Cellulosomes localise on the surface of membrane vesicles from the cellulolytic bacterium Clostridium thermocellum

ABSTRACTMembrane vesicles released from bacteria contribute to cell–cell communication by carrying various cargos such as proteins, nucleic acids and signaling molecules. Cellulolytic bacteria have been isolated from many environments, yet the function of membrane vesicles for cellulolytic ability has been rarely described. Here, we show that a Gram-positive cellulolytic bacterium Clostridium thermocellum released membrane vesicles, each approximately 50–300 nm in diameter, into the broth. The observations with immunoelectron microscopy also revealed that cellulosomes, which are carbohydrate-active enzyme complexes that give C. thermocellum high cellulolytic activity, localized on the surface of the membrane vesicles. The membrane vesicles collected by ultracentrifugation maintained the cellulolytic activity. Supplementation with the biosurfactant surfactin or sonication treatment disrupted the membrane vesicles in the exoproteome of C. thermocellum and significantly decreased the degradation activity of the exoproteome for microcrystalline cellulose. However, these did not affect the degradation activity for soluble carboxymethyl cellulose. These results suggest a novel function of membrane vesicles: C. thermocellum releases cellulolytic enzymes on the surface of membrane vesicles to enhance the cellulolytic activity of C. thermocellum for crystalline cellulose.

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Growth and cellulolytic activity of Cellulomonas flavigena

Canadian Journal of Microbiology ◽

10.1139/m81-193 ◽

1981 ◽

Vol 27 (12) ◽

pp. 1260-1266 ◽

Cited By ~ 23

Author(s):

B. H. Kim ◽

J. W. T. Wimpenny

Keyword(s):

Free Enzyme ◽

Crystalline Cellulose ◽

Exponential Growth Phase ◽

Cellulolytic Bacterium ◽

Cellulolytic Activity ◽

Mineral Salts ◽

Cellulomonas Flavigena ◽

Rate Limiting Step ◽

Rate Limiting ◽

Hydrolysis Of

Growth factor requirements, growth kinetics, and the ability to produce the enzyme cellulase were examined in the cellulolytic bacterium Cellulomonas flavigena KIST 321. The organism was found to require only thiamine for growth in mineral salts medium containing simple sugars or cellulose. Growth rates on various carbohydrates suggested that disruption of the crystalline structure was the rate-limiting step in the utilization of crystalline cellulose, and hydrolysis of the polymer itself was as rapid as the uptake of the hydrolytic product. When the organism was grown on cellulose the cellulolytic activity appeared to be bound to the cell at the beginning of the exponential growth phase: only after this did cell-free enzyme activity appear. The cell-free enzyme appeared to be unstable, and its activity decreased at the beginning of the stationary phase.

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Purification and characterization of endoglucanase Ss from Clostridium thermocellum

Biochemical Journal ◽

10.1042/bj2790067 ◽

1991 ◽

Vol 279 (1) ◽

pp. 67-73 ◽

Cited By ~ 18

Author(s):

U Fauth ◽

M P M Romaniec ◽

T Kobayashi ◽

A L Demain

Keyword(s):

Clostridium Thermocellum ◽

Hydrophobic Interaction Chromatography ◽

Cellulolytic Enzymes ◽

Crystalline Cellulose ◽

Optimum Temperature ◽

Purification And Characterization ◽

Specific Antibodies ◽

Optimum Ph ◽

Exchange Adsorption

The extracellular cellulolytic enzymes of the thermophilic anaerobe Clostridium thermocellum occur as a protein complex or aggregate known as the cellulosome. By using a combination of ion-exchange, adsorption and hydrophobic-interaction chromatography, it was possible to isolate from extracellular broth a specific endoglucanase of interest without the use of denaturants. The endoglucanase was identified as the cellulosomal subunit Ss by the use of specific antibodies. The enzyme has an Mr of 83,000, an isoelectric point of 3.55, optimum pH of 6.6 and optimum temperature of 70 degrees C. It hydrolyses CM-cellulose and, at a higher rate, the cellodextrins, cellotetraose and cellopentaose, but does not hydrolyse a crystalline cellulose such as Avicel. Cellobiose and cellotriose are also immune to attack. It differs from endoglucanases previously isolated by others and a 76,000-Mr endoglucanase recently isolated in this laboratory.

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Metabolome Analysis of Constituents in Membrane Vesicles for Clostridium thermocellum Growth Stimulation

Microorganisms ◽

10.3390/microorganisms9030593 ◽

2021 ◽

Vol 9 (3) ◽

pp. 593

Author(s):

Shunsuke Ichikawa ◽

Yoichiro Tsuge ◽

Shuichi Karita

Keyword(s):

Growth Rate ◽

Clostridium Thermocellum ◽

Consolidated Bioprocessing ◽

Membrane Vesicle ◽

Single Gene ◽

Membrane Vesicles ◽

Growth Stimulation ◽

Cellulosic Biomass ◽

Cellulolytic Bacterium ◽

Metabolome Analysis

The cultivation of the cellulolytic bacterium, Clostridium thermocellum, can have cost-effective cellulosic biomass utilizations, such as consolidated bioprocessing, simultaneous biological enzyme production and saccharification. However, these processes require a longer cultivation term of approximately 1 week. We demonstrate that constituents of the C. thermocellum membrane vesicle fraction significantly promoted the growth rate of C. thermocellum. Similarly, cell-free Bacillus subtilis broth was able to increase C. thermocellum growth rate, while several B. subtilis single-gene deletion mutants, e.g., yxeJ, yxeH, ahpC, yxdK, iolF, decreased the growth stimulation ability. Metabolome analysis revealed signal compounds for cell–cell communication in the C. thermocellum membrane vesicle fraction (ethyl 2-decenoate, ethyl 4-decenoate, and 2-dodecenoic acid) and B. subtilis broth (nicotinamide, indole-3-carboxaldehyde, urocanic acid, nopaline, and 6-paradol). These findings suggest that the constituents in membrane vesicles from C. thermocellum and B. subtilis could promote C. thermocellum growth, leading to improved efficiency of cellulosic biomass utilization.

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Isolation of Cellulolytic Bacterium Staphylococcus sp. JC20 from the Intestine of Octopus (Octopus sp.) for Fish Probiotic Candidate

Jurnal Perikanan Universitas Gadjah Mada ◽

10.22146/jfs.39525 ◽

2019 ◽

Vol 21 (2) ◽

pp. 93 ◽

Cited By ~ 1

Author(s):

Indah Istiqomah ◽

Imelda Novita Atitus ◽

Ahmad Fauzi Rohman ◽

Alim Isnansetyo

Keyword(s):

Digestive Tract ◽

Agar Plate ◽

Acid Resistance ◽

Cellulolytic Bacterium ◽

Cellulolytic Activity ◽

Cellulolytic Bacteria ◽

Fish Pathogens ◽

Antagonist Activity ◽

16S Rdna Gene ◽

Antibiotics Sensitivity

Aim of this study was to isolate, characterize, and identify cellulolytic bacteria from the digestive tract of marine vertebrates and invertebrates as a candidate of fish probiotics. The bacteria were isolated from the digestive tract and grown on a cellulose agar plate. The bacteria were screened based on the cellulolytic activity, acid resistance, antagonist activity against fish pathogens, antibiotics sensitivity, ability to live in fish digestive tract and non-pathogenic test. Selected bacterium was identified molecularly, based on the 16S rDNA gene sequences, and phenotipically. A total of 14 bacteria demonstrated celulolitic index of 1.1-1.8. The bacteria with cellulolytic index of > 1.6 were screened by the selection criteria, resulted a selected strain, JC20 isolate which was isolated from the digestive tract of octopus (Octopus sp.). The selected bacterium was sensitive to antibiotics, resists to acidic environment, able to live in the fish digestive tract, and non-pathogen. Thus, the bacterium was potential for further characterization as fish probiotics candidate. Molecular and phenotypic identification revealed that JC20 isolate was Staphylococcus sp.

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Interplay between Clostridium thermocellum Family 48 and Family 9 Cellulases in Cellulosomal versus Noncellulosomal States

Applied and Environmental Microbiology ◽

10.1128/aem.00009-10 ◽

2010 ◽

Vol 76 (10) ◽

pp. 3236-3243 ◽

Cited By ~ 55

Author(s):

Yael Vazana ◽

Sarah Moraïs ◽

Yoav Barak ◽

Raphael Lamed ◽

Edward A. Bayer

Keyword(s):

Proximity Effect ◽

Clostridium Thermocellum ◽

Crystalline Cellulose ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Cellulolytic Bacterium ◽

Carbohydrate Binding Module ◽

Wild Type ◽

Designer Cellulosome ◽

Targeting Effect

ABSTRACT 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.

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Cellulolytic ability of a promising Irpex lacteus (Basidiomycota: Polyporales) strain from the subtropical rainforest of Misiones province, Argentina

Revista de Biología Tropical ◽

10.15517/rbt.v66i3.30589 ◽

2018 ◽

Vol 66 (3) ◽

pp. 1034 ◽

Cited By ~ 1

Author(s):

Ernesto Martin Giorgio ◽

Laura Lidia Villalba ◽

Gerardo Lucio Robledo ◽

Pedro Dario Zapata ◽

Mario Carlos Saparrat

Keyword(s):

Agar Medium ◽

Technological Development ◽

White Rot Fungi ◽

White Rot ◽

Cellulolytic Enzymes ◽

Crystalline Cellulose ◽

Its Sequence ◽

Cellulolytic Activity ◽

Irpex Lacteus ◽

Misiones Province

The cellulolytic activity of fungi growing in the subtropical rainforest of Misiones (Argentina) represents a challenge in the technological development of the production of cellulosic bioethanol in the region using native sources. These fungi are promising to obtain sustainable enzyme cocktails using their enzymes. Cellulolytic ability of 22 white-rot fungi isolated from the subtropical rainforest of Misiones-Argentina in agar medium with two types of cellulosic substrates, carboxy-methylcellulose or crystalline cellulose, were comparatively analyzed, and the activity of two cellulolytic enzymes was evaluated in liquid medium. Although all isolates were able to grow and degrade both substrates in agar medium, and to produce total cellulase Filter paper (FPase) and endo-β-1,4-glucanase (EG) activities in broth, the isolate Irpex sp. LBM 034 showed the greatest enzymatic levels (FPase, 65.45 U L-1; EG, 221.21 U L-1). Therefore, the ITS sequence of this fungus was sequenced and analyzed through a phylogenetic analysis. These results indicate that the isolate LBM 034, corresponding to Irpex lacteus, has a promising cellulolytic ability and enzymes such as EG useful in sustainable saccharification of cellulosic materials in the region.

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Genome Sequence of the Anaerobic, Thermophilic, and Cellulolytic Bacterium “Anaerocellum thermophilum” DSM 6725

Journal of Bacteriology ◽

10.1128/jb.00256-09 ◽

2009 ◽

Vol 191 (11) ◽

pp. 3760-3761 ◽

Cited By ~ 65

Author(s):

Irina A. Kataeva ◽

Sung-Jae Yang ◽

Phuongan Dam ◽

Farris L. Poole ◽

Yanbin Yin ◽

...

Keyword(s):

Genome Sequence ◽

Plant Biomass ◽

Biomass Conversion ◽

Cellulolytic Enzymes ◽

Crystalline Cellulose ◽

Cellulolytic Bacterium ◽

Strictly Anaerobic ◽

Caldicellulosiruptor Saccharolyticus ◽

Sugar Utilization ◽

Untreated Plant

ABSTRACT “Anaerocellum thermophilum” DSM 6725 is a strictly anaerobic bacterium that grows optimally at 75°C. It uses a variety of polysaccharides, including crystalline cellulose and untreated plant biomass, and has potential utility in biomass conversion. Here we report its complete genome sequence of 2.97 Mb, which is contained within one chromosome and two plasmids (of 8.3 and 3.6 kb). The genome encodes a broad set of cellulolytic enzymes, transporters, and pathways for sugar utilization and compared to those of other saccharolytic, anaerobic thermophiles is most similar to that of Caldicellulosiruptor saccharolyticus DSM 8903.

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Diversity and cellulolytic activity of culturable bacteria isolated from the gut of higher termites (Odontotermes sp.) in eastern Thailand

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d220831 ◽

2021 ◽

Vol 22 (8) ◽

Author(s):

Parima Boontanom ◽

Aiya Chantarasiri

Keyword(s):

Rrna Gene ◽

Cellulolytic Bacterium ◽

Cellulolytic Activity ◽

Culturable Bacteria ◽

Optimum Temperature ◽

Cellulolytic Bacteria ◽

Neutral Ph ◽

Cmcase Activity ◽

Pcr Rflp ◽

The 16S Rrna Gene

Abstract. Boontanom P, Chantarasiri A. 2021. Diversity and cellulolytic activity of culturable bacteria isolated from the gut of higher termites (Odontotermes sp.) in eastern Thailand. Biodiversitas 22: 3349-3357. Cellulolytic bacteria are vital symbionts associated with the gut of all higher termites. Odontotermes termites are a higher termite widely found in Thailand. However, information concerning the diversity of cellulolytic bacteria in this termite gut remains inadequate. The aim of this study is to isolate and identify the culturable cellulolytic bacteria from the Odontotermes gut collected from eastern Thailand. The crude cellulases produced from the most active cellulolytic bacterium were further characterized. Thirty-two cellulolytic bacteria were isolated and subsequently classified by PCR-RFLP of the 16S rRNA gene. A total of 10 different RFLP patterns were obtained belonging to five bacterial genera, namely Acinetobacter, Bacillus, Citrobacter, Paenibacillus, and Serratia. The B. cereus strain TWV503 was considered to be the most active cellulolytic bacterium based on the CMC agar method. B. cereus strain TWV503 showed CMCase activity at 2.190 ± 0.063 U/mL of CMCase and 0.276 ± 0.031 U/mL of FPase. The optimum temperature and pH for CMCase activity were 50°C and the neutral pH ranging from 7.0 to 8.0, respectively. CMCase activity remained stable at up to 70°C and neutral pH ranging from 7.0 to 8.0 for 24 hours of incubation. This study revealed novel information related to cellulolytic bacteria isolated from the gut of Odontotermes termites collected from Thailand.

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EndB, a Multidomain Family 44 Cellulase from Ruminococcus flavefaciens 17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another R. flavefaciens Protein via a Dockerin Domain

Applied and Environmental Microbiology ◽

10.1128/aem.67.10.4426-4431.2001 ◽

2001 ◽

Vol 67 (10) ◽

pp. 4426-4431 ◽

Cited By ~ 32

Author(s):

Marco T. Rincón ◽

Sheila I. McCrae ◽

James Kirby ◽

Karen P. Scott ◽

Harry J. Flint

Keyword(s):

Amino Acids ◽

Catalytic Domain ◽

Cellulolytic Enzymes ◽

Crystalline Cellulose ◽

Glycoside Hydrolase Family ◽

Ruminococcus Flavefaciens ◽

Cellulose Binding Module ◽

Strong Candidate ◽

Cellulose Binding ◽

Enzyme Complexes

ABSTRACT The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase geneendB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression inEscherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945–1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex.

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Isolation, Cellulase Activity Test and Molecular Identification of Selected Cellulolytic Bacteria Indigenous Rice Bran

Indonesian Journal of Chemistry ◽

10.22146/ijc.26783 ◽

2018 ◽

Vol 18 (3) ◽

pp. 514 ◽

Cited By ~ 1

Author(s):

Akyunul Jannah ◽

Aulanni`am Aulanni`am ◽

Tri Ardyati ◽

Suharjono Suharjono

Keyword(s):

Rice Bran ◽

Waste Product ◽

Cellulase Activity ◽

Raw Material ◽

Cellulolytic Enzymes ◽

Cellulolytic Activity ◽

Cellulolytic Bacteria ◽

Endoglucanase Activity ◽

Activity Test ◽

Rice Milling

Rice bran is the waste product of rice milling which is abundant in Indonesia, it can be used as a raw material for the manufacture of bioethanol by fermentation. Before being fermented, rice bran must be hydrolyzed into glucose by biomass degrading. This study was aimed to isolate indigenous cellulolytic bacteria from rice bran as producer of cellulolytic enzymes and resulted in 22 bacterial isolates that demonstrated cellulolytic activity being identified. Among them, BE 8 and BE 14 isolates showed the highest endoglucanase activity at 2.16 and 1.31 U/mL respectively. Identification of the 16S rDNA showed that BE 8 belongs to Bacillus subtilis and BE 14 in Bacillus cereus.

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