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Journal of Bacteriology, December 2002, p. 6437-6447, Vol. 184, No. 23
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.23.6437-6447.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Impact of Phosphorylation of Specific Residues in the Tyrosine Autokinase, Wzc, on Its Activity in Assembly of Group 1 Capsules in Escherichia coli

Anne Paiment, Jennifer Hocking, and Chris Whitfield*

Department of Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Received 21 May 2002/ Accepted 26 August 2002

WzcCPS 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 WzcCPS C terminus is essential for HMW CPS assembly. Overexpression of WzbCPS (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 WzcCPS phosphorylation for capsule assembly. In this study, the tyrosine-rich region was dissected in an attempt to identify residues critical for WzcCPS phosphorylation and/or capsule expression. Site-directed mutagenesis demonstrated that no single tyrosine residue in this region is sufficient for detectable phosphorylation of WzcCPS 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 WzcCPS 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 WzcCPS. Tyrosine 569, a residue shown to modulate the in vitro phosphorylation of WzcCA from E. coli K-12, was also mutated. The derivative with this mutation still functioned in capsule assembly. Quantitation of K30CPS from this mutant revealed no difference in the amount of polymer produced. Finally, dithiobis(succinimidylpropionate) cross-linking was used to confirm that WzcCPS forms complexes in vivo, independent of the phosphorylation state of the protein.

* Corresponding author. Mailing address: Department of Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1. Phone: (519) 824-4120, ext. 3478. Fax: (519) 837-1802. E-mail: cwhitfie{at}uoguelph.ca.

Journal of Bacteriology, December 2002, p. 6437-6447, Vol. 184, No. 23
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.23.6437-6447.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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