achieved by (i) 60% ammonium sulfate precipitation, (ii) heat treatment, (iii) Sephadex G-100 gel filtration, and (iv) preparative polyacrylamide gel electrophoresis (PAGE). The purity was ascertained by sodium dodecyl sulphate (SDS) – PAGE and Western blot analysis. Positive reaction to periodic acid – Schiff s reagent revealed its glycoprotein nature, and mannose was identified as a major sugar component. The specificity of the polyclonal antibody raised against electrophoretically purified complementary protein in rabbit was confirmed by dot immunobinding and Western blot analyses. Immunofluorescence microscopy revealed surface localization of the protein on the germ tubes of P. virginiana. Fluorescence was also observed at the surface of the germinated spores and hyphae of the host M. pusilla, after treatment with complementary protein from P. virginiana, primary antibody prepared against the complementary protein, and FITC – goat anti-rabbit IgG conjugate.Key words: biotrophic mycoparasite, cell surface agglutinin, glycoprotein immunobinding, immunofluorescence, mucoraceous host.

scholarly journals Disruption of gingipain oligomerization into non-covalent cell-surface attached complexes

2012 ◽  
Vol 393 (9) ◽  
pp. 971-977 ◽  
Author(s):  
Maryta Sztukowska ◽  
Florian Veillard ◽  
Barbara Potempa ◽  
Matthew Bogyo ◽  
Jan J. Enghild ◽  
...  
Keyword(s):  
Cell Surface ◽  
Porphyromonas Gingivalis ◽  
Catalytic Domain ◽  
Proteolytic Processing ◽  
Growth Media ◽  
Conserved Motif ◽  
His Tag ◽  
Hexahistidine Tag

Abstract RgpA and Kgp gingipains are non-covalent complexes of endoprotease catalytic and hemagglutinin-adhesin domains on the surface of Porphyromonas gingivalis. A motif conserved in each domain has been suggested to function as an oligomerization motif. We tested this hypothesis by mutating motif residues to hexahistidine or insertion of hexahistidine tag to disrupt the motif within the Kgp catalytic domain. All modifications led to the secretion of entire Kgp activity into the growth media, predominantly in a form without functional His-tag. This confirmed the role of the conserved motif in correct posttranslational proteolytic processing and assembly of the multidomain complexes.

Cell surface characteristics of Mortierella species and their interaction with a mycoparasite

1984 ◽  
Vol 30 (3) ◽  
pp. 290-298 ◽  
Author(s):  
M. S. Manocha
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Characteristics ◽  
Protein Patterns ◽  
Periodic Acid Schiff ◽  
The Difference ◽  
Chitin And Chitosan

Cell surface characteristics of three Mortierella species differing in their response to a mycoparasite, Piptocephalis virginiana, were examined. Their cell wall composition was typical of mucoraceous fungi with chitin and chitosan as major polysaccharides. Electron microscopy revealed that the mycoparasite penetrated and formed haustoria in the hyphae of susceptible hosts, M. pusilla and M. isabellina. The failure of the parasite to establish contact and penetrate a hypha of the nonhost, M. candelabrum, was not due to cell wall thickness, rigidity, or chitin contents. Markedly different protein patterns obtained from crude alkali extracts of host and nonhost cell walls by sodium dodecyl sulfate – polyacrylamide gel electrophoresis might explain the difference in host and nonhost response to the mycoparasite. Whereas most of the bands differed only in intensity after staining with either Coomassie blue or periodic acid – Schiff reagent, there were two distinct bands of glycoproteins (76 000 and 74 000) observed in the host species which were absent in the nonhost species.

scholarly journals LysPBC2, a Novel Endolysin Harboring aBacillus cereusSpore Binding Domain

2018 ◽  
Vol 85 (5) ◽  
Author(s):  
Minsuk Kong ◽  
Hongjun Na ◽  
Nam-Chul Ha ◽  
Sangryeol Ryu
Keyword(s):  
Cell Wall ◽  
Catalytic Domain ◽  
Binding Activity ◽  
Binding Domain ◽  
Lytic Activity ◽  
Content Type ◽  
Cell Wall Binding ◽  
A Cell ◽  
Cell Wall Binding Domain

ABSTRACTTo control the spore-forming human pathogenBacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolatedB. cereusphage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against allBacillus,Listeria, andClostridiumspecies tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds toB. cereusspores but not to vegetative cells ofB. cereus. Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells ofB. cereuscompared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.IMPORTANCEBacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of aBacillus cereusphage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controllingB. cereus.

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