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ENZYMES AND PROTEINS

A Scaffoldin of the Bacteroides cellulosolvens Cellulosome That Contains 11 Type II Cohesins

Shi-You Ding, Edward A. Bayer, David Steiner, Yuval Shoham, Raphael Lamed
Shi-You Ding
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot,
Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv, and
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Edward A. Bayer
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot,
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David Steiner
Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv, and
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Yuval Shoham
Department of Food Engineering and Biotechnology and Institute of Catalysis Science and Technology, Technion—Israel Institute of Technology, Haifa, Israel
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Raphael Lamed
Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv, and
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DOI: 10.1128/JB.182.17.4915-4925.2000
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  • Fig. 1.
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    Fig. 1.

    Identification of scaffoldin-like polypeptides fromB. cellulosolvens. Bc, Coomassie brilliant blue-stained SDS-PAGE-separated proteins from concentrated cell-free culture fluids; Ab, Western blot analysis using antibodies specific for the scaffoldin subunit from C. thermocellum; GSI, blotted protein bands cross-reacting with the GS-I lectin from G. simplicifolia. The relative molecular weights (103) of the designated bands are indicated.

  • Fig. 2.
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    Fig. 2.

    Domain organization of CipBc and overview of sequencing strategy. (A) Domain architecture of CipBc. The polypeptide chain includes 11 cohesins (Coh-1 through Coh-11), an internal CBD, linkers (black), a single C-terminal dockerin domain (D), and an N-terminal signal peptide (stripes). Restriction enzyme sites (E,EcoRI; H, HindIII; P, PstI; S,SacI) and a DNA scale bar are shown. (B) A 1.2-kb PCR fragment obtained with degenerate primers based on peptide sequencing. (C) Top, a 2.8-kb fragment obtained from a SacI genomic library; C1 to C3, subclones of SacI fragment C obtained by using HindIII. (D and E) Respective 2.5- and 2.2-kb fragments, amplified by two-step inverse PCR fromPstI-digested and self-ligated genomic DNA. (F) A 0.8-kb PCR product. (G and H) Respective 2- and 2.8-kb fragments, amplified by genomic-walking PCR from the PstI-pUC19 minigenomic library. (I) A 0.5-kb fragment, amplified from the EcoRI-pUC19 minigenomic library. (J and K) Respective 1.1- and 1.2-kb PCR fragments, obtained from genomic DNA. (B to K) An “F” label on a primer indicates forward; “R” indicates reverse direction. Two primers shown on one end indicate two-step PCR. Arrows indicate the location and direction of primers. Dotted lines indicate a cyclic DNA fragment. Broken lines indicate the pUC19 vector. For primer sequences, see Table 1.

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    Fig. 3.

    Nucleotide and deduced amino acid sequences of theB. cellulosolvens scaffoldin subunit (CipBc). The presumed beginning of each cohesin, CBD, or dockerin domain is labeled. The signal sequence is shown in italics, and the intermodular linker sequences are underlined. Primer sequences are boxed, and their directionality is indicated by an arrow.

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    Fig. 4.

    Assignment of the B. cellulosolvens cohesins as type II cohesins. (A) Phylogenetic analysis of CipBc cohesin sequences. The type I cohesins include those from the other known scaffoldins and two other cellulase-binding surface proteins (OlpA fromC. thermocellum and OrfX from C. cellulolyticum). In addition to the CipBc cohesins, type II cohesins include the anchoring proteins from C. thermocellum and a putative anchoring protein from A. cellulolyticus. See Materials and Methods for sources of the sequences and abbreviations used in this and subsequent figures. The scale bar in this and subsequent figures indicates percentage (0.1) of amino acid substitutions. (B) Alignment of CipBc cohesin sequences versus types I and II cohesins (Coh-I and Coh-II, respectively). The positions of the β strands, known from the crystal structure of type I cohesins from C. thermocellum, are also shown. The sequence for cohesin 5 (Bc_coh-5) is shown as representative of the 11 B. cellulosolvens cohesins. The conserved sequence identities, similarities, and gaps of the B. cellulosolvens cohesins coincide with those of the type II cohesins.

  • Fig. 5.
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    Fig. 5.

    Relationship of the CipBc CBD to other scaffoldin and nonscaffoldin family III CBDs. (A) Sequence alignment of portions of selected family III CBDs, encompassing β-strands 4 through 7 (enumerated arrows). The CipBc CBD and the recently sequencedAcetivibrio CipV CBD (7) are compared to other known scaffoldin CBDs from family IIIa and nonscaffoldin family IIIb CBDs. Shaded residues indicate proposed cellulose-binding residues (39), and numbers refer to presumed positions on the mature CipBc protein. Dashes indicate gaps. (B) Phylogenetic analysis of the family III CBDs. Scaffoldin CBDs are shown as squares. The weighted centroid is shown as a shaded circle on the branch connecting the family IIIb and IIIc CBDs. This analysis is based on a similar analysis of family III CBDs (7).

  • Fig. 6.
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    Fig. 6.

    Relationship of the CipBc C-terminal dockerin with other dockerins of scaffoldin and nonscaffoldin origin. (A) Sequence alignment of the dockerin domain from CipBc with the type II dockerin from the CipA C. thermocellum scaffoldin and their relationship to selected type I dockerins from various cellulosomal enzyme subunits. Presumed calcium-binding residues are shaded, and proposed recognition residues are indicated in bold. (B) Phylogenetic analysis of selected dockerins. The dockerins included in panel A for sequence alignment are circled. Scaffoldin-borne dockerins are indicated by squares. The scale bar indicates percentage (0.1) of amino acid substitutions.

  • Fig. 7.
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    Fig. 7.

    Identification of cohesin-binding polypeptides derived from cellobiose-grown cells of B. cellulosolvens. (A) Coomassie brilliant blue-stained SDS-PAGE-separated proteins from cellulose-bound extracellular fraction. (B) Blot of gel in panel A, transferred electrophoretically onto nitrocellulose strips, probed with the His-tagged cohesin 5, and stained immunochemically, using an anti-His-tag antibody. Lanes: Bc, cellulose-adsorbed B. cellulosolvens proteins; Ct, purified cellulosome from C. thermocellum; Coh, purified recombinant type II cohesin 5, containing a His tag. The relative molecular weights (103) of the designated bands are indicated.

Tables

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  • Table 1.

    Primers used in this study

    NameNucleotide sequenceaLocationbComments
    SEQ3-FATNTTYGGNMGNACNTAYATGAAYYTInitially unspecified cohesinDegenerate primer derived from initial protease digest of candidate scaffoldin
    CBD-RTGWKYRWARTTWSWCCAGTCCBDDegenerate primer designed on the basis of known family III CBD sequences
    En-FTTRTCNACRTCRAADATCCAN terminusDegenerate primer that aligned with N-terminal portion of signal sequence
    24C-RCATATTCAGGAGCTGATGCATCoh-2To amplify N terminus of CipBc
    24N-RTTTTCTGCTGCTCCAGAATTCCoh-2To amplify N terminus of CipBc
    H706N-RGTCTGTGATGGTGGTAGTGALinker between Coh-3 and Coh-4Sequencing
    H706N-FATTGGTTCAGGTGTAACAGCLinker between Coh-3 and Coh-4Sequencing
    COH-FnCAGCTCGAGGGTTCAGGAGTAGTATCAACTCoh-5Cohesin expression
    COH-RnGTGGATCCTTACTATCCGTTTATTGAAGAAGCCTGCoh-5Cohesin expression
    COH-FAGACCATGGGTTCAGGAGTAGTAGCAACCoh-5Cohesin expression
    COH-RAGTCTCGAGTCCGTTTATTGAAGAAGCCTGCoh-5Cohesin expression, inverse PCR
    12F1TTAACAGCGACTTGAAGTTCBDInverse PCR, sequencing
    12R2AGCTTGCTGCTGCACCATTCBDInverse PCR, sequencing
    12R1GATCATATGTTGATGATCCTGCBDInverse PCR, sequencing
    12F2CAATTCAGGGTAGAGTTGCACBDInverse PCR, sequencing
    SC-FGGTTCAACATCCATTAAGTTAGCoh-6Genomic-walking PCR
    3601-FATGGAGTTCAGTTGGACAATCoh-7Sequencing
    4041-FACTGAAACTAAAGTATTATTGAACoh-8Sequencing
    C-R2CAACAACTGGAGCATTAACTLinker between Coh-10 and Coh-11Genomic-walking PCR
    C-R1ATCATTATCAATAGCTGTCAACoh-11Genomic-walking PCR
    C-F1TCACAAGGTGTATTGAACTTCoh-11Genomic-walking PCR
    C-F2GAACCTTTCAGCATACAGAGCoh-11Genomic-walking PCR
    M13-R1AGCGGATAACAATTTCACACAGGApUC19Genomic-walking PCR
    M13-R2AACAGCTATGACCATGATTACGpUC19Genomic-walking PCR
    M13-F1GTTTTCCCAGTCACGACGTTGpUC19Genomic-walking PCR
    M13-F2TGTAAAACGACGGCCAGTpUC19Genomic-walking PCR
    • ↵a Abbreviations for degenerate nucleotides: K, G or T; Y, C or T; W, A or T; R, A or G; S, C or G. N represents A, C, G, or T.

    • ↵b The exact locations of relevant primers are shown in Fig. 3.

  • Table 2.

    Sequence of peptide fragment from cellulose-binding cell-derived components of B. cellulosolvens and its similarity to a conserved segment of family 48 glycosyl hydrolases

    BacteriumEnzymeSequence of fragment
    Bacteroides cellulosolvens“CelS”aWIFDVDNWYK
    Clostridium thermocellum CelSaWLMDVDNWYG
    Clostridium cellulolyticum CelFaWILDVDNWYG
    Clostridium josui CelDaWLLDVDNWYG
    Clostridium cellulovorans ExgSaWLLDVDNWYG
    Caldocellum saccharolyticum CelAWLMDVDNWYG
    Anaerocellum thermophilum CelAWLMDVDNWYG
    Clostridium stercorarium CbhWLLDVDNWYG
    Cellulomonas fimi CbhBWLADVDNIYG
    • ↵a Cellulosomal enzyme.

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A Scaffoldin of the Bacteroides cellulosolvens Cellulosome That Contains 11 Type II Cohesins
Shi-You Ding, Edward A. Bayer, David Steiner, Yuval Shoham, Raphael Lamed
Journal of Bacteriology Sep 2000, 182 (17) 4915-4925; DOI: 10.1128/JB.182.17.4915-4925.2000

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A Scaffoldin of the Bacteroides cellulosolvens Cellulosome That Contains 11 Type II Cohesins
Shi-You Ding, Edward A. Bayer, David Steiner, Yuval Shoham, Raphael Lamed
Journal of Bacteriology Sep 2000, 182 (17) 4915-4925; DOI: 10.1128/JB.182.17.4915-4925.2000
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KEYWORDS

Bacterial Proteins
bacteroides
Carrier Proteins
Cellulase
Glycoproteins

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