The poly (A) polymerase pcnB modulates virulence and resistance in Klebsiella pneumoniae by differentially regulating chromosomal mRNA stability and plasmid copy number

<i>Klebsiella pneumoniae</i> is a leading cause of healthcare-associated infections, with emerging strains exhibiting both multidrug resistance and hypervirulence, largely mediated by plasmid-encoded genes. Poly (A) polymerase I, encoded by <i>pcnB</i>, plays a key role in RNA degradation and plasmid copy number control, yet its global regulatory impact in <i>K. pneumoniae</i> remains unclear. Here, we constructed a <i>pcnB</i> deletion mutant in <i>K. pneumoniae</i> ATCC 13883 using CRISPR-Cas9 and examined its effects on chromosomal virulence factors and plasmid-borne resistance. Deletion of <i>pcnB</i> impaired bacterial growth and metabolic activity, reduced biofilm formation, but unexpectedly enhanced siderophore and exopolysaccharide production via upregulation of chromosomal virulence genes. In contrast, <i>pcnB</i> deletion drastically reduced the copy number and stability of a ColE1-type plasmid carrying a spectinomycin resistance gene (<i>aadA</i>), leading to decreased <i>aadA</i> expression and a twofold reduction in antibiotic resistance. These findings reveal the dual role of <i>pcnB</i> as a repressor of chromosomal virulence genes and an activator of plasmid maintenance, highlighting its potential as a novel target for anti-virulence and anti-resistance strategies.