Genetic characterization of periplasmic folding factors and proteases responsible for the maintenance of outer membrane integrity in Escherichia coli

Abstract

The asymmetric outer membrane (OM) of gram-negative bacteria such as Escherichia coli serves as an essential barrier against harmful environmental factors. The OM contains β-barrel proteins called outer membrane proteins (OMPs) that allow for the influx and efflux of solutes. OMPs, such as LptD, are assembled by the Bam complex at the OM. LptD is responsible for assembling lipopolysaccharides (LPS), which are crucial for maintaining OM impermeability.Thus, LptD is an essential OMP. Several chaperone proteins have been implicated in LptD assembly, including SurA, Skp, and FkpA. We used synthetic phenotypes to better understand the relationship between these chaperones and LptD. We report that the PPIase activity of FkpA does not appear to be important for LptD assembly. Analyses from suppressor screens of synthetic phenotypes revealed an interesting parallel between BaeSR and CpxAR regulatory systems. We also demonstrated a possible role for the chaperone protein, Spy, as an adapter for the BaeSR system. Analysis of our yciM suppressor mutation demonstrated that a partial loss-of-function of YciM leads to LpxC overexpression, but that this can be counterbalanced in a Δskp mutant due to polar effects on lpxD. Lastly, we report important findings regarding differences in substrate specificity and localization of periplasmic proteases DegP and BepA.