Abstract
The indiscriminate use of antibiotics has exacerbated the clinical challenge posed by Klebsiella pneumoniae, with persisters often evading antibiotic treatment. To investigate the mechanisms underlying persister formation, we identified the phosphate transporter gene phoU as differentially expressed during persister emergence and recovery. Using CRISPR-Cas9 gene editing, we created a ∆phoU knockout strain in K. pneumoniae ATCC 700603, along with its complemented (CphoU) and various overexpression strains (OE phoU, OE metE, and OE phoA). While growth rates and antibiotic susceptibility remained unchanged in the ∆phoU strain, its capacity to form persisters under levofloxacin and tobramycin stress was significantly reduced. Metabolomic and transcriptomic analyses linked phoU deletion to downregulation of metE (involved in methionine synthesis) and phoA (encoding alkaline phosphatase). ∆phoU also exhibited impaired biofilm formation and reduced extracellular polymeric substance (EPS) production compared to WT, CphoU, and the OE metE and OE phoA overexpression strains. Complementation of metE or phoA partially restored both biofilm formation and persister levels. Importantly, the reduction in persisters is primarily attributed to metabolic defects in planktonic cells-specifically, impaired methionine synthesis and stress response-rather than to reduced biofilm mass, which is considered a parallel phenotype. We conclude that PhoU promotes K. pneumoniae persistence by transcriptionally upregulating metE and phoA, thereby supporting the stress response capacity of planktonic cells. This study elucidates a novel pathway for persister formation and identifies PhoU and its downstream targets as potential therapeutic vulnerabilities.