Bacterial strains and plasmids.

<div><p><i>Klebsiella pneumoniae</i> belongs to the group of bacterial pathogens causing the majority of antibiotic-resistant nosocomial infections worldwide; however, the molecular mechanisms underlying post-translational regulation of its physiology are poorly understood. Here we perform a comprehensive analysis of <i>Klebsiella</i> phosphoproteome, focusing on HipA, a Ser/Thr kinase involved in antibiotic tolerance in <i>Escherichia coli</i>. We show that overproduced <i>K</i>. <i>pneumoniae</i> HipA (HipA<sub>kp</sub>) is toxic to both <i>E</i>. <i>coli</i> and <i>K</i>. <i>pneumoniae</i> and its toxicity can be rescued by overproduction of the antitoxin HipB<sub>kp</sub>. Importantly, HipA<sub>kp</sub> overproduction leads to increased tolerance against ciprofloxacin, a commonly used antibiotic in the treatment of <i>K</i>. <i>pneumoniae</i> infections. Proteome and phosphoproteome analyses in the absence and presence of ciprofloxacin confirm that HipA<sub>kp</sub> has Ser/Thr kinase activity, auto-phosphorylates at S150, and shares multiple substrates with HipA<sub>ec</sub>, thereby providing a valuable resource to clarify the molecular basis of tolerance and the role of Ser/Thr phosphorylation in this human pathogen.</p></div>