Abstract
ABSTRACTThe genus Rickettsia encompasses a diverse group of obligate intracellular bacteria that are highly virulent disease agents of mankind as well as symbionts of arthropods. Native plasmids of Rickettsia amblyommatis (AaR/SC) have been used as models to construct shuttle vectors for genetic manipulation of several Rickettsia species. Here we report on the isolation of the complete plasmid (pRM658B) from Rickettsia monacensis (IrR/Munich) mutant Rmona658B and the construction of shuttle vectors based on pRM. To identify regions essential for replication, we made vectors containing the dnaA and parA genes of pRM with varying portions of the region surrounding these genes and a selection-reporter cassette conferring resistance to spectinomycin and expression of green fluorescent protein. Rickettsia amblyommatis (AaR/SC), R. monacensis (IrR/Munich), Rickettsia bellii (RML 369-C), Rickettsia parkeri (Tate’s Hell), and Rickettsia montanensis (M5/6) were successfully transformed with shuttle vectors containing pRM parA and dnaA. PCR assays targeting pRM regions not included in the vectors revealed that native pRM was retained in R. monacensis transformants. Determination of native pRM copy number using a plasmid-encoded gene (RM_p5) in comparison to chromosomal encoded gltA indicated reduced copy numbers in R. monacensis transformants. In transformed R. monacensis, native pRM and shuttle vectors with homologous parA and dnaA formed native plasmid-shuttle vector complexes. These studies provide insight on the maintenance of plasmids and shuttle vectors in rickettsiae.IMPORTANCEThis paper describes a new series of plasmid shuttle vectors for the transformation of rickettsiae.Shuttle vectors based on parA and dnaA sequences of the plasmid pRM can be used to transform diverse rickettsia as well as its native host Rickettsia monacensis.Our results provide insight into the maintenance of rickettsial-based shuttle vectors in rickettsiae.
Publisher
Cold Spring Harbor Laboratory