Examination of Rickettsial Host Range for Shuttle Vectors Based on dnaA and parA Genes from the pRM Plasmid of Rickettsia monacensis

Nicole Burkhardt, Lisa D. Price, Xinru Wang, Chan C. Heu, Gerald D Baldridge, Ulrike G. Munderloh, Timothy J. Kurtti

Research output: Contribution to journalArticlepeer-review

Abstract

The 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 various 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-carried gene (RM_p5) in comparison to chromosomally carried gltA indicated reduced copy numbers in R. monacensis transformants. In transformed R. monacensis strains, 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.

Original languageEnglish (US)
Pages (from-to)e0021022
JournalApplied and environmental microbiology
Volume88
Issue number7
DOIs
StatePublished - Apr 12 2022

Bibliographical note

Funding Information:
This research was supported by grants (R01 AI049424 and R01 A1081690) to U.G.M. from the U.S. National Institutes of Health. We thank Roderick Felsheim for technical advice and assistance.

Publisher Copyright:
© 2022 Burkhardt et al.

Keywords

  • host range
  • plasmids
  • Rickettsia
  • Rickettsia amblyommatis
  • Rickettsia monacensis
  • Rickettsia plasmids
  • shuttle plasmids
  • shuttle vector
  • transformation

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural

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