PelX is a UDP-N-acetylglucosamine C4-epimerase involved in Pel polysaccharide-dependent biofilm formation

Pel is an N-acetylgalactosamine rich polysaccharide that contributes to the structure and function of Pseudomonas aeruginosa biofilms. The pelABCDEFG operon is highly conserved among diverse bacterial species, and thus Pel may be a widespread biofilm determinant. Previous annotation of pel gene clusters led us to identify an additional gene, pelX, that is found adjacent to pelABCDEFG in over 100 different bacterial species. The pelX gene is predicted to encode a member of the short-chain dehydrogenase/reductase (SDR) superfamily of enzymes, but its potential role in Pel-dependent biofilm formation is unknown. Herein, we have used Pseudomonas protegens Pf-5 as a model to understand PelX function as P. aeruginosa lacks a pelX homologue in its pel gene cluster. We find that P. protegens forms Pel-dependent biofilms, however, despite expression of pelX under these conditions, biofilm formation was unaffected in a ΔpelX strain. This observation led to our identification of the pelX paralogue, PFL_5533, which we designate pgnE, that appears to be functionally redundant to pelX. In line with this, a ΔpelX ΔpgnE double mutant was substantially impaired in its ability to form Pel-dependent biofilms. To understand the molecular basis for this observation, we determined the structure of PelX to 2.1A resolution. The structure revealed that PelX resembles UDP-N-acetylglucosamine (UDP-GlcNAc) C4-epimerases and, using 1H NMR analysis, we show that PelX catalyzes the epimerization between UDP-GlcNAc and UDP-GalNAc. Taken together, our results demonstrate that Pel-dependent biofilm formation requires a UDP-GlcNAc C4-epimerase that generates the UDP-GalNAc precursors required by the Pel synthase machinery for polymer production.