Klebsiella pneumoniae MGH78578 contains ~500 uncharacterized signaling proteins and in this study, we characterized the biological functions of a novel eukaryotic-like serine/threonine kinase; ESTK (KpnK). Studies demonstrated that KpnK undergoes autophosphorylation within the pH range 7.0-7.5 at 37°C in a time- and concentration- dependent manner, with Mn(2+) as its cofactor. The ΔkpnK mutant exhibited higher sensitivity to gastrointestinal and oxidative stresses. Deletion of kpnK resulted in a two to threefold increased susceptibility towards imipenem, cefepime, ceftriaxone and ceftazidime. Our study has provided overall evidence for the involvement of ESTK in regulating bacterial physiology, stress response and drug resistance. This report has unmasked the occurrence of Ser/Thr kinase mediated signaling for the first time in K. pneumoniae. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.