title: Expression of an epitope-tagged virulence protein in Rickettsia parkeri using transposon insertion.
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.
authors.affiliation: Department of Molecular & Cell Biology, University of California, Berkeley, California, United States of America. welch@berkeley.edu
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Welch
authors.forename: Shawna C O
authors.initials: SC
authors.lastname: Reed
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Lamason
authors.forename: Alisa W
authors.initials: AW
authors.lastname: Serio
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D005455:Fluorescent Antibody Technique; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology*; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D020161:Physical Chromosome Mapping; D010957:Plasmids / Q000235:genetics; D012281:Rickettsia / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D014709:Vero Cells; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C491314:RickA protein, Rickettsia; D037521:Virulence Factors