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Applied and Environmental Microbiology, January 2007, p. 524-534, Vol. 73, No. 2
0099-2240/07/$08.00+0     doi:10.1128/AEM.01450-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Functional Analysis of Burkholderia cepacia Genes bceD and bceF, Encoding a Phosphotyrosine Phosphatase and a Tyrosine Autokinase, Respectively: Role in Exopolysaccharide Biosynthesis and Biofilm Formation{triangledown}

Ana S. Ferreira, Jorge H. Leitão, Sílvia A. Sousa, Ana M. Cosme, Isabel Sá-Correia, and Leonilde M. Moreira*

Institute for Biotechnology and Bioengineering, Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal

Received 23 June 2006/ Accepted 6 November 2006

The biosynthesis of the exopolysaccharide (EPS) cepacian by Burkholderia cepacia complex strains requires the 16.2-kb bce cluster of genes. Two of the clustered genes, bceD and bceF, code for two proteins homologous to phosphotyrosine phosphatases and tyrosine kinases, respectively. We show experimental evidence indicating that BceF is phosphorylated on tyrosine and that the conserved lysine residue present at position 563 in the Walker A ATP-binding motif is required for this autophosphorylation. It was also proved that BceD is capable of dephosphorylating the phosphorylated BceF. Using the artificial substrate p-nitrophenyl phosphate (PNPP), BceD exhibited a Vmax of 8.8 µmol of PNPP min–1 mg–1 and a Km of 3.7 mM PNPP at 30°C. The disruption of bceF resulted in the abolishment of cepacian accumulation in the culture medium, but 75% of the parental strain's EPS production yield was still registered for the bceD mutant. The exopolysaccharide produced by the bceD mutant led to less viscous solutions and exhibited the same degree of acetylation as the wild-type cepacian, suggesting a lower molecular mass for this mutant biopolymer. The size of the biofilm produced in vitro by bceD and bceF mutant strains is smaller than the size of the biofilm formed by the parental strain, and this phenotype was confirmed by complementation assays, indicating that BceD and BceF play a role in the establishment of biofilms of maximal size.

* Corresponding author. Mailing address: Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Phone: (351) 218419031. Fax: (351) 218419199. E-mail: lmoreira{at}ist.utl.pt.

{triangledown} Published ahead of print on 17 November 2006.

Applied and Environmental Microbiology, January 2007, p. 524-534, Vol. 73, No. 2
0099-2240/07/$08.00+0     doi:10.1128/AEM.01450-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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