Morphogenesis of the Escherichia coli cell envelope

The E. coli cell envelope is composed of the inner membrane, periplasm and outer membrane (OM). The cell envelope provides physical robustness to the cell and functions as a permeability barrier, while undergoing changes during growth. This thesis addresses the transport of lipopolysaccharides (LPSs) to the OM and the cell-cycle-dependent behaviour of Pal (attaches the OM to the septal peptidoglycan).
When the transport of LPS to the OM is inhibited cells form deeply constricted filaments. The divisome components FtsZ and FtsN accumulate and depart from constriction sites. Pal, however, accumulates but does not depart. The constriction sites contain unseparated peptidoglycan and the overall transport of proteins to the periplasm seems impaired.
Several fluorescence-based detection tools for the Lpt machinery were explored. Antibodies against LptA were found to be suitable for immunolocalisation. Fusions of fluorescent proteins to LptC, LptD, LptE, LptF and LptG localised correctly. The interaction between mCherry-LptF and mKO-LptG could be detected with FRET but suffered from replicability issues.
Balancing the expression of sfTq2^ox and mNG in the cytoplasm of E. coli results in a donor-acceptor pair for which FRET in a large dynamic range can be detected. An improvement from currently 1.5-25 % (mCherry-mKO) to 1.5-66 % (mNG-sfTq2^ox).
Innate Pal was studied with immunolabeling. Pal’s retention at the new pole persists up to 40% of the cell division cycle, and its abundance changes during the cell division cycle. The periplasmic protease Prc may indirectly regulate this, but the small RNA MicA regulates Pal in a cell division cycle-independent manner.