Understanding the role of the lipoprotein DolP in cell envelope Biogenesis

Boelter, Gabriela (2023). Understanding the role of the lipoprotein DolP in cell envelope Biogenesis. University of Birmingham. Ph.D.

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Abstract

The bacterial cell envelope protects the cell against environmental threats, maintains the cell shape and contributes to metabolism and growth. The Gram-negative bacterial envelope is composed of three-layers, the outer membrane (OM), a peptidoglycan (PG) layer, and the inner membrane. The coordination of proteins involved in cell growth and septation is essential to avoid cell lyses. The aim of this thesis is to study the lipoprotein, DolP (formerly YraP) and its role in the cell envelope biogenesis of the Gram-negative bacterium Escherichia coli. DolP is a dual-BON domain lipoprotein localised in the OM. Studies have suggested that DolP might be a component of the BAM (β-barrel assembly machinery) complex and be a player in cell division. The BAM complex is formed by a β-barrel lipoprotein BamA and four accessory components, BamB, BamC, BamD and BamE. To test the first hypothesis, we deleted the non-essential genes bamB, bamC and bamE in a ΔdolP background. We observed a reduction in cell fitness and increase in the number of lysed cells in ΔbamBΔdolP and ΔbamCΔdolP mutants compared to the single mutants. The results suggest that DolP impacts the OM proteins assembly machinery. The second hypothesis is based on a study that suggested that DolP is an upstream regulator of NlpD. NlpD is the activator of the amidase AmiC. Amidases cleave the shared PG layer of adjunct cells to separate into daughter cells. In E. coli, amidases (AmiA, AmiB and AmiC) are regulated by NlpD, EnvC or ActS. To verify DolP’s link to this process, we first observed that DolP does not regulate amidases activity in vitro and does not interact with NlpD in pull-down and MST (MicroScale Thermophoresis) assays. In addition, the mutant ΔdolP did not phenocopied ΔnlpD in a range of envelope stresses. Next, we tested the morphology of a panel of double deletion mutants of amidases and amidase regulators with dolP. The analysis showed that ΔamiAΔdolP and ΔenvCΔdolP mutants present longer chain length compared to their parental strains indicating a role for DolP in cell division. In conclusion, we suggest that DolP might not be a NlpD regulator. However, DolP may impact daughter cell separation by interacting with AmiA and AmiC, or by a yet unknown mechanism.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):