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.
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 V(max) of 8.8 mumol of PNPP min(-1) mg(-1) and a K(m) of 3.7 mM PNPP at 30 degrees 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.
Ferreira
Ana S
AS
Institute for Biotechnology and Bioengineering, Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
Leitão
Jorge H
JH
Sousa
Sílvia A
SA
Cosme
Ana M
AM
Sá-Correia
Isabel
I
Moreira
Leonilde M
LM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D018441:Biofilms / Q000254:growth & development*; D016956:Burkholderia cepacia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; C487212:cepacian; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases