Impaired BrkA Processing in a geneX Mutant Background May Compromise the Outer Membrane Integrity of Escherichia coli

Abstract

Autotransporters such as BrkA require coordinated folding and insertion into the outer membrane (OM) of Escherichia coli, a process facilitated by periplasmic chaperones, including SurA. This study investigated the relationship between BrkA maturation and OM integrity in strains annotated as surA knockouts from the Keio collection. Two independent isolates of the Keio collection surA knockout strain JW0052 E. coli (JW0052-O and JW0052-T), obtained from separate laboratories, were analyzed alongside wild-type BW25113 E. coli. Despite their annotation, PCR analysis revealed that surA was not removed in either of the JW0052 strains. To assess BrkA processing and OM stability, the strains were transformed with a BrkA-expressing plasmid (pPALMC1) and evaluated via western blotting and growth assays under SDS-EDTA stress conditions. While JW0052-O exhibited BrkA protein processing comparable to wild-type, JW0052-T showed impaired BrkA cleavage and heightened sensitivity to membrane stress when transformed with pPALMC1. These defects suggest that an unknown gene, geneX, presents an uncharacterized mutation, designated geneX^mut, that may disrupt OM homeostasis independently of surA. This work underscores the importance of knockout validation and highlights the potential role of additional factors in autotransporter maturation and OM integrity.