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Journal of Bacteriology, June 2009, p. 3950-3964, Vol. 191, No. 12
0021-9193/09/$08.00+0     doi:10.1128/JB.00016-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Development of a mariner-Based Transposon and Identification of Listeria monocytogenes Determinants, Including the Peptidyl-Prolyl Isomerase PrsA2, That Contribute to Its Hemolytic Phenotype{triangledown}

Jason Zemansky,1 Benjamin C. Kline,1 Joshua J. Woodward,1 Jess H. Leber,1,{dagger} Hélène Marquis,3 and Daniel A. Portnoy1,2*

Department of Molecular and Cellular Biology,1 School of Public Health, University of California, Berkeley, California 94720-3202,2 Department of Microbiology and Immunology, Cornell University, Ithaca, New York 148533

Received 6 January 2009/ Accepted 6 April 2009

Listeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the intracellular pathogen Listeria monocytogenes. In order to identify factors that control the production, activity, or secretion of this essential virulence factor, we constructed a Himar1 mariner transposon delivery system and screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately 200 hypohemolytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular growth defects. Eight mutants with a phenotype were identified, and they contained insertions in the following genes: lmo0964 (similar to yjbH), lmo1268 (clpX), lmo1401 (similar to ymdB), lmo1575 (similar to ytqI), lmo1695 (mprF), lmo1821 (similar to prpC), lmo2219 (prsA2), and lmo2460 (similar to cggR). Some of these genes are involved in previously unexplored areas of research with L. monocytogenes: the genes yjbH and clpX regulate the disulfide stress response in Bacillus subtilis, and the prpC phosphatase has been implicated in virulence in other gram-positive pathogens. Here we demonstrate that prsA2, an extracytoplasmic peptidyl-prolyl cis/trans isomerase, is critical for virulence and contributes to the folding of LLO and to the activity of another virulence factor, the broad-range phospholipase C (PC-PLC). Furthermore, although it has been shown that prsA2 expression is linked to PrfA, the master virulence transcription factor in L. monocytogenes pathogenesis, we demonstrate that prsA2 is not directly controlled by PrfA. Finally, we show that PrsA2 is involved in flagellum-based motility, indicating that this factor likely serves a broad physiological role.

* Corresponding author. Mailing address: Department of Molecular & Cell Biology, 510 Barker Hall no. 3202, University of California, Berkeley, Berkeley, CA 94720-3202. Phone: (510) 643-3926. Fax: (510) 643-6334. E-mail: portnoy{at}berkeley.edu

{triangledown} Published ahead of print on 17 April 2009.

{dagger} Present address: Department of Microbiology, University of Chicago, Chicago, IL 60637.

Journal of Bacteriology, June 2009, p. 3950-3964, Vol. 191, No. 12
0021-9193/09/$08.00+0     doi:10.1128/JB.00016-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Roy, H., Ibba, M. (2009). Broad Range Amino Acid Specificity of RNA-dependent Lipid Remodeling by Multiple Peptide Resistance Factors. J. Biol. Chem. 284: 29677-29683 [Abstract] [Full Text]  
  • Alonzo, F. III, Port, G. C., Cao, M., Freitag, N. E. (2009). The Posttranslocation Chaperone PrsA2 Contributes to Multiple Facets of Listeria monocytogenes Pathogenesis. Infect. Immun. 77: 2612-2623 [Abstract] [Full Text]  


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