ScaC, an adaptor protein carrying a novel cohesin that expands the dockerin-binding repertoire of the Ruminococcus flavefaciens 17 cellulosome - PubMed (original) (raw)

ScaC, an adaptor protein carrying a novel cohesin that expands the dockerin-binding repertoire of the Ruminococcus flavefaciens 17 cellulosome

Marco T Rincón et al. J Bacteriol. 2004 May.

Abstract

A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His(6)-tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex.

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Figures

FIG.1.

Diagrams showing the disposition on the genome, the domain organization, and the sequence of the R. flavefaciens scaC gene. (A) Gene organization of the structural scaffoldin proteins in R. flavefaciens 17. (B) Sequence strategy and domain architecture. scaC was discovered to be upstream of scaA by PCR walking extension with primers ScaAcoh9r (9r) and M13f (see Table 1 and Materials and Methods for details) from a pUC18 R. flavefaciens 17 plasmid library. The sequence was completed by using the additional internal primers ScaAcoh11r (11r), ScaAcoh10f (10f), and ScaAcoh10r (10r). Domains within ScaC are indicated. The solid box indicates a linker sequence, and the striped boxes indicate N-terminal signal peptides (SP). The positions of the ScaC cohesin and dockerin are indicated. (C) Nucleotide sequence and sequence of the gene product (top) of scaC. Shine-Dalgarno sequences (SD) and the predicted cleavage site that removes the signal peptide (▾) are indicated. The F-hand repeat motifs typical of the Ca2+-binding loop of dockerin domains are indicated by open boxes. The putative cohesin domain is indicated by a solid box. The initial sequence coding for ScaA is indicated by a striped box.

FIG.2.

Relationship of R. flavefaciens ScaC-borne cohesin and dockerin domains to previously described cellulosomal domains. (A) Phylogenetic analysis of the ScaC cohesin relative to the known type I, II, and III cohesins. The ScaC cohesin maps on a separate branch, distinct from all other cohesins. The ScaC cohesin emanates from the central branch close to the branching point of the three A. cellulolyticus ScaC cohesins and approximately equidistant from the type I cohesins and the point of deflection that separates the type II cohesins from the type III cohesins. (B) Phylogenetic analysis of the ScaC dockerin relative to other enzyme and scaffoldin-borne dockerins. The ScaC dockerin (solid square) maps among the EndB-like dockerins from R. flavefaciens enzymes (solid circles) but distinct from the dockerins of CesA, XynE, and XynX. The other known R. flavefaciens scaffoldin-borne dockerin (ScaA) and the dockerins of C. thermocellum (Clotm), A. cellulolyticus (Acece), and B. cellulosolvens (Bacce) are indicated by open squares. Other dockerin-borne enzymes include enzymes from Ruminococcus albus (Rumal-EgV, Rumal-EgVI, Rumal-EgVII) and Ruminococcus sp. (Rumsp-Xyn1) and a selection of enzymes from C. thermocellum and mesophilic clostridia (C. cellulolyticum, C. cellulovorans, and C. josui). For a list of the proteins and their accession numbers, see references , , and . Scale bars = 0.1% amino acid substitutions.

FIG. 3.

Multiple-sequence alignment of the cohesin domain from R. flavefaciens ScaC with cohesin domains from other cellulolytic bacteria. RUMFL-ScaC, R. flavefaciens ScaC (AJ585075); RUMFL-ScaB, R. flavefaciens ScaB (accession no. tr:Q9AE52); RUMFL-ScaA, R. flavefaciens ScaA (tr:Q9AE53); ACECE-ScaC, A. cellulolyticus ScaC (tr:Q7WYN2); CLOTH-CipA, C. thermocellum CipA (sp:Q06851); ACECE-CipV, A. cellulolyticus CipV (tr:Q9RPLO); CLOCE-CipC, C. cellulolyticum CipC (sp:Q45996); CLOJO-CipA, C. josui CipA (sp:O82830); CLOCL-CbpA. C. cellulovorans CbpA (sp:P38058); CLOAC-0910, C. acetobutylicum 0910 (tr:Q977Y4). Amino acids that are conserved in sequences are highlighted. The alignment was constructed by using ClustalW (

http://www2.ebi.ac.uk/clustalw/

) and was edited by using GeneDoc (

http://www.psc.edu/biomed/genedoc

FIG. 4.

SDS-PAGE of recombinant protein probes. His6-tagged proteins were overexpressed in E. coli (see Materials and Methods for details). The recombinant product was purified by nickel affinity chromatography and was separated by SDS-PAGE. Proteins were stained with Coomassie blue. Lane M contained a protein molecular size marker. The predicted molecular masses of the protein constructs are 16.7, 23.2, and 33.9 kDa for His6-ScaC-Doc, His6-ScaC-Coh, and His6-ScaC-mat, respectively. The faint bands above the protein constructs appear to be products of dimerization. The numbers on the left indicate the positions of the protein molecular mass markers (in kilodaltons).

FIG. 5.

One-dimensional Western blot analysis of recombinant cohesin domains from ScaC and ScaA. Cell surface-associated proteins from R. flavefaciens 17 grown on birchwood xylan (lanes X) or crystalline cellulose (Avicel) (lanes C) were separated by SDS-PAGE. Proteins were blotted onto a PVDF membrane and incubated after blocking with recombinant His6-ScaC-Coh or His6-ScaA-Coh. Cohesin-dockerin interactions were exposed by incubating the membranes with Ni-conjugated peroxidase to detect the polyhistidine tag of the recombinant proteins following enhanced chemiluminescence. The numbers on the left indicate the positions of the protein molecular mass markers (in kilodaltons).

FIG. 6.

2D gel electrophoresis of cell surface-associated protein fraction of R. flavefaciens 17 grown on birchwood xylan. (A) Colloidal Coomassie blue-stained gel. The positions of the enzyme EndB and the structural cellulosomal proteins ScaA and ScaC are indicated. The numbers at bottom indicate the pH gradient of the first separation. The numbers on the right indicate the positions of the protein markers for the second separation. (B) 2D Western blot analysis performed with a similar gel after blotting onto a PVDF membrane and probing with His6-ScaA-Coh (28) . The positions of EndB and ScaC are indicated. (C) 2D Western blot analysis performed with a similar gel after the protein spots were blotted onto a PVDF membrane and probed with His6-ScaC-Coh. Cohesin-dockerin interactions were exposed as described in the legend to Fig. 5.

FIG. 7.

Spot test analysis of ScaC-mediated cohesin-dockerin interactions. (A and B) Recombinant His6-tagged proteins were spotted onto a nylon membrane and revealed by interaction with Ni-conjugated peroxidase. (C) The membrane was incubated with the biotinylated ScaC-mat construct, and cohesin-dockerin interactions revealed by a second incubation with peroxidase-conjugated streptavidin. (D) The membrane was incubated with the ScaC-Doc construct, and cohesin-dockerin interactions were revealed by a second incubation with HRP-conjugated S protein, which recognized the S tag present only in the recombinant ScaC-Doc construct. Spots 1 to 6, dockerin domains from EndB, XynB, CesA, XynE, XynX, and ScaA, respectively; spots 7 and 8, X domains from ScaA and ScaB, respectively; spots 9 to 14, cohesins 1, 2, 3, 4, 6, and 7 from ScaB, respectively; spot 15, ScaA-Coh2.

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ScaC, an adaptor protein carrying a novel cohesin that expands the dockerin-binding repertoire of the Ruminococcus flavefaciens 17 cellulosome - PubMed (original) (raw)