Rickettsial ompB promoter regulated expression of GFPuv in transformed Rickettsia montanensis.

PloS One
Gerald D BaldridgeUlrike G Munderloh

Abstract

Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, alpha-proteobacteria that have historically been associated with blood-feeding arthropods. Certain species cause typhus and spotted fevers in humans, but others are of uncertain pathogenicity or may be strict arthropod endosymbionts. Genetic manipulation of rickettsiae should facilitate a better understanding of their interactions with hosts. We transformed a species never associated with human disease, Rickettsia montanensis, by electroporation with a TN5 transposon (pMOD700) containing green fluorescent protein (GFPuv) and chloramphenicol acetyltransferase (CAT) genes under regulation of promoters cloned from the Rickettsia rickettsii ompB gene, and isolated a Chloramphenicol-resistant GFP-fluorescent rickettsiae population (Rmontanensis700). The Rmontanensis700 rickettsiae contained a single transposon integrated near an acetyl-CoA acetyltransferase gene in the rickettsial chromosome. Northern blots showed that GFPuv and CAT mRNAs were both expressed as two transcripts of larger and smaller than predicted length. Western immunoblots showed that Rmontanensis700 and E. coli transformed with a plasmid containing the pMOD700 transposon bot...Continue Reading

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Citations

Jan 2, 2016·Journal of Medical Entomology·Jonathan D OliverUlrike G Munderloh
Mar 19, 2013·Zoonoses and Public Health·H Wood, H Artsob
Nov 4, 2015·Current Protocols in Microbiology·Sean P RileyJuan J Martinez
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Sep 14, 2021·Pathogens and Disease·Jason R Hunt, Jason A Carlyon

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Methods Mentioned

BETA
gene knockout
Protein Assay
electrophoresis

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