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Journal of Bacteriology, February 2003, p. 1001-1009, Vol. 185, No. 3
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.3.1001-1009.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Involvement of a Protein Tyrosine Kinase in Production of the Polymeric Bioemulsifier Emulsan from the Oil-Degrading Strain Acinetobacter lwoffii RAG-1

David Nakar and David L. Gutnick*

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Science, Tel Aviv University, Tel Aviv 69978, Israel

Received 1 July 2002/ Accepted 21 October 2002

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 Vmax of 12 µmol of PNPP min-1 mg-1 and a Km 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.

* Corresponding author. Mailing address: Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Science, Tel Aviv University, Tel Aviv 69978, Israel. Phone: 972-3-6409834. Fax: 972-3-6409407. E-mail: davidg{at}post.tau.ac.il.

Journal of Bacteriology, February 2003, p. 1001-1009, Vol. 185, No. 3
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.3.1001-1009.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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