Enzymatic profiling of cellulosomal enzymes from the human gut bacterium, Ruminococcus champanellensis, reveals a fine‐tuned system for cohesin‐dockerin recognition

Ruminococcus champanellensis is considered a keystone species in the human gut that degrades microcrystalline cellulose efficiently and contains the genetic elements necessary for cellulosome production. The basic elements of its cellulosome architecture, mainly cohesin and dockerin modules from sca...

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Published in	Environmental microbiology Vol. 18; no. 2; pp. 542 - 556
Main Authors	Moraïs, Sarah, David, Yonit Ben, Bensoussan, Lizi, Duncan, Sylvia H, Koropatkin, Nicole M, Martens, Eric C, Flint, Harry J, Bayer, Edward A
Format	Journal Article
Language	English
Published	England Blackwell Science 01.02.2016 Blackwell Publishing Ltd Wiley Subscription Services, Inc
Subjects	Bacteria Bacterial Proteins - metabolism beta-glucanase Cell Cycle Proteins - metabolism cellulases Cellulose Cellulose - metabolism cellulosome Cellulosomes - enzymology Chromosomal Proteins, Non-Histone - metabolism Cohesins digestive system Enzymatic activity enzyme activity enzyme-linked immunosorbent assay Enzymes Gastrointestinal Microbiome - physiology Glycoside Hydrolases - metabolism glycosides hemicellulose Humans intestinal microorganisms Keystone species Molecular Sequence Data Multienzyme Complexes - metabolism Ruminococcus Ruminococcus - enzymology Ruminococcus - genetics xylanases
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ISSN	1462-2912 1462-2920
DOI	10.1111/1462-2920.13047

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Summary:	Ruminococcus champanellensis is considered a keystone species in the human gut that degrades microcrystalline cellulose efficiently and contains the genetic elements necessary for cellulosome production. The basic elements of its cellulosome architecture, mainly cohesin and dockerin modules from scaffoldins and enzyme‐borne dockerins, have been characterized recently. In this study, we cloned, expressed and characterized all of the glycoside hydrolases that contain a dockerin module. Among the 25 enzymes, 10 cellulases, 4 xylanases, 3 mannanases, 2 xyloglucanases, 2 arabinofuranosidases, 2 arabinanases and one β‐glucanase were assessed for their comparative enzymatic activity on their respective substrates. The dockerin specificities of the enzymes were examined by ELISA, and 80 positives out of 525 possible interactions were detected. Our analysis reveals a fine‐tuned system for cohesin–dockerin specificity and the importance of diversity among the cohesin–dockerin sequences. Our results imply that cohesin–dockerin pairs are not necessarily assembled at random among the same specificity types, as generally believed for other cellulosome‐producing bacteria, but reveal a more organized cellulosome architecture. Moreover, our results highlight the importance of the cellulosome paradigm for cellulose and hemicellulose degradation by R. champanellensis in the human gut.
Bibliography:	http://dx.doi.org/10.1111/1462-2920.13047 ark:/67375/WNG-G5TBS1H7-N Israel Strategic Alternative Energy Foundation (I-SAEF) United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel Fig. S1. Purity of the recombinant enzymes after Ni-NTA purification as assessed by SDS-PAGE gels (10% acrylamide). Fig. S2. Comparative proteome of (A) cellobiose and (B) filter paper cellulose-grown Ruminococcus champanellensis 18P13. Spot F1 = Cel9F and Spot F2 = Cel48A. Fig. S3. New division of R. champanellensis Groups 3 and 4 dockerins. The dockerins of Groups 3 and 4 were re-divided based on the finding of the alternative-binding mode (Fig. 4). Positions of the putative cohesin recognition residues are highlighted in cyan for the first helix and in yellow for the second helix. Proteins highlighted in green were examined in our previous study (Ben David et al., 2015), and proteins highlighted in blue were topics of the present study. Fig. S4. Affinity-based ELISA with Group 2 enzymes. The dockerin-containing enzymes were coated at 1 μg ml−1, and the CBMs fused to CohH, CohI, CohA2, CohB1/B2/B3, CohB4, CohB5/B6 or CohCc (from Clostridium cellulolyticum as negative control) were used at 100 ng ml−1. Reactions were performed at least three times in triplicate; standard deviations are indicated. Fig. S5. Affinity-based ELISA with Groups 3 and 4 enzymes. The dockerin-containing enzymes were coated at 1 μg ml−1, and the CBMs fused to CohC, CohD, CohH or CohCc (from C. cellulolyticum as negative control) were used at 100 ng ml−1. Reactions were performed at least three times in triplicate; standard deviations are indicated. Fig. S6. R. champanellensis dockerin Group 2 alignment. The 17 dockerin sequences of R. champanellensis were aligned, using bioinformatics-based criteria. Dockerins selected for this study are highlighted in blue and those highlighted in green were also assayed in our previous study (Ben David et al., 2015) (see Table 1 for GI number of the parent proteins). Positions of calcium binding residues are shown in cyan, and putative recognition residues are shown in yellow. Protein names highlighted green were examined in our previous study (Ben David et al., 2015), and protein names highlighted in blue were topics of the present study. Table S1. Primers used in the study (restrictions sites represented in upper cases). ArticleID:EMI13047 Israel Science Foundation (ISF) - No. 1349 Israel Science Foundation BBSRC - No. BB/L009951/1 Scottish Government Food, Land and People program istex:8A07540B72D516E21D227CBF42D7504B77B747C9 Society for Applied Microbiology ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23
ISSN:	1462-2912 1462-2920
DOI:	10.1111/1462-2920.13047