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PLoS One. 2012;7(5):e37310. doi: 10.1371/journal.pone.0037310. Epub 2012 May 18.

Expression of an epitope-tagged virulence protein in Rickettsia parkeri using transposon insertion.

Author information

1
Department of Molecular & Cell Biology, University of California, Berkeley, California, United States of America. welch@berkeley.edu

Abstract

Despite recent advances in our ability to genetically manipulate Rickettsia, little has been done to employ genetic tools to study the expression and localization of Rickettsia virulence proteins. Using a mariner-based Himar1 transposition system, we expressed an epitope-tagged variant of the actin polymerizing protein RickA under the control of its native promoter in Rickettsia parkeri, allowing the detection of RickA using commercially-available antibodies. Native RickA and epitope-tagged RickA exhibited similar levels of expression and were specifically localized to bacteria. To further facilitate protein expression in Rickettsia, we also developed a plasmid for Rickettsia insertion and expression (pRIE), containing a variant Himar1 transposon with enhanced flexibility for gene insertion, and used it to generate R. parkeri strains expressing diverse fluorescent proteins. Expression of epitope-tagged proteins in Rickettsia will expand our ability to assess the regulation and function of important virulence factors.

PMID:
22624012
PMCID:
PMC3356282
DOI:
10.1371/journal.pone.0037310
[Indexed for MEDLINE]
Free PMC Article

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