Role of PelF in pel polysaccharide biosynthesis in Pseudomonas aeruginosa.
Appl Environ Microbiol
; 79(9): 2968-78, 2013 May.
Article
em En
| MEDLINE
| ID: mdl-23435893
ABSTRACT
Pseudomonas aeruginosa produces three exopolysaccharides, Psl, Pel, and alginate, that play vital roles in biofilm formation. Pel is a glucose-rich, cellulose-like exopolysaccharide. The essential Pel biosynthesis proteins are encoded by seven genes, pelA to pelG. Bioinformatics analysis suggests that PelF is a cytosolic glycosyltransferase. Here, experimental evidence was provided to support this PelF function. A UDP-glucose dehydrogenase-based assay was developed to quantify UDP-glucose. UDP-glucose was proposed as the substrate for PelF. The isogenic pelF deletion mutant accumulated 1.8 times more UDP-glucose in its cytosol than the wild type. This suggested that PelF, which was found localized in the cystosol, uses UDP-glucose as substrate. Additionally, in vitro experiments confirmed that PelF uses UDP-glucose as substrate. To analyze the functional roles of conserved residues in PelF, site-directed mutagenesis was performed. The presence of the EX7E motif is characteristic for various glycosyltransferase families, and in PelF, E405/E413 are the conserved residues in this motif. Replacement of E405 with A resulted in a reduction of PelF activity to 30.35% ± 3.15% (mean ± standard deviation) of the wild-type level, whereas replacement of the second E, E413, with A did not produce a significant change in the activity of PelF. Moreover, replacement of both E residues did not result in a loss of PelF function, but replacement of the conserved R325 or K330 with A resulted in a complete loss of PelF activity. Overall, our data show that PelF is a soluble glycosyltransferase that uses UDP-glucose as the substrate for Pel synthesis and that conserved residues R325 and K330 are important for the activity of PelF.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Polissacarídeos
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Pseudomonas aeruginosa
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Proteínas de Bactérias
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Regulação Bacteriana da Expressão Gênica
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Glicosiltransferases
Tipo de estudo:
Prognostic_studies
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article