Bernhardt, Thomas GDubois, Laurent2026-03-0420262026-02-272026Dubois, Laurent. 2026. Investigating the spatiotemporal control of lipopolysaccharide transport to the outer membrane in Escherichia coli. Doctoral Dissertation, Harvard University Graduate School of Arts and Sciences.32403782https://dash.harvard.edu/handle/1/42731185The Gram-negative cell envelope consists of an inner membrane (IM), an outer membrane (OM) and a thin layer of peptidoglycan (PG) in between the two membranes. The IM has phospholipids in its inner and outer leaflets, whereas the OM is asymmetric with phospholipids (PL) in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. The bacterial cell envelope is essential for the cell, as it gives the bacteria its shape and acts as a protective barrier inhibiting the entry of external antimicrobial factors such as antibiotics and phages. Many of the proteins involved in cell envelope biogenesis are known, however it is less well understood how synthesis of the different cell envelope components is coordinated. Here we investigated the transport of LPS to the OM and how it is regulated during cell division. First, we showed that fluorescent wheat germ agglutinin (WGA) binds specifically to N-acetylglucosamine (GlcNAc)-modified LPS (GlcNAcLPS) in the model organism E. coli MG1655. Previous studies showed that WGA binds to GlcNAc, but this sugar is present in different molecules in the cell envelope. The binding site for fluorescently labeled WGA (FL-WGA) on cells therefore remained unclear. We showed that in intact cells, FL-WGA does not bind to PG as previously thought but instead labels GlcNAcLPS. We used this discovery to develop an E. coli strain in which transport of newly synthesized GlcNAcLPS to the OM could be tracked. Using this tool, we showed that nascent LPS is inserted into the OM at dispersed locations in the cell cylinder during cell elongation and at the division site during cell division. A similar pattern of labeling was previously observed for protein insertion into the OM and for new PG synthesis, indicating that LPS transport is likely to be coordinated with these processes. While studying the effect of different cell division inhibitors on LPS transport at the division site, we discovered that the division-specific PG synthesis inhibitor cephalexin blocks LPS transport at midcell. Such a block was not observed when assembly of the division machinery (divisome) was blocked in cephalexin treated cells by expression of the division inhibitor SulA. Thus, inhibition of LPS transport at the division site in cephalexin treated cells, requires assembly of the divisome. We hypothesized that PL is used to expand the OM at midcell when LPS transport is blocked by cephalexin treatment. Accordingly, cephalexin treatment rendered cells sensitive to the detergent sodium dodecyl sulfate (SDS) to which cells with an intact OM are normally resistant. These results reveal that beta-lactam antibiotics like cephalexin not only affect PG synthesis but also disrupt the permeability barrier of the OM. Additionally, these findings indicate that LPS transport requires proper PG synthesis at the division site and that the two processes are likely to be coordinated by a mechanism that remains to be elucidated.application/pdfenBiologyMicrobiologyGeneticsThesis or Dissertation2026-03-040000-0003-0963-4029