<b>Amino acid starvation and iron limitation facilitate the biofilm formation of </b><b><i>Klebsiella pneumoniae</i></b><b> within urine</b>

Biofilm formation is a critical virulence mechanism in pathogens.<i> Klebsiella pneumoniae</i> enhances biofilm formation in urine, a key factor implicated in catheter-associated and urinary tract infections. Here we found that compared to nutrient-rich media, <i>K. pneumoniae</i> exhibited heightened biofilm production in urine. Transposon-insertion sequencing identified 19 fitness genes crucial for urinary adaptation, primarily involved in <i>de novo</i> biosynthesis of arginine, methionine, and isoleucine, indicating urine represents an amino acid-starved (AAS) environment. AAS modulated c-di-GMP signaling, upregulating eight diguanylate cyclase (DGC) genes, four phosphodiesterase (PDE) genes, and four dual-function genes, to promote biofilm formation. Furthermore, transcriptomics revealed urine-induced activation of exopolysaccharide (EPS) biosynthesis genes (lipopolysaccharides, capsules, peptidoglycan, and the enterobacterial common antigen). Iron limitation in urine further amplified EPS production and biofilm development. In conclusion, this study identifies AAS-driven c-di-GMP signaling and iron restriction as central mechanisms underlying <i>K. pneumoniae</i> biofilm formation in urine, offering potential targets for anti-biofilm vaccine strategies.