LolA and LolB are conserved in Bacteroidetes and are crucial for gliding motility and Type IX secretion.

Abstract

In Gram-negative bacteria, lipoproteins are major components of the outer membrane (OM) where they play a variety of roles, from the involvement in membrane biogenesis to virulence. Bacteroidetes, a widespread phylum of Gram-negative bacteria, including free-living organisms, commensals and pathogens, encode an exceptionally high number of outer membrane lipoproteins. These proteins are crucial in this phylum mainly because they are key components of SUS-like nutrient acquisition systems as well as of the Type 9 secretion (T9SS) and gliding motility machineries. The transport of lipoproteins to the OM has mainly been studied in E. coli and relies on the Lol system, composed of the inner membrane extraction machinery LolCDE, the periplasmic carrier LolA and the OM lipoprotein LolB. While most Lol proteins are essential and conserved across Gram-negative bacteria, to date, no LolB homologs have been identified outside of {gamma}- and {beta}-proteobacteria. How lipoproteins reach and are inserted in the OM of Bacteroidetes is not known. Here we identified LolB homologs in Bacteroidetes and disclosed the co-existence of several LolA and LolB in several species. We provide evidence that one LolA (LolA1) and one LolB (LolB1) of F. johnsoniae are devoted to targeting gliding and T9SS lipoproteins to the OM. A proteomic analysis of the OM composition of the lolA1 and lolB1 mutants supports this evidence. Furthermore, we show that, while LolB1 and LolA1 have conserved functions in Bacteroidetes, they are functionally different from their E. coli counterparts. We also show that surface lipoprotein transport is LolA and LolB independent. Finally, the finding that, in the absence of LolA and LolB homologs, lipoproteins still localize to the OM, suggests the presence in Bacteroidetes of yet unidentified LolAB-alternative lipoprotein transport pathways. In conclusion, Bacteroidetes have evolved different and more complex lipoprotein transport pathways than other Gram-negative bacteria and further research is required to uncover their complexity. SignificanceIn Gram-negative bacteria, lipoproteins are key components of the outer membrane (OM), essential for functions like membrane biogenesis and virulence. Bacteroidetes, a widespread phylum, encode a high number of OM lipoproteins crucial for nutrient acquisition, Type IX secretion, and gliding motility. While lipoprotein transport in E. coli depends on the Lol system, LolB homologs were previously unidentified outside {gamma}- and {beta}-proteobacteria. Here we identify LolB homologs in Bacteroidetes, revealing the co-existence of multiple LolA and LolB proteins in various species. In F. johnsoniae, LolA1 and LolB1 specifically target gliding and Type 9 secretion system lipoproteins to the OM. Despite this, lipoproteins still localize to the OM without LolA and LolB, suggesting alternative transport pathways. These findings indicate that Bacteroidetes have evolved more complex lipoprotein transport mechanisms than other Gram-negative bacteria, requiring further research to fully understand them.