PgaR is a positive regulator of the pgaABCD biosynthetic operon in Klebsiella pneumoniae

The biosynthetic locus encoding the exopolysaccharide poly-N-acetyl-glucosamine (PNAG) is widely conserved across bacteria, including the World Health Organization critical-priority pathogen Klebsiella pneumoniae (Kp). In Kp, PNAG synthesis is mediated by the pgaABCD operon, yet its lineage-specific regulation remains incompletely understood. Using a comparative genomics approach to interrogate the pgaABCD locus across the high-risk clonal Kp complex 258 (CC258) lineage, we identified a previously uncharacterized positive transcriptional regulator located immediately upstream of pgaA , which we designate pgaR . Phylogenetic analysis revealed recurrent evolutionary events affecting this regulatory region, including repeated deletion or truncation of pgaR and a G > A substitution upstream of the pgaR start codon. Functional characterization demonstrated that loss of pgaR abolishes pgaABCD expression and PNAG production, whereas the upstream G > A substitution drives PNAG hyperproduction. Under in vitro growth conditions, Kp produce extensive extracellular PNAG networks. Using a prototype hypervirulent strain to model the role of PNAG in vivo indicated that it is dispensable for virulence in murine pneumonia and peritonitis models. In contrast, PNAG hyper-production significantly attenuated virulence and reduced disease severity. Collectively, these findings identify PgaR as a novel upstream gene regulator of the pgaABCD operon and reveal a previously unrecognized lineage-specific layer of PNAG regulation in Kp. This demonstrates that opposing PNAG phenotypes, loss and hyper-production, have independently and repeatedly emerged among clinical CC258 isolates, highlighting selective pressures acting on this locus.