Revisiting the functions of periplasmic chaperones in the quality control of the autotransporter Ag43 using a phenotypically homogeneous Escherichia coli strain

Antigen 43 is a surface-displayed autotransporter protein that mediates bacterial self-association and pathogenicity. The quality control factors that facilitate Ag43 crossing the periplasm and inserting into the outer membrane remain enigmatic, mostly because Ag43 is phase variable and associated with heterologous phenotypes, which obscures the mutational effects of potential quality control factors. Here, we describe a screening method that allowed us to isolate a subpopulation of Escherichia coli that consistently displays an Ag43-mediated autoaggregation phenotype. Based on this subpopulation, we analyzed how disruptions of known periplasmic chaperones affect Ag43 biogenesis. We found that only the disruption of surA reduced Ag43 levels and abolished the autoaggregation phenotype of cells, but surA disruption did not affect the phase-variable expression of agn43 . Using purified proteins, we showed that SurA effectively protected the β-barrel domain of Ag43 from aggregation. In contrast, the previously reported Ag43 biogenesis factor OsmY showed weak chaperoning effects on Ag43 only in the absence of SurA. Our results shed light on the roles of different periplasmic chaperones in Ag43 biogenesis and provide a methodology applicable to the study of other phase-variable proteins. • An Ag43+, Fim- E. coli strain eliminates phenotypic heterogeneity caused by the phase variation property of agn43 . • Chaperones SurA and OsmY serve as obligatory and auxiliary quality control factors for Ag43 biogenesis, respectively. • Knockout of surA in the E. coli K-12 MG1655 strain abrogates the Ag43-mediated autoaggregation phenotype.