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.