Involvement of a Protein Tyrosine Kinase in Production of the Polymeric Bioemulsifier Emulsan from the Oil-Degrading StrainAcinetobacter lwoffiiRAG-1

Abstract

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...

Autophosphorylation of the Escherichia coli Protein Kinase Wzc Regulates Tyrosine Phosphorylation of Ugd, a UDP-glucose Dehydrogenase

“…Thus, The UDP-glucose dehydrogenase RkpK of S. meliloti is involved in the synthesis of capsular polysaccharides (40), whereas similar proteins encoded by the udgH gene of Xanthomonas campestris or hasB of Streptococcus pyogenes are required for the biosynthesis of xanthan gum (41) and hyaluronic acid (42), respectively. Similarly, the synthesis of alginate, a polysaccharide produced by Pseudomonas aeruginosa and Azotobacter vinelandii, depends on the UDP-glucose dehydrogenase AlgD (43,44), and the ugd gene product of Acinetobacter lwoffii is involved in emulsan biosynthesis (20). Also, mutations in the UDP-glucose dehydrogenase-encoding cap3A gene of S. pneumoniae are responsible for a change from the encapsulated to unencapsulated phenotype (45).…”

“…Actually, PTKs and PTPs have been shown to play an important role in the production and/or processing of capsular polysaccharides and exopolysaccharides (19,20). For example, in Escherichia coli the kinase Wzc and the phosphatase Wzb have been reported to participate in the biosynthesis of the exopolysaccharide colanic acid (21).…”

Characterization of a Eukaryotic-Like Tyrosine Protein Kinase Expressed by the Shiga Toxin-Encoding Bacteriophage 933W

“…Many of these PKs are required for the synthesis of capsular polysaccharides and exopolysaccharides in a variety of gram-negative and gram-positive bacteria (26,36,38,62). They resemble eukaryotic PKs because they have conserved residues that are important for catalysis in eukaryotic PKs (14).…”

“…Most of the known prokaryotic Tyr PKs are members of a family of membrane-bound autokinases that share a distinct mechanism of autophosphorylation (14,26,38,42,62). Many of these PKs are required for the synthesis of capsular polysaccharides and exopolysaccharides in a variety of gram-negative and gram-positive bacteria (26,36,38,62).…”

Wzy-Dependent Bacterial Capsules as Potential Drug Targets

“…The phosphoregulatory system in particular seems to be well suited for this, given the many studies that have shown its central role in CPS production, and its presence across a wide range of bacterial pathogens [72][73][74]95]. Of the two processes, phosphorylation and dephosphorylation, the effects of BY-kinases are the better studied and have been the focus of recent reviews [66,77].…”