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Journal of Bacteriology, December 2006, p. 8360-8367, Vol. 188, No. 24
0021-9193/06/$08.00+0     doi:10.1128/JB.01237-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Frederick F. Correia,1 Anthony D'Onofrio,1 Tomas Rejtar,2 Lingyun Li,2 Barry L. Karger,2 Kira Makarova,3 Eugene V. Koonin,3 and Kim Lewis1*

Department of Biology and Antimicrobial Discovery Center, Northeastern University, Boston, Massachusetts 02115,1 Barnett Institute and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115,2 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 208943

Received 7 August 2006/ Accepted 5 October 2006

Overexpression of the HipA protein of the HipBA toxin/antitoxin module leads to multidrug tolerance in Escherichia coli. HipA is a "toxin" that causes reversible dormancy, whereas HipB is an antitoxin that binds HipA and acts as a transcriptional repressor of the hipBA operon. Comparative sequence analysis shows that HipA is a member of the phosphatidylinositol 3/4-kinase superfamily. The kinase activity of HipA was examined. HipA was autophosphorylated in the presence of ATP in vitro, and the purified protein appeared to carry a single phosphate group on serine 150. Thus, HipA is a serine kinase that is at least partially phosphorylated in vivo. Overexpression of HipA caused inhibition of cell growth and increase in persister formation. Replacing conserved aspartate 309 in the conserved kinase active site or aspartate 332 in the Mg2+-binding site with glutamine produced mutant proteins that lost the ability to stop cellular growth upon overexpression. Replacing serine 150 with alanine yielded a similarly inactive protein. The mutant proteins were then examined for their ability to increase antibiotic tolerance. Cells overexpressing wild-type HipA were highly tolerant to cefotaxime, a cell wall synthesis inhibitor, to ofloxacin, a fluoroquinolone inhibitor of DNA gyrase, and to topoisomerase IV and were almost completely resistant to killing by mitomycin C, which forms DNA adducts. The mutant proteins did not protect cells from cefotaxime or ofloxacin and had an impaired ability to protect from mitomycin C. Taken together, these results suggest that the protein kinase activity of HipA is essential for persister formation.

* Corresponding author. Mailing address: Northeastern University, Department of Biology, 405 Mugar Hall, 360 Huntington Avenue, Boston, MA 02115. Phone: (617) 373-8238. Fax: (617) 373-3724. E-mail: k.lewis{at}neu.edu.

{triangledown} Published ahead of print on 13 October 2006.

Journal of Bacteriology, December 2006, p. 8360-8367, Vol. 188, No. 24
0021-9193/06/$08.00+0     doi:10.1128/JB.01237-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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