Amino acid starvation and iron limitation facilitate the biofilm formation of Klebsiella pneumoniae within urine

Biofilm formation is a critical virulence mechanism in pathogens such as <i>Klebsiella pneumoniae</i>, a Gram-negative, encapsulated bacterium that has emerged as a zoonotic threat capable of infecting both humans and animals. Its biofilm-forming ability is closely associated with catheter-related and urinary tract infections. Given its potential to cross species barriers and cause significant public health concern, elucidating the environmental cues and conserved molecular pathways driving biofilm formation is essential for developing cross-species prevention strategies. Here we found that <i>K. pneumoniae</i> exhibited significantly greater biofilm-forming efficiency in urine than in nutrient-rich medium under comparable biomass conditions. Transposon-insertion sequencing (Tn-seq) identified 19 fitness genes essential for optimal growth in urine, most involved in the <i>de novo</i> biosynthesis of amino acids, particularly arginine, methionine, and isoleucine. Urine represents an amino acid-starved (AAS) environment for <i>K. pneumoniae</i>, modulating c-di-GMP signaling to promote biofilm formation. Eight diguanylate cyclase (DGC, c-di-GMP synthesis) genes, four phosphodiesterase (PDE, c-di-GMP degradation) genes, and four DGC + PDE genes were significantly regulated in response to urine. Furthermore, transcriptomic analysis comparing <i>K. pneumoniae</i> grown in urine with that grown in M9 medium revealed significant activation of genes associated with exopolysaccharide (EPS) biosynthesis, including those encoding lipopolysaccharides (LPS), capsules, peptidoglycan, and enterobacterial common antigen (ECA). Notably, <i>K. pneumoniae</i> increases EPS biosynthesis under the iron-limited conditions in urine, further promoting biofilm development. In conclusion, AAS-mediated c-di-GMP signaling and iron limitation are key drivers of biofilm formation by <i>K. pneumoniae</i> in urine, providing mechanistic insights that may guide strategies to disrupt biofilm formation.