TagR, a newly identified member of the MarR family of transcriptional regulators, represses the NRPS operon in Klebsiella oxytoca

Abstract

ABSTRACT Toxigenic Klebsiella oxytoca strains produce the pyrrolobenzodiazepine enterotoxins tilimycin (TM) and tilivalline (TV), which contribute to the development of antibiotic-associated hemorrhagic colitis. The biosynthesis of these toxins depends on the nonribosomal peptide synthetase (NRPS) operon located within the til pathogenicity island. Although several global and signal-responsive regulators of NRPS operon expression have been identified, the regulatory network governing enterotoxin biosynthesis remains incompletely characterized. In this study, we identified a previously unrecognized transcriptional regulator encoded within the til pathogenicity island of K. oxytoca. This protein, designated TagR (Tilivalline-associated genes repressor), is a member of the MarR family and acts as a negative regulator of NRPS operon expression. Structural prediction, molecular dynamics simulations, and biochemical analyses demonstrated that TagR exhibits the characteristic architecture of MarR family regulators and forms a stable homodimer. Deletion of tagR led to significant upregulation of the NRPS-associated genes npsA, thdA, and npsB, while complementation restored transcriptional repression. Electrophoretic mobility shift assays confirmed that TagR binds directly and specifically to the regulatory region upstream of the NRPS operon, supporting a mechanism of direct transcriptional repression. Consistent with these findings, loss of TagR significantly increased the cytotoxicity of K. oxytoca culture supernatants toward HeLa cells. Collectively, these results identify TagR as a direct repressor of the NRPS operon and expand the regulatory framework governing enterotoxin biosynthesis in toxigenic K. oxytoca. This study provides new insight into the transcriptional control of virulence-associated genes and establishes TagR as a previously unrecognized component of the regulatory network controlling TM and TV production. IMPORTANCE Elucidating the mechanisms by which toxigenic Klebsiella oxytoca regulates enterotoxin production is critical for understanding the pathogenesis of antibiotic-associated hemorrhagic colitis. TagR is identified as a previously unrecognized MarR family regulator that directly represses the nonribosomal peptide synthetase (NRPS) operon responsible for tilimycin (TM) and tilivalline (TV) biosynthesis. This discovery uncovers a novel regulatory mechanism governing toxin production and offers new perspectives on virulence regulation in this emerging intestinal pathogen. Elucidating the mechanisms by which toxigenic Klebsiella oxytoca regulates enterotoxin production is critical for understanding the pathogenesis of antibiotic-associated hemorrhagic colitis. TagR is identified as a previously unrecognized MarR family regulator that directly represses the nonribosomal peptide synthetase (NRPS) operon responsible for tilimycin (TM) and tilivalline (TV) biosynthesis. This discovery uncovers a novel regulatory mechanism governing toxin production and offers new perspectives on virulence regulation in this emerging intestinal pathogen.