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Journal of Bacteriology, September 2004, p. 6265-6276, Vol. 186, No. 18
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.18.6265-6276.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Novel Mutation within the Central Listeria monocytogenes Regulator PrfA That Results in Constitutive Expression of Virulence Gene Products

Kendy K. Y. Wong and Nancy E. Freitag*

Seattle Biomedical Research Institute, and Departments of Pathobiology and Microbiology, University of Washington, Seattle, Washington

Received 30 January 2004/ Accepted 9 June 2004

The PrfA protein of Listeria monocytogenes functions as a key regulatory factor for the coordinated expression of many virulence genes during bacterial infection of host cells. PrfA activity is controlled by multiple regulatory mechanisms, including an apparent requirement for either the presence of a cofactor or some form of posttranslational modification that regulates the activation of PrfA. In this study, we describe the identification and characterization of a novel PrfA mutation that results in constitutive activation of the PrfA protein. The PrfA L140F mutation was found to confer high-level expression of PrfA-regulated genes and to be functionally dominant over the wild-type allele. The presence of the PrfA L140F mutation resulted in the aggregation of L. monocytogenes in broth culture and, unlike previously described prfA mutations, appeared to be slightly toxic to the bacteria. High-level PrfA-dependent gene expression showed no additional increase in L. monocytogenes strains containing an additional copy of prfA L140F despite a >4-fold increase in PrfA protein levels. In contrast, the introduction of multiple copies of the wild-type prfA allele to L. monocytogenes resulted in a corresponding increase in PrfA-dependent gene expression, although overall expression levels remained far below those observed for PrfA L140F strains. These results suggest a hierarchy of PrfA regulation, such that the relative levels of PrfA protein present within the cell correlate with the levels of PrfA-dependent gene expression when the protein is not in its fully activated state; however, saturating levels of the protein are then quickly reached when PrfA is converted to its active form. Regulation of the PrfA activation status must be an important facet of L. monocytogenes survival, as mutations that result in constitutive PrfA activation may have deleterious consequences for bacterial physiology.

* Corresponding author. Mailing address: Seattle Biomedical Research Institute, 307 Westlake Ave. N., Suite 500, Seattle, WA 98109-5219. Phone: (206) 256-7345. Fax: (206) 256-7229. E-mail: nancy.freitag{at}sbri.org.

Journal of Bacteriology, September 2004, p. 6265-6276, Vol. 186, No. 18
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.18.6265-6276.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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