The cellulosome system of Acetivibrio cellulolyticus includes a novel type of adaptor protein and a cell surface anchoring protein - PubMed (original) (raw)
The cellulosome system of Acetivibrio cellulolyticus includes a novel type of adaptor protein and a cell surface anchoring protein
Qi Xu et al. J Bacteriol. 2003 Aug.
Abstract
A scaffoldin gene cluster was identified in the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. The previously described scaffoldin gene, cipV, encodes an N-terminal family 9 glycoside hydrolase, a family 3b cellulose-binding domain, seven cohesin domains, and a C-terminal dockerin. The gene immediately downstream of cipV was sequenced and designated scaB. The protein encoded by this gene has 942 amino acid residues and a calculated molecular weight of 100,358 and includes an N-terminal signal peptide, four type II cohesions, and a C-terminal dockerin. ScaB cohesins 1 and 2 are very closely linked. Similar, but not identical, 39-residue Thr-rich linker segments separate cohesin 2 from cohesin 3 and cohesin 3 from cohesin 4, and an 84-residue Thr-rich linker connects the fourth cohesin to a C-terminal dockerin. The scaC gene downstream of scaB codes for a 1,237-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module. A long, ca. 550-residue linker separates the third cohesin and the SLH module of ScaC and is characterized by an 18-residue Pro-Thr-Ala-Ser-rich segment that is repeated 27 times. The calculated molecular weight of the mature ScaC polypeptide (excluding the signal peptide) is 124,162. The presence of the cohesins and the conserved SLH module implies that ScaC acts as an anchoring protein. The ScaC cohesins are on a separate branch of the phylogenetic tree that is close to, but distinct from, the type I cohesins. Affinity blotting with representative recombinant probes revealed the following specific intermodular interactions: (i) an expressed CipV cohesin binds selectively to an enzyme-borne dockerin, (ii) a representative ScaB cohesin binds to the CipV band of the cell-free supernatant fraction, and (iii) a ScaC cohesin binds to the ScaB dockerin. The experimental evidence thus indicates that CipV acts as a primary (enzyme-recognizing) scaffoldin, and the protein was also designated ScaA. In addition, ScaB is thought to assume the role of an adaptor protein, which connects the primary scaffoldin (ScaA) to the cohesin-containing anchoring scaffoldin (ScaC). The cellulosome system of A. cellulolyticus thus appears to exhibit a special type of organization that reflects the function of the ScaB adaptor protein. The intercalation of three multiple cohesin-containing scaffoldins results in marked amplification of the number of enzyme subunits per cellulosome unit. At least 96 enzymes can apparently be incorporated into an individual A. cellulolyticus cellulosome. The role of such amplified enzyme incorporation and the resultant proximity of the enzymes within the cellulosome complex presumably contribute to the enhanced synergistic action and overall efficient digestion of recalcitrant forms of cellulose. Comparison of the emerging organization of the A. cellulolyticus cellulosome with the organizations in other cellulolytic bacteria revealed the diversity of the supramolecular architecture.
Figures
FIG. 1.
Scheme showing the positions on the genome and the domain organization of the scaB and scaC genes of A. cellulolyticus. The two genes are located in tandem immediately downstream of scaA (cipV). scaB and scaC both contain multiple copies of cohesin domains (numbered). scaB harbors a typical dockerin domain at its C terminus. The first two cohesins of scaB and all three scaC cohesins are closely attached, with short or no identifiable linker sequences. In contrast, the linker segments that connect the other modules in scaB are relatively long. The scaC linker sequence that connects cohesin 3 to the SLH module is particularly long and is characterized by an 18-residue repeated sequence. ORF, open reading frame.
FIG. 2.
Relationship of the A. cellulolyticus cohesins and dockerins to previously described domains. (A) Phylogenetic analysis of the ScaB and ScaC cohesins relative to the known type I, II, and III cohesins. All four ScaB cohesins map together on a separate branch of the type II cohesins. The ScaC cohesins form a new group, which emanates from the central branch and is distinct from the other cohesin types. Scale bar = 0.1% amino acid substitutions. (B) Phylogenetic analysis of the dockerins of ScaB and the GH9B enzyme. Scaffoldin-based dockerins are indicated by squares.(C) Sequence alignment of the ScaB and the GH9B dockerin domains and their relationships to selected type I dockerins from various cellulosomal scaffoldin and enzyme subunits. Presumed calcium-binding residues are indicated by a blue background, and proposed recognition residues are indicated by a yellow background. With the exception of the domains derived from the proteins described in this paper, the sources of sequences used are described in references , , and . Abbreviations: Acece, A. cellulolyticus; Bacce_, B. cellulosolvens;_ Cloce_, C. cellulolyticus;_ Clocl_, C. cellulovorans;_ Clotm_, C. thermocellum;_ Rumfl_, R. flavefaciens_.
FIG. 3.
Multiple-sequence alignment of the SLH module of A. cellulolyticus ScaC (Acece-ScaC) and the three C. thermocellum anchoring proteins (Clotm-OlpA, Clotm-OlpB, and Clotm-Orf2p). The degrees of conservation are indicated as follows: asterisks indicate that all sequences are identical, colons indicate that the residues are conserved, and dots indicate that residues are semiconserved, as defined by the EBI server (
http://www2.ebi.ac.uk/clustalw/
).
FIG. 4.
Affinity blotting of cell-derived proteins, performed with representative recombinant cohesins from ScaA, ScaB, and ScaC as the probes. A. cellulolyticus cells were grown on cellobiose and separated by centrifugation into supernatant (Sup) and pellet (Cell) fractions. The supernatant fraction was further fractionated by adsorption onto amorphous (phosphoric acid-treated) cellulose prior to subsequent electrophoresis. Samples (cell-associated pellet or cellulose-adsorbed supernatant) were subjected to SDS-PAGE (Gel) and were blotted onto nitrocellulose membranes (Blots). Gels were stained with Coomassie brilliant blue. The blots were probed with different recombinant protein samples, and labeled bands were detected by chemiluminescence by using peroxidase-conjugated, anti-His tag antibody. The probes were CohA5 (the fifth cohesin of ScaA), CohB1 (the first cohesin of ScaB), and CohC3 (the third cohesin of ScaC). Lane Std contained prestained protein molecular weight markers.
FIG. 5.
Affinity blotting of selected dockerin-containing fusion proteins performed with recombinant cohesins from ScaA and ScaC. Dockerins from ScaB and the cellulosomal GH9B enzyme were fused to G. stearothermophilus xylanase T6, and the resultant fusion proteins (Xyn-DocB and Xyn-DocGH9, respectively) were expressed in an appropriate E. coli host cell system. The fusion proteins were subjected to SDS-PAGE (Gel), transferred to nitrocellulose membranes (Blots), and probed with the ScaA and ScaC cohesins (CohA5 and CohC3, respectively), as described in the legend to Fig. 4. Lane Std contained prestained protein molecular weight markers.
FIG. 6.
Schematic representation of the proposed cell surface disposition of the A. cellulolyticus cellulosomal components identified. The GH9B enzyme and other putative dockerin-containing enzymes are incorporated into the ScaA scaffoldin by virtue of their interaction with the ScaA cohesins. ScaB plays the role of an adaptor protein that mediates between the dockerin of the primary scaffoldin, ScaA, and the cohesins of the anchoring scaffoldin, ScaC. The entire complex appears to be cell associated via the resident SLH module of ScaC.
References
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- Bayer, E. A., Y. Shoham, and R. Lamed. September 2000, revision date. Cellulose-decomposing prokaryotes and their enzyme systems. In M. Dworkin, S. Falkow, E. Rosenberg, K.-H. Schleifer, and E. Stackebrandt (ed.), The prokaryotes: an evolving electronic resource for the microbiological community, 3rd ed., release 3.7. [Online.] http://link.springer.de/link/service/books/10125/index.htm. Springer-Verlag, New York, N.Y.
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