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


RESULTS
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 both expressed GFPuv proteins of the predicted molecular weight.


CONCLUSIONS
Long-standing barriers to transformation of rickettsiae have been overcome by development of transposon-based rickettsial transformation vectors. The ompB promoter may be the most problematic of the four promoters so far employed in those vectors.
Baldridge
Gerald D
GD
Department of Entomology, University of Minnesota, St Paul, Minnesota, United States of America. baldr001@umn.edu
Burkhardt
Nicole Y
NY
Oliva
Adela S
AS
Kurtti
Timothy J
TJ
Munderloh
Ulrike G
UG
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017931:DNA Primers; D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012281:Rickettsia / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D004251:DNA Transposable Elements; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D015500:Chloramphenicol O-Acetyltransferase