Abstract
The β-barrel assembly machinery (BAM) complex in Escherichia coli contains key transporter and chaperone proteins that catalyze the folding and outer membrane (OM) insertion of β-barrel outer membrane proteins (OMPs). One of the OMPs reliant on the BAM complex is the BrkA virulence factor and type V secretion system originating in Bordetella pertussis. Recent knockout studies have suggested that two nonessential BAM proteins, BamB and BamE, play key roles in BrkA secretion and membrane stability. This study aims to characterize the importance of the remaining and understudied nonessential BAM protein, BamC, in BrkA secretion and the maintenance of membrane stability. To determine and contextualize the importance of BamC, we studied single-gene knockouts of, bamC, alongside previously studied bamB and bamE knockouts. We initially confirmed the BAM subunit knockouts in JW2462 (∆bamC), JW2496 (∆bamB), and JW2598 (∆bamE) through PCR amplification and Sanger sequencing. We then assessed cleaved and uncleaved BrkA expression by Western blot analysis, which demonstrated that knocking out bamC reduces secreted BrkA. Using growth curve analysis and ethylenediaminetetraacetic acid (EDTA) minimum inhibitory concentration (MIC) assays, we showed that knocking out BamC had no effect on E. coli growth rates and, in contrast to BamB and BamE knockouts, showed a minimal reduction in membrane stability relative to the wild-type (WT). Taken together, these results suggest that BamC plays a key role in the secretion of BrkA, and plays a minimal role in the maintenance of membrane stability.