title: Role of PelF in pel polysaccharide biosynthesis in Pseudomonas aeruginosa.
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.
authors: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
authors: Aamir
authors: A
authors: Ghafoor
authors: Zoe
authors: Z
authors: Jordens
authors: Bernd H A
authors: BH
authors: Rehm
mesh_terms: D000464:Alginates / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D020723:Glucuronic Acid / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006603:Hexuronic Acids / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D020723:Glucuronic Acid; D014533:Uridine Diphosphate Glucose Dehydrogenase; D016695:Glycosyltransferases; D014532:Uridine Diphosphate Glucose