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

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 <i>Klebsiella pneumoniae</i> BamA paralog, can functionally replace BamA; however, native <i>bamK</i> 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 <i>bamK</i> in <i>K. pneumoniae</i>. Selections for <i>K. pneumoniae</i> mutants resistant to a BamA-targeting antibacterial macrocyclic peptide, PTB1-1, yield on-target and off-target mutations. On-target <i>bamA</i> mutations overlap with previously described positions in the PTB1-1 binding site. Off-target mutations map to three distinct genes, <i>oqxR</i>, <i>ramR</i>, and <i>baeS</i>. Transcriptomics analyses indicate that mutations in <i>baeS,</i> a sensor histidine kinase, activate <i>bamK</i> expression, and these mutants render <i>bamA</i> dispensable for bacterial growth. Our results demonstrate that BamK can functionally substitute for the essential function of BamA <i>in vitro</i> and <i>in vivo,</i> and <i>bamK</i> 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 <i>Klebsiella</i> 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 <i>bamK</i> expression to overcome the loss of BamA or its function both <i>in vitro</i> and <i>in vivo</i>. These findings provide important insights into BamK, <i>Klebsiella</i> biology, gram-negative stress responses, and targeting outer membrane protein folding as an antibacterial strategy.