Using fluorescently labeled wheat germ agglutinin to track lipopolysaccharide transport to the outer membrane in .

The cell envelope of gram-negative bacteria consists of two membranes sandwiching the peptidoglycan (PG) cell wall. The outer membrane (OM) contains integrated beta-barrel proteins and has an outer leaflet composed of lipopolysaccharide (LPS). LPS is transported from the inner membrane where it is made to the OM surface by the Lpt system. In the polarly elongating alpha-proteobacterium , LPS transport has been localized to the polar growth zone and division site. However, LPS transport has not been tracked in live proteobacteria like that elongate by dispersed incorporation of envelope material along their cell body. Here, we report an investigation into the binding target of fluorescently labeled wheat germ agglutinin (FL-WGA) on cells that led to the development of a method for visualizing LPS transport. We show that instead of PG or enterobacterial common antigen for which FL-WGA labeling has been used to detect in the past, this probe recognizes LPS modified with a terminal N-acetylglucosamine formed by the defective O-antigen synthesis pathway of laboratory strains of . This finding enabled the construction of mutants inducible for LPS modification that were used together with FL-WGA labeling to track LPS transport to the cell surface. We show that new LPS is inserted throughout the cell cylinder and at the division site, but not at the cell poles. A similar pattern was observed previously for PG synthesis and OM protein insertion in , suggesting that LPS transport to the OM is coordinated with these processes.IMPORTANCEGram-negative bacteria like are surrounded by a multilayered cell envelope that includes an outer membrane (OM) responsible for their high intrinsic resistance to antibiotics. The outer leaflet of this membrane is composed of a glycolipid called lipopolysaccharide (LPS). Here, we report the development of an imaging method to track the transport of LPS to the outer membrane. The results indicate that transport occurs throughout the cell cylinder and at the division site, but not at the cell poles. A similar pattern was observed previously when cell wall synthesis and the insertion of proteins into the OM were tracked. Our results therefore suggest that LPS transport to the OM is coordinated with other essential processes that underly gram-negative cell envelope biogenesis.