title: Impact of phosphorylation of specific residues in the tyrosine autokinase, Wzc, on its activity in assembly of group 1 capsules in Escherichia coli.
abstract: Wzc(CPS) is a tyrosine autokinase essential for the assembly of a high-molecular-weight (HMW) group 1 capsular polysaccharide (CPS) in Escherichia coli. Homologues of Wzc participate in the formation of CPS and exopolysaccharides in a variety of gram-positive and gram-negative bacteria. Phosphorylation of tyrosine residues in the Wzc(CPS) C terminus is essential for HMW CPS assembly. Overexpression of Wzb(CPS) (phosphatase) in a wild-type background caused a 3.7-fold decrease in the amount of cell-associated K30 CPS produced, confirming the importance of Wzc(CPS) phosphorylation for capsule assembly. In this study, the tyrosine-rich region was dissected in an attempt to identify residues critical for Wzc(CPS) phosphorylation and/or capsule expression. Site-directed mutagenesis demonstrated that no single tyrosine residue in this region is sufficient for detectable phosphorylation of Wzc(CPS) in vivo or for HMW CPS expression. Furthermore, no single tyrosine residue is essential for phosphorylation or capsule assembly, since removal of any one tyrosine residue has no detectable effect. Altering combinations of tyrosine residues (from two to five) led to Wzc(CPS) derivatives that were still competent for phosphorylation but that could not support assembly of HMW CPS, showing that phosphorylation of Wzc per se is not an accurate measure of its ability to function in capsule assembly. One interpretation of these data is that the overall level of phosphorylation in this region, rather than the precise combination of residues accessible to phosphorylation, is important for the activity of Wzc(CPS). Tyrosine 569, a residue shown to modulate the in vitro phosphorylation of Wzc(CA) from E. coli K-12, was also mutated. The derivative with this mutation still functioned in capsule assembly. Quantitation of K30(CPS) from this mutant revealed no difference in the amount of polymer produced. Finally, dithiobis(succinimidylpropionate) cross-linking was used to confirm that Wzc(CPS) forms complexes in vivo, independent of the phosphorylation state of the protein.
authors.affiliation: Department of Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
authors.forename: Anne
authors.initials: A
authors.lastname: Paiment
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hocking
authors.forename: Chris
authors.initials: C
authors.lastname: Whitfield
mesh_terms: D016667:Bacterial Capsules / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C417168:WZC protein, Bacteria; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C499615:wzc protein, E coli