Novel organization and divergent dockerin specificities in the cellulosome system of Ruminococcus flavefaciens - PubMed (original) (raw)

Novel organization and divergent dockerin specificities in the cellulosome system of Ruminococcus flavefaciens

Marco T Rincon et al. J Bacteriol. 2003 Feb.

Abstract

The DNA sequence coding for putative cellulosomal scaffolding protein ScaA from the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17 was completed. The mature protein exhibits a calculated molecular mass of 90,198 Da and comprises three cohesin domains, a C-terminal dockerin, and a unique N-terminal X domain of unknown function. A novel feature of ScaA is the absence of an identifiable cellulose-binding module. Nevertheless, native ScaA was detected among proteins that attach to cellulose and appeared as a glycosylated band migrating at around 130 kDa. The ScaA dockerin was previously shown to interact with the cohesin-containing putative surface-anchoring protein ScaB. Here, six of the seven cohesins from ScaB were overexpressed as histidine-tagged products in E. coli; despite their considerable sequence differences, each ScaB cohesin specifically recognized the native 130-kDa ScaA protein. The binding specificities of dockerins found in R. flavefaciens plant cell wall-degrading enzymes were examined next. The dockerin sequences of the enzymes EndA, EndB, XynB, and XynD are all closely related but differ from those of XynE and CesA. A recombinant ScaA cohesin bound selectively to dockerin-containing fragments of EndB, but not to those of XynE or CesA. Furthermore, dockerin-containing EndB and XynB, but not XynE or CesA, constructs bound specifically to native ScaA. XynE- and CesA-derived probes did however bind a number of alternative R. flavefaciens bands, including an approximately 110-kDa supernatant protein expressed selectively in cultures grown on xylan. Our findings indicate that in addition to the ScaA dockerin-ScaB cohesin interaction, at least two distinct dockerin-binding specificities are involved in the novel organization of plant cell wall-degrading enzymes in this species and suggest that different scaffoldins and perhaps multiple enzyme complexes may exist in R. flavefaciens.

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Figures

FIG. 1.

FIG. 1.

ScaA sequencing strategy and domain architecture. The sequence of ScaA was completed by PCR walking, by sequencing extended products produced with ScaAcoh5r (∼0.8 kb) and then with 7r (∼0.9 kb) (Table 1; also see Materials and Methods). Additional primers and amplifications were used to obtain sequences for both strands and to confirm the relationship of the ScaA N terminus with unique ScaA dockerin, and ScaB sequences and their positions are also indicated. Domains within the ScaA protein are shown as follows: X, N terminal domain of unknown function; 1, 2, and 3, cohesin domains; Doc, dockerin. Solid boxes indicates threonine-rich linkers and striped boxes indicate N-terminal signal peptide.

FIG. 2.

FIG. 2.

Detection of native ScaA with antibodies raised against purified, recombinant ScaA-Coh2. Proteins from supernatants of cellobiose-grown R. flavefaciens 17 cultures were separated by SDS-PAGE and analyzed by Coomassie blue staining (lane 1) or glycostaining (lane 2). A duplicate sample was transferred to PVDF membranes and probed with anti-ScaA-Coh2 antibodies (lane 3). Numbers at left are molecular masses in kilodaltons.

FIG. 3.

FIG. 3.

Electron micrograph showing the distribution of ScaA, detected by immunogold labeling, in cellulose-grown R. flavefaciens 17 at late stationary phase.

FIG. 4.

FIG. 4.

Recognition of a common 130-kDa protein band by different cohesin domains from ScaB. Total R. flavefaciens 17 supernatant proteins (approximately 7 μg) from a cellulose-grown culture were loaded onto successive lanes of an SDS-PAGE gel, separated, and subjected to Western blotting. Membrane strips containing the electrophoretically transferred material were then incubated with each of the designated cohesin probes (standardized by His tag detection, corresponding to approximately 10 to 50 μg of protein). Binding was detected by chemiluminescence using peroxidase-conjugated anti-His tag antibody. Lanes are marked 1 through 7 according to the cohesin probe used. Lane Rf, profile of Coomassie-stained R. flavefaciens supernatant proteins. Numbers at left are molecular masses in kilodaltons.

FIG. 5.

FIG. 5.

Affinity blotting of dockerin-containing constructs from CesA, XynE, and EndB using recombinant ScaA-Coh2. Dockerin-containing fragments of the three enzymes and the ScaA-Coh2 cohesin domain, were constructed as six-His-tagged fusions (Table 1), and the products were purified. The enzyme constructs were separated by SDS PAGE, transferred to a PVDF membrane, and probed with biotinylated ScaA-Coh2 (right-hand panel). Coomassie-stained lanes from the same gel are shown in the left-hand panel. Significant fragmentation of the recombinant proteins was observed in all preparations. The estimated molecular masses for the CesA, XynE and EndB constructs are 45,427, 41,625, and 87,040 Da, respectively. EndB fragmented into two major fractions, of which the full-length construct and the smaller (∼50-kDa) fragment were labeled by the cohesin-containing probe. No labeling of XynE or CesA was observed. Numbers at left are molecular weights (MW) in thousands.

FIG. 6.

FIG. 6.

Affinity blotting of cell-associated proteins from R. flavefaciens 17 by dockerin-containing constructs from CesA, XynE, and EndB (a) and XynB (b). Cultures were grown on either microcrystalline cellulose (C) or birch wood xylan (X) as the energy source. Proteins were separated in successive lanes of the same SDS-PAGE gel before transfer to a PVDF membrane. The molecular size of the XynB product used as probe was 44,782 Da. The blots were probed with the indicated biotinylated protein construct, and labeled bands were detected by chemiluminescence using peroxidase-conjugated streptavidin. A band of around 45 kDa (not shown in the figure), representing a native biotinylated protein present in R. flavefaciens cells, was detected in all samples and was used as an internal standard. The EndB-induced labeling of the 130-kDa band derived from both cellulose- and xylan-grown cultures was confirmed by immunochemical staining using a nonbiotinylated construct and anti-His-Tag antibodies (not shown). Numbers at left and right are molecular weights (MW) in thousands.

FIG. 7.

FIG. 7.

Binding of biotinylated CesA, XynE, and EndB probes to extracellular material of R. flavefaciens 17 cultures grown on either cellulose (C) or birchwood xylan (X). Concentrated culture supernatants were subjected to SDS-PAGE (gel). Duplicate samples were transferred to membranes and probed using the designated biotinylated construct. Numbers at left are molecular weights (MW) in thousands.

FIG. 8.

FIG. 8.

Schematic representation of dockerin-cohesin interactions involved in cellulosome organization in R. flavefaciens 17. Three different specificities of cohesin-dockerin interaction are shown: between the ScaA dockerin and ScaB cohesins (a), between the ScaA cohesins and enzymes with EndB-type dockerins (b), and between the cohesins of a putative 110-kDa scaffoldin (ScaX) and the CesA and XynE dockerins (c). Although ScaB is a cell-associated protein, the proposed interaction of the C-terminal X domain with the cell surface awaits experimental verification.

FIG. 9.

FIG. 9.

Phylogenetic relationship of R. flavefaciens dockerin domains. Dockerins from the indicated R. flavefaciens enzymes (designated Rumfl-EndA, etc.) are shown as filled circles. Scaffoldin-borne dockerins from R. flavefaciens (Rumfl-ScaA), C. thermocellum (Clotm-CipA), A. cellulolyticus (Acece-CipV), and B. cellulosolvens (Bacce-CipBc) are shown as squares. Other dockerin-borne enzymes are from R. albus (Rumal-EgV, etc.), Ruminococcus sp. (Rumsp-Xyn1), and a selection of enzymes from C. thermocellum and mesophilic clostridia (C. cellulolyticum, C. cellulovorans, and C. josui). For a precise list of the proteins and their accession numbers, consult Materials and Methods. The scale bar indicates the percentage (0.1) of amino acid substitutions.

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