The MarR-family regulator TilR links biosynthesis of enterotoxin tilimycin with physiologic adaptation in Klebsiella oxytoca

Members of the Klebsiella oxytoca species complex (KoSC) are ubiquitous gut colonizers that produce the DNA-damaging enterotoxin tilimycin (TM) as part of their virulence-related program. A MarR-like transcriptional regulator, herein designated TilR, controls expression of the divergently transcribed aroX- and NRPS-operons responsible for TM production. Deletion of tilR resulted in pronounced induction of both operons, elevated toxin production, and increased cytotoxicity – establishing TilR as the key repressor of TM biosynthesis. TilR is encoded downstream of the NRPS operon and binds a 20 bp motif within the aroX-NRPS intergenic region, spatially separated from the predicted promoters driving toxin gene expression. Analysis of global transcriptome profiles revealed that TilR, the most highly expressed of the seven MarRs in KoSC, links toxin biosynthesis to a broader transcriptional network involved in nutrient acquisition, stress adaptation, and central metabolism. In addition, TM production coincided with induction of multiple virulence-associated genes involved in adhesion, biofilm formation, and iron acquisition. Phylogenetic and structural analyses revealed that TilR is distinct from canonical MarRs and absent from members of the closely related, competing Klebsiella pneumoniae species complex (KpSC). These findings identify TilR as an evolutionarily specialized global regulator that coordinates TM biosynthesis with broader metabolic, stress-adaptive, and virulence-associated transcriptional responses.