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Elaborate cellulosome architecture of Acetivibrio cellulolyticus revealed by selective screening of cohesin-dockerin interactions.

Hamberg Y, Ruimy-Israeli V, Dassa B, Barak Y, Lamed R, Cameron K, Fontes CM, Bayer EA, Fried DB - PeerJ (2014)

Bottom Line: Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry.Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction.The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Chemistry, The Weizmann Institute of Science , Rehovot , Israel.

ABSTRACT
Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry. Owing to the recalcitrance of crystalline cellulose to enzymatic degradation, it is necessary to design economical methods of liberating the fermentable sugars required for bioethanol production. One route towards unlocking the potential of cellulosic waste lies in a highly complex class of molecular machines, the cellulosomes. Secreted mainly by anaerobic bacteria, cellulosomes are structurally diverse, cell surface-bound protein assemblies that can contain dozens of catalytic components. The key feature of the cellulosome is its modularity, facilitated by the ultra-high affinity cohesin-dockerin interaction. Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction. As opposed to previous approaches, the present study utilized proteins contained in unpurified whole-cell extracts. This strategy was made possible due to an experimental design that allowed for the relevant proteins to be "purified" via targeted affinity interactions as a function of the binding assay. The approach thus represents a new strategy, appropriate for future medium- to high-throughput screening of whole genomes, to determine the interactions between cohesins and dockerins. We have selected the cellulosome of Acetivibrio cellulolyticus for this work due to its exceptionally complex cellulosome systems and intriguing diversity of its cellulosomal modular components. Containing 41 cohesins and 143 dockerins, A. cellulolyticus has one of the largest number of potential cohesin-dockerin interactions of any organism, and contains unusual and novel cellulosomal features. We have surveyed a representative library of cohesin and dockerin modules spanning the cellulosome's total cohesin and dockerin sequence diversity, emphasizing the testing of unusual and previously-unknown protein modules. The screen revealed several novel cell-bound cellulosome architectures, thus expanding on those previously known, as well as soluble cellulose systems that are not bound to the bacterial cell surface. This study sets the stage for screening the entire complement of cellulosomal components from A. cellulolyticus and other organisms with large cellulosome systems. The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs.

No MeSH data available.


Representative histogram showing cohesin library screen results.Normalized interaction intensities between the A. cellulolyticus CBM-Coh library and XDoc-Xyn10. CBM-Cohs A3 though P are shown along with negative controls (CBM alone, C. thermocellum CBM-CohA2, Ruminococcus flavefaciens CBM-CohE), and the positive control (Xyn-CBM). CohF1 was found to have the highest experimental interaction intensity and was normalized to an interaction value of one. The dashed line indicates the threshold for positive interaction, determined by the CBM negative control.
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fig-6: Representative histogram showing cohesin library screen results.Normalized interaction intensities between the A. cellulolyticus CBM-Coh library and XDoc-Xyn10. CBM-Cohs A3 though P are shown along with negative controls (CBM alone, C. thermocellum CBM-CohA2, Ruminococcus flavefaciens CBM-CohE), and the positive control (Xyn-CBM). CohF1 was found to have the highest experimental interaction intensity and was normalized to an interaction value of one. The dashed line indicates the threshold for positive interaction, determined by the CBM negative control.

Mentions: The intensity of each cohesin–dockerin interaction was normalized for protein expression by dividing each spot’s Cy3 fluorescence value by its Cy5 fluorescence value. Cohesin-dockerin interaction intensities were calculated for the highest two-protein dilutions, and the averaged values were graphed in a histogram. A sample histogram showing the XDoc-Xyn10 interaction with the 21 cohesin library and the control proteins is presented (Fig. 6). The interaction intensity of the highest-interacting cohesin (CohE7) is arbitrarily set to a value of 1. In this representative experiment, seven cohesins (CohA4, CohB4, CohD1, CohD3, CohF1, CohM1 and CohP) were found to interact to varying extents with XDocXyn10. The averaged cohesin–dockerin interaction intensities for all the interactions were completed by repeating this process with all 15 dockerins.


Elaborate cellulosome architecture of Acetivibrio cellulolyticus revealed by selective screening of cohesin-dockerin interactions.

Hamberg Y, Ruimy-Israeli V, Dassa B, Barak Y, Lamed R, Cameron K, Fontes CM, Bayer EA, Fried DB - PeerJ (2014)

Representative histogram showing cohesin library screen results.Normalized interaction intensities between the A. cellulolyticus CBM-Coh library and XDoc-Xyn10. CBM-Cohs A3 though P are shown along with negative controls (CBM alone, C. thermocellum CBM-CohA2, Ruminococcus flavefaciens CBM-CohE), and the positive control (Xyn-CBM). CohF1 was found to have the highest experimental interaction intensity and was normalized to an interaction value of one. The dashed line indicates the threshold for positive interaction, determined by the CBM negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4217186&req=5

fig-6: Representative histogram showing cohesin library screen results.Normalized interaction intensities between the A. cellulolyticus CBM-Coh library and XDoc-Xyn10. CBM-Cohs A3 though P are shown along with negative controls (CBM alone, C. thermocellum CBM-CohA2, Ruminococcus flavefaciens CBM-CohE), and the positive control (Xyn-CBM). CohF1 was found to have the highest experimental interaction intensity and was normalized to an interaction value of one. The dashed line indicates the threshold for positive interaction, determined by the CBM negative control.
Mentions: The intensity of each cohesin–dockerin interaction was normalized for protein expression by dividing each spot’s Cy3 fluorescence value by its Cy5 fluorescence value. Cohesin-dockerin interaction intensities were calculated for the highest two-protein dilutions, and the averaged values were graphed in a histogram. A sample histogram showing the XDoc-Xyn10 interaction with the 21 cohesin library and the control proteins is presented (Fig. 6). The interaction intensity of the highest-interacting cohesin (CohE7) is arbitrarily set to a value of 1. In this representative experiment, seven cohesins (CohA4, CohB4, CohD1, CohD3, CohF1, CohM1 and CohP) were found to interact to varying extents with XDocXyn10. The averaged cohesin–dockerin interaction intensities for all the interactions were completed by repeating this process with all 15 dockerins.

Bottom Line: Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry.Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction.The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Chemistry, The Weizmann Institute of Science , Rehovot , Israel.

ABSTRACT
Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry. Owing to the recalcitrance of crystalline cellulose to enzymatic degradation, it is necessary to design economical methods of liberating the fermentable sugars required for bioethanol production. One route towards unlocking the potential of cellulosic waste lies in a highly complex class of molecular machines, the cellulosomes. Secreted mainly by anaerobic bacteria, cellulosomes are structurally diverse, cell surface-bound protein assemblies that can contain dozens of catalytic components. The key feature of the cellulosome is its modularity, facilitated by the ultra-high affinity cohesin-dockerin interaction. Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction. As opposed to previous approaches, the present study utilized proteins contained in unpurified whole-cell extracts. This strategy was made possible due to an experimental design that allowed for the relevant proteins to be "purified" via targeted affinity interactions as a function of the binding assay. The approach thus represents a new strategy, appropriate for future medium- to high-throughput screening of whole genomes, to determine the interactions between cohesins and dockerins. We have selected the cellulosome of Acetivibrio cellulolyticus for this work due to its exceptionally complex cellulosome systems and intriguing diversity of its cellulosomal modular components. Containing 41 cohesins and 143 dockerins, A. cellulolyticus has one of the largest number of potential cohesin-dockerin interactions of any organism, and contains unusual and novel cellulosomal features. We have surveyed a representative library of cohesin and dockerin modules spanning the cellulosome's total cohesin and dockerin sequence diversity, emphasizing the testing of unusual and previously-unknown protein modules. The screen revealed several novel cell-bound cellulosome architectures, thus expanding on those previously known, as well as soluble cellulose systems that are not bound to the bacterial cell surface. This study sets the stage for screening the entire complement of cellulosomal components from A. cellulolyticus and other organisms with large cellulosome systems. The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs.

No MeSH data available.