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December 4, 2006Journal of BacteriologyOpen Access

Kinase Activity of Overexpressed HipA Is Required for Growth Arrest and Multidrug Tolerance in Escherichia coli

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Authors

FCF F CorreiaNortheastern UniversityADAnthony D’OnofrioMerck & Co., Inc., Rahway, NJ, USA (United States)TRTomáš RejtarNovartis (China)

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Implication

In vitro study demonstrates that HipA kinase activity mediates growth arrest and persister formation in Escherichia coli, indicating that phosphorylation drives bacterial multidrug tolerance.

Key Points

  • Determine whether the predicted kinase activity of the toxin HipA is necessary for inducing cellular growth arrest and multidrug tolerance in Escherichia coli.
  • Assayed in vitro autophosphorylation of purified HipA with ATP and mapped the phosphorylation site to serine 150.
  • Generated site-directed mutations in the kinase active site (D309Q), Mg2+-binding site (D332Q), and phosphorylation site (S150A) to test growth inhibition upon overexpression.
  • Assessed bacterial survival and persister formation following challenge with cefotaxime, ofloxacin, and mitomycin C.
  • HipA functioned as a serine kinase autophosphorylated at Ser150 in the presence of ATP.
  • Mutations targeting conserved catalytic residues (D309Q, D332Q) or the autophosphorylation site (S150A) completely abolished HipA-mediated growth arrest.
  • Wild-type HipA overexpression conferred high tolerance against cefotaxime, ofloxacin, and mitomycin C, whereas kinase-deficient mutants failed to provide antibiotic protection.

Cite This Study

Correia et al. (2006) studied this question.

synapsesocial.com/papers/6a6f2e3dce524a4339c334afhttps://doi.org/10.1128/jb.01237-06
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