This Article Full Text Full Text (PDF) Other Versions of this Article: JB.01237-06v1 188/24/8360    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Correia, F. F. Articles by Lewis, K. Search for Related Content PubMed PubMed Citation Articles by Correia, F. F. Articles by Lewis, K.

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

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.

Published ahead of print on 13 October 2006.

This article has been cited by other articles:

• Ishikawa, H. O., Takeuchi, H., Haltiwanger, R. S., Irvine, K. D. (2008). Four-jointed Is a Golgi Kinase That Phosphorylates a Subset of Cadherin Domains. Science 321: 401-404 [Abstract] [Full Text]  
• Rice, K. C., Bayles, K. W. (2008). Molecular Control of Bacterial Death and Lysis. Microbiol. Mol. Biol. Rev. 72: 85-109 [Abstract] [Full Text]  
• Golby, P., Hatch, K. A., Bacon, J., Cooney, R., Riley, P., Allnutt, J., Hinds, J., Nunez, J., Marsh, P. D., Hewinson, R. G., Gordon, S. V. (2007). Comparative transcriptomics reveals key gene expression differences between the human and bovine pathogens of the Mycobacterium tuberculosis complex. Microbiology 153: 3323-3336 [Abstract] [Full Text]  
• Khoo, S. K., Loll, B., Chan, W. T., Shoeman, R. L., Ngoo, L., Yeo, C. C., Meinhart, A. (2007). Molecular and Structural Characterization of the PezAT Chromosomal Toxin-Antitoxin System of the Human Pathogen Streptococcus pneumoniae. J. Biol. Chem. 282: 19606-19618 [Abstract] [Full Text]  
• Li, Y., Zhang, Y. (2007). PhoU Is a Persistence Switch Involved in Persister Formation and Tolerance to Multiple Antibiotics and Stresses in Escherichia coli. Antimicrob. Agents Chemother. 51: 2092-2099 [Abstract] [Full Text]