The BaeSR two-component system activates bamK , a paralog of the essential β-barrel foldase bamA , in Klebsiella pneumoniae

β-Barrel outer membrane proteins (OMPs) perform critical functions in gram-negative bacteria including outer membrane biogenesis, nutrient acquisition, and efflux. OMPs are folded by the β-barrel assembly machinery (BAM) with BamA as a central and essential component. BamK, a Klebsiella pneumoniae BamA paralog, can functionally replace BamA; however, native bamK expression and a role for BamK have not been reported. Here, we identify mutations in a two-component signal transduction system that activate expression of bamK in K. pneumoniae. Selections for K. pneumoniae mutants resistant to a BamA-targeting antibacterial macrocyclic peptide, PTB1-1, yield on-target and off-target mutations. On-target bamA mutations overlap with previously described positions in the PTB1-1 binding site. Off-target mutations map to three distinct genes, oqxR, ramR, and baeS. Transcriptomics analyses indicate that mutations in baeS, a sensor histidine kinase, activate bamK expression, and these mutants render bamA dispensable for bacterial growth. Our results demonstrate that BamK can functionally substitute for the essential function of BamA in vitro and in vivo, and bamK expression is controlled by a two-component signal transduction system that responds to envelope stress in gram-negative bacteria. These findings provide insight into possible roles for BamK and considerations for future efforts to develop antibiotics targeting OMP folding.IMPORTANCEThe complex envelope of gram-negative bacteria is a critical structure. It is assembled and maintained by multiple essential pathways, all of which, including the β-barrel assembly machinery (BAM) complex, have been the focus of novel antibiotic discovery efforts. Species in the genus Klebsiella encode a paralog to the BAM complex component BamA, called BamK, but the importance and role of this protein has remained a mystery. Leveraging mutants resistant to a recently discovered BamA inhibitor, we describe how activation of the BaeSR envelope stress response system can activate bamK expression to overcome the loss of BamA or its function both in vitro and in vivo. These findings provide important insights into BamK, Klebsiella biology, gram-negative stress responses, and targeting outer membrane protein folding as an antibacterial strategy.