Abstract
The lipopolysaccharide (LPS) is essential for most Gram-negative bacteria as it is a main component of the outer membrane (OM). In the pathogen Brucella abortus, smooth LPS (S-LPS) is crucial for virulence. Being part of the Rhizobiales, Brucella displays unipolar growth and its LPS was shown to be incorporated at the growth sites, i.e. the new pole and the constriction site. When synthesized, the rough LPS (R-LPS) is anchored to the cytoplasmic leaflet of the inner membrane (IM) and is then flipped to the periplasmic leaflet by MsbA, an essential ABC transporter. The O-antigen is independently flipped to the periplasmic leaflet of the IM and is then ligated by a periplasmic ligase to a fraction of the R-LPS, giving S-LPS. S-LPS and R- LPS are translocated by the essential LPS translocation machinery (Lpt). LptDE and LptB2CFG form the OM and IM complexes, respectively. Nothing is known about the localization of MsbA or the Lpt complex, and the O-antigen ligase remains unidentified in Brucella. Here, we showed that the IM complex (LptC and LptF) is localized at the growth sites, while LptD is dispersed all over the OM. We hypothesize that LPS could be translocated only when the entire complex is formed at the growth sites and that a fraction of LptD would be inactive. Surprisingly, MsbA was mainly localized at the old pole and time-lapse microscopy suggestedthat MsbA was able to move during growth. Based on this, we proposed that MsbA would be mobile in the IM, actively flipping LPS. Then, LPS would diffuse in the IM until it encounters the Lpt pathway. In addition, we identified WadA as the main O-antigen ligase in Brucella spp. WadA presents a N-terminal glycosyltransferase domain, that was shown to add the last glucose onto the R-LPS core, and a C-terminal predictedto be an O-antigen ligase domain. We showed that deleting the biggest periplasmic loop of the O-antigen ligase domain generates a rough phenotype, that could be restored to smooth by adding a copy of the O-antigen ligase domain only. This work highlights a new class of bifunctional O- antigen ligase.
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