Identification of FkpA as a Key Quality Control Factor for the Biogenesis of Outer Membrane Proteins under Heat Shock Conditions

Abstract

The outer membrane proteins (OMPs) of Gram-negative bacterial cells, as well as the mitochondrion and chloroplast organelles, possess unique and highly stable ␤-barrel structures. Biogenesis of OMPs in Escherichia coli involves such periplasmic chaperones as SurA and Skp. In this study, we found that the ⌬surA ⌬skp double-deletion strain of E. coli, although lethal and defective in the biogenesis of OMPs at the normal growth temperature, is viable and effective at the heat shock temperature. We identified FkpA as the multicopy suppressor for the lethal phenotype of the ⌬surA ⌬skp strain. We also demonstrated that the deletion of fkpA from the ⌬surA cells resulted in only a mild decrease in the levels of folded OMPs at the normal temperature but a severe decrease as well as lethality at the heat shock temperature, whereas the deletion of fkpA from the ⌬skp cells had no detectable effect on OMP biogenesis at either temperature. These results strongly suggest a functional redundancy between FkpA and SurA for OMP biogenesis under heat shock stress conditions. Mechanistically, we found that FkpA becomes a more efficient chaperone for OMPs under the heat shock condition, with increases in both binding rate and affinity. In light of these observations and earlier reports, we propose a temperature-responsive OMP biogenesis mechanism in which the degrees of functional importance of the three chaperones are such that SurA > Skp > FkpA at the normal temperature but FkpA > SurA > Skp at the heat shock temperature.

Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins

“…2 shows that the individual ΔdegP, Δskp, and ΔfkpA simulations display fOMP and free and aggregated uOMP concentration profiles similar to WT profiles under normal growth conditions. These results are consistent with the modest phenotypic effects observed in genetic studies of the null strains for each corresponding chaperone (11).…”

“…In contrast, SurA is the only single periplasmic chaperone that is known to cause a significant change in fOMP density when depleted (11,13). Our simulations capture this genetic finding (Fig.…”

“…The importance of SurA in OMP biogenesis arises from its dual functions, both of which are incorporated in OMPBioM: (i) SurA can bind to uOMP, thereby preventing aggregation, and (ii) SurA can facilitate the folding of uOMP (13)(14)(15)(16)(17)22). Because SurA plays a prominent role in OMP folding, the loss of SurA in vivo results in the induction of the σ E stress response (11,13), a regulatory mechanism caused by the accumulation of unfolded protein in the periplasm. For ΔsurA, activation of this envelope stress response results in an increase in chaperone expression and a reduction in uOMP expression; these experimental observations are incorporated into our ΔsurA simulation (SI Appendix, Table S3) (26,27).…”

“…1 to simulate genetic mutants lacking specific chaperones. Four distinct single-chaperone-null strain simulations (ΔdegP, Δskp, ΔfkpA, or ΔsurA) were independently performed and compared with the WT simulations and with known biological phenotypes (11)(12)(13). Fig.…”

“…Many in vitro and in vivo studies have investigated various components involved in this pathway (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). However, in vitro experiments typically characterize individual components in isolation, and in vivo studies are often incapable of deciphering how specific components are responsible for observed phenomena.…”