A scaffoldin of the Bacteroides cellulosolvens cellulosome that contains 11 type II cohesins - PubMed (original) (raw)

A scaffoldin of the Bacteroides cellulosolvens cellulosome that contains 11 type II cohesins

S Y Ding et al. J Bacteriol. 2000 Sep.

Abstract

A cellulosomal scaffoldin gene, termed cipBc, was identified and sequenced from the mesophilic cellulolytic anaerobe Bacteroides cellulosolvens. The gene encodes a 2,292-residue polypeptide (excluding the signal sequence) with a calculated molecular weight of 242,437. CipBc contains an N-terminal signal peptide, 11 type II cohesin domains, an internal family III cellulose-binding domain (CBD), and a C-terminal dockerin domain. Its CBD belongs to family IIIb, like that of CipV from Acetivibrio cellulolyticus but unlike the family IIIa CBDs of other clostridial scaffoldins. In contrast to all other scaffoldins thus far described, CipBc lacks a hydrophilic domain or domain X of unknown function. The singularity of CipBc, however, lies in its numerous type II cohesin domains, all of which are very similar in sequence. One of the latter cohesin domains was expressed, and the expressed protein interacted selectively with cellulosomal enzymes, one of which was identified as a family 48 glycosyl hydrolase on the basis of partial sequence alignment. By definition, the dockerins, carried by the cellulosomal enzymes of this species, would be considered to be type II. This is the first example of authentic type II cohesins that are confirmed components of a cellulosomal scaffoldin subunit rather than a cell surface anchoring component. The results attest to the emerging diversity of cellulosomes and their component sequences in nature.

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Figures

FIG. 1

Identification of scaffoldin-like polypeptides from B. 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

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, _Eco_RI; H, _Hin_dIII; P, _Pst_I; S, _Sac_I) 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 _Sac_I genomic library; C1 to C3, subclones of _Sac_I fragment C obtained by using _Hin_dIII. (D and E) Respective 2.5- and 2.2-kb fragments, amplified by two-step inverse PCR from _Pst_I-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 _Pst_I-pUC19 minigenomic library. (I) A 0.5-kb fragment, amplified from the _Eco_RI-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.

FIG. 3

Nucleotide and deduced amino acid sequences of the B. 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.

FIG. 3

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 from C. 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. 4

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 sequenced Acetivibrio 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

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

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.

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A scaffoldin of the Bacteroides cellulosolvens cellulosome that contains 11 type II cohesins - PubMed (original) (raw)