Cellulosomes are multi-enzyme complexes that orchestrate the efficient degradation of cellulose and related plant cell wall polysaccharides. The complex is maintained by the high-affinity protein-protein interaction between two complementary modules: the cohesin and the dockerin. In order to characterize the interaction between different cohesins and dockerins, we have developed matching fusion-protein systems, which harbor either the cohesin or the dockerin component. For this purpose, corresponding plasmid cassettes were designed, which encoded for the following carrier proteins: (i) a thermostable xylanase with an appended His-tag; and (ii) a highly stable cellulose-binding module (CBM). The resultant xylanase-dockerin and CBM-cohesin fusion products exhibited high expression levels of soluble protein. The expressed, affinity-purified proteins were extremely stable, and the functionality of the cohesin or dockerin component was retained. The fusion protein system was used to establish a sensitive and reliable, semi-quantitative enzyme-linked affinity assay for determining multiple samples of cohesin-dockerin interactions in microtiter plates. A variety of cohesin-dockerin systems, which had been examined previously using other methodologies, were revisited applying the affinity-based enzyme assay, the results of which served to verify the validity of the approach.

The cohesive cellulosome complex is sustained by the high-affinity cohesin-dockerin interaction. In previous work [J. Biol. Chem. 276 (2001) 9883], we demonstrated that a single Thr-to-Leu replacement in the Clostridium thermocellum dockerin component differentiates between non-recognition and highaffinity recognition by the interspecies rival cohesin from C. cellulolyticum. In this report, we show that a single Asp-toAsn substitution on the cohesin counterpart also disrupts normal recognition of the dockerin. The Asp34 carboxyl group of the cohesin appears to play a central role in the resultant hydrogenbonding network as an acceptor of two crucial hydrogen bonds from Ser45 of the dockerin domain. The results underscore the fragile nature of the intermolecular contact interactions that maintain this very high-affinity protein-protein interaction.

The genes associated with the biosynthesis of the polymeric bioemulsifier emulsan, produced by the oil-degrading Acinetobacter lwoffii RAG-1 are clustered within a 27-kbp region termed the wee cluster. This report demonstrates the involvement of two genes of the wee cluster of RAG-1, wzb and wzc, in emulsan biosynthesis. The two gene products, Wzc and Wzb were overexpressed and purified. Wzc exhibited ATP-dependent autophosphorylating protein tyrosine kinase activity. Wzb was found to be a protein tyrosine phosphatase capable of dephosphorylating the phosphorylated Wzc. Using the synthetic substrate p-nitrophenyl phosphate (PNPP) Wzb exhibited a V max of 12 mol of PNPP min ؊1 mg ؊1 and a K m of 8 mM PNPP at 30°C. The emulsifying activity of mutants lacking either wzb or wzc was 16 and 15% of RAG-1 activity, respectively, suggesting a role for the two enzymes in emulsan production. Phosphorylation of Wzc was found to occur within a cluster of five tyrosine residues at the C terminus. Colonies from a mutant in which these five tyrosine residues were replaced by five phenylalanine residues along with those of a second mutant, which also lacked Wzb, exhibited a highly viscous colony consistency. Emulsan activity of these mutants was 25 and 24% of that of RAG-1, respectively. Neither of these mutants contained cell-associated emulsan. However, they did produce an extracellular high-molecular-mass galactosamine-containing polysaccharide. A model is proposed in which subunit polymerization, translocation and release of emulsan are all associated and coregulated by tyrosine phosphorylation.The hydrocarbon-degrading organism Acinetobacter lwoffii RAG-1 produces an extracellular, polymeric, galactosaminecontaining lipoheteropolysaccharide bioemulsifier (molecular mass, 10 3 kDa), termed emulsan (31, 43). Because of its properties as an emulsion stabilizer, emulsan has been studied extensively and its chemical composition, physical properties, physiology, and fermentation along with its industrial applications have been reviewed (5,16,17). Briefly, emulsan is composed of a linear repeating trisaccharide subunit consisting of N-acyl-D-galactosamine, N-acyl-L-galactosamine uronic acid and 2,4-diamino-6-deoxy-D-glucosamine (2, 16, 43). The amphipathic properties of emulsan are due in part to the presence of about 15% fatty acids covalently bound to the water-soluble biopolymer in both ester and amide linkages (2). During growth on minimal medium, the biopolymer accumulates on the cell surface of exponential-phase RAG-1 cells as a minicapsule and is released into the medium as a protein-polysaccharide complex as the cells approach stationary phase (11,29). The protein-free polysaccharide termed apoemulsan is less effective than emulsan in forming emulsions with hydrophobic substrates, although it does stabilize preformed emulsions (35,42). An exocellular esterase of RAG-1, cloned and sequenced in Escherichia coli, was found to enhance the emulsification of very hydrophobic substrates by apoemulsan in a fashion which did not de...

A cluster (27 kbp) of genes responsible for the biosynthesis of the amphipathic, polysaccharide bioemulsifier emulsan from the oil-degrading Acinetobacter lwoffii RAG-1 was isolated and characterized. The complete sequence of this cluster, termed wee, consisted of 20 ORFs. One set of 17 ORFs was transcribed in one direction, while a second set of three ORFs, 607 bp upstream of the first, was transcribed in the opposite direction. Mutations in either of the two regions caused defects in emulsan production, yielding specific activities of 5-14 % of parental emulsifying activity. Putative functions could be assigned to proteins involved in production of nucleotide amino sugar precursors, transglycosylation, transacetylation, polymerization and transport. However, no JUMPstart or ops sequences, normally found associated with some polysaccharide biosynthetic gene clusters, were identified. Evidence is presented suggesting that the bioemulsifier may be a member of the group 1 or group 4 polysaccharides.

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