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Journal of Bacteriology, May 2005, p. 3359-3368, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3359-3368.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Defective O-Antigen Polymerization in tolA and pal Mutants of Escherichia coli in Response to Extracytoplasmic Stress

Enrique D. Vinés,1 Cristina L. Marolda,1 Aran Balachandran,1 and Miguel A. Valvano1,2*

Departments of Microbiology and Immunology,1 Medicine, University of Western Ontario, London, Ontario N6A 5C1, Canada2

Received 28 December 2004/ Accepted 31 January 2005

We have previously shown that the TolA protein is required for the correct surface expression of the Escherichia coli O7 antigen lipopolysaccharide (LPS). In this work, {Delta}tolA and {Delta}pal mutants of E. coli K-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding the Klebsiella pneumoniae O1 LPS gene cluster), or pWQ802 (encoding the genes necessary for the synthesis of Salmonella enterica O:54). Both {Delta}tolA and {Delta}pal mutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression of K. pneumoniae O1 LPS and S. enterica O:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in a wzy (polymerase)-dependent manner (like those of E. coli O7 and O16) but not for O antigens assembled by wzy-independent pathways (like K. pneumoniae O1 and S. enterica O:54). Furthermore, we show that the reduced surface expression of O16 LPS in {Delta}tolA and {Delta}pal mutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intact tolA and pal genes, respectively. Using derivatives of W3110{Delta}tolA and W3110{Delta}pal containing lacZ reporter fusions to fkpA and degP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extracytoplasmic stress responses in tol+ and pal+ genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.

* Corresponding author. Mailing address: Departments of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada. Phone: (1)5196613996. Fax: (1) 5196613499. E-mail: mvalvano{at}uwo.ca.

Journal of Bacteriology, May 2005, p. 3359-3368, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3359-3368.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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