Functional characterization of MigA and WapR: putative rhamnosyltransferases involved in outer core oligosaccharide biosynthesis of Pseudomonas aeruginosa

Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada; Institute for Biological Sciences, National Research Council, Ottawa, ON, K1A 0A6, Canada; Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA

^* To whom correspondence should be addressed. Email: jlam{at}uoguelph.ca.

	Abstract

Pseudomonas aeruginosa lipopolysaccharide (LPS) contains two glycoforms of core oligosaccharide (OS): one is capped with O-antigen through an -1,3-linked L-rhamnose (L-Rha), while the other is uncapped and contains an -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 of both genes were generated. The wapR mutant produces LPS lacking O antigen, and addition of wapR in trans complemented this defect. The migA mutant produces LPS with a truncated outer core, and showed no reactivity to the 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 core-plus-one O-repeat). This was due to overexpression of MigA in the complemented strain causing an increase in the proportion of the uncapped core OS, thereby diminishing the amount of core-plus-one O-repeat, indicating a regulatory role for MigA. 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 -1,3-L-Rha. In contrast, uncapped core OS from the migA mutant lacked -1,6-L-Rha. These results provide evidence that WapR is the -1,3-rhamnosyltransferase while MigA is the -1,6-rhamnosyltransferase.