Construction of a Chloramphenicol‑Resistant Bioluminescent Reporter for Monitoring Envelope Stress in Keio Collection Escherichia coli Knockout Strains

Gram-negative bacteria rely on periplasmic chaperones such as SurA and Skp, and on DegP, a dual-function protease and chaperone, to ensure proper outer membrane protein (OMP) biogenesis​. SurA primarily delivers unfolded OMPs to the β-barrel assembly machinery (BAM complex)​. BrkA, a 103-kDa autotransporter OMP from Bordetella pertussis, is processed into a 73-kDa passenger (α-domain) and a 30-kDa β-barrel domain that anchors in the outer membrane​. Envelope stress can cause uncleaved BrkA to accumulate, but whether this misfolding activates the CpxAR two-component response remains unclear. We initially hypothesized that a surA knockout in Escherichia coli would exacerbate BrkA misfolding and activate the Cpx pathway. However, PCR and Sanger sequencing revealed that the Keio JW0052 strain retained surA. We therefore redirected our study to examine the effects of IPTG-induced BrkA overexpression in wild-type E. coli on growth, BrkA processing, and envelope stress activation. Western blotting showed IPTG-dependent BrkA expression, with passenger cleavage detected only at 1 mM IPTG. Co-transformation of the PcpxP::luxCDABE reporter plasmid pJW1 with the BrkA-expression plasmid pPALMC1 abolished luminescence and reduced growth by ~50%, suggesting metabolic burden or interference with reporter activity. Dose-dependent suppression of luminescence was also observed with IPTG treatment alone, even in the absence of BrkA. Additionally, we engineered a chloramphenicol-resistant reporter plasmid (pRAAMS) to enable future studies in the kanamycin-resistant Keio strains. These findings provide a validated single-plasmid system for Cpx pathway monitoring in E. coli BW25113 and highlight the complexity of stress regulation.