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Journal of Bacteriology, March 2008, p. 1857-1865, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01546-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Functional Characterization of MigA and WapR: Putative Rhamnosyltransferases Involved in Outer Core Oligosaccharide Biosynthesis of Pseudomonas aeruginosa{triangledown}

Karen K. H. Poon,1,{dagger},{ddagger} Erin L. Westman,1,{dagger} Evgeny Vinogradov,2 Shouguang Jin,3 and Joseph S. Lam1*

Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada,1 Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0A6, Canada,2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 326103

Received 25 September 2007/ Accepted 21 December 2007

Pseudomonas aeruginosa lipopolysaccharide (LPS) contains two glycoforms of core oligosaccharide (OS); one form is capped with O antigen through an {alpha}-1,3-linked L-rhamnose (L-Rha), while the other is uncapped and contains an {alpha}-1,6-linked L-Rha. Two genes in strain PAO1, wapR (PA5000) and migA (PA0705), encode putative glycosyltransferases associated with core biosynthesis. We propose that WapR and MigA are the rhamnosyltransferases responsible for the two linkages of L-Rha to the core. Knockout mutants with mutations in both genes were generated. The wapR mutant produced LPS lacking O antigen, and addition of wapR in trans complemented this defect. The migA mutant produced LPS with a truncated outer core and showed no reactivity to outer core-specific monoclonal antibody (MAb) 5C101. Complementation of this mutant with migA restored reactivity of the LPS to MAb 5C101. Interestingly, LPS from the complemented migA strain was not reactive to MAb 18-19 (specific for the core-plus-one O repeat). This was due to overexpression of MigA in the complemented strain that caused an increase in the proportion of the uncapped core OS, thereby decreasing the amount of the core-plus-one O repeat, indicating that MigA has a regulatory role. The structures of LPS from both mutants were elucidated using nuclear magnetic resonance spectroscopy and mass spectrometry. The capped core of the wapR mutant was found to be truncated and lacked {alpha}-1,3-L-Rha. In contrast, uncapped core OS from the migA mutant lacked {alpha}-1,6-L-Rha. These results provide evidence that WapR is the {alpha}-1,3-rhamnosyltransferase, while MigA is the {alpha}-1,6-rhamnosyltransferase.

* Corresponding author. Mailing address: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada. Phone: (519) 824-4120, ext. 53823. Fax: (519) 837-1802. E-mail: jlam{at}uoguelph.ca

{triangledown} Published ahead of print on 4 January 2008.

{dagger} K.K.H.P. and E.L.W. contributed equally to this work.

{ddagger} Present address: Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive N.W., Calgary, AB T2N 4N1, Canada.

Journal of Bacteriology, March 2008, p. 1857-1865, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01546-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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