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

Pel is anN-acetylgalactosamine rich polysaccharide that contributes to the structure and function ofPseudomonas aeruginosabiofilms. ThepelABCDEFGoperon is highly conserved among diverse bacterial species, and thus Pel may be a widespread biofilm determinant. Previous annotation ofpelgene clusters led us to identify an additional gene,pelX, that is found adjacent topelABCDEFGin over 100 different bacterial species. ThepelXgene 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 usedPseudomonas protegensPf-5 as a model to understand PelX function asP. aeruginosalacks apelXhomologue in itspelgene cluster. We find thatP. protegensforms Pel-dependent biofilms, however, despite expression ofpelXunder these conditions, biofilm formation was unaffected in a ΔpelXstrain. This observation led to our identification of thepelXparalogue, PFL_5533, which we designatepgnE, that appears to be functionally redundant topelX. In line with this, a ΔpelXΔpgnEdouble 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 2.1Å resolution. The structure revealed that PelX resembles UDP-N-acetylglucosamine (UDP-GlcNAc) C4-epimerases and, using1H 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.