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

doi:10.1128/aem.00210-22

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

Entomology

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

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 language	English (US)
Journal	Applied and environmental microbiology
Volume	88
Issue number	7
DOIs	https://doi.org/10.1128/aem.00210-22
State	Published - Apr 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

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

Publisher link

10.1128/aem.00210-22

Find It

Find It at U of M Libraries

Cite this

@article{b91a59082ce04df693ef53c17036aaac,

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

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.",

keywords = "Rickettsia, Rickettsia amblyommatis, Rickettsia monacensis, Rickettsia plasmids, host range, plasmids, shuttle plasmids, shuttle vector, transformation",

author = "Nicole Burkhardt and Price, {Lisa D.} and Xinru Wang and Heu, {Chan C.} and Baldridge, {Gerald D} and Munderloh, {Ulrike G.} and Kurtti, {Timothy J.}",

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: {\textcopyright} 2022 Burkhardt et al.",

year = "2022",

month = apr,

doi = "10.1128/aem.00210-22",

language = "English (US)",

volume = "88",

journal = "Applied and environmental microbiology",

issn = "0099-2240",

publisher = "American Society for Microbiology",

number = "7",

}

TY - JOUR

T1 - Examination of Rickettsial Host Range for Shuttle Vectors Based on dnaA and parA Genes from the pRM Plasmid of Rickettsia monacensis

AU - Burkhardt, Nicole

AU - Price, Lisa D.

AU - Wang, Xinru

AU - Heu, Chan C.

AU - Baldridge, Gerald D

AU - Munderloh, Ulrike G.

AU - Kurtti, Timothy J.

N1 - 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.

PY - 2022/4

Y1 - 2022/4

N2 - 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.

AB - 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.

KW - Rickettsia

KW - Rickettsia amblyommatis

KW - Rickettsia monacensis

KW - Rickettsia plasmids

KW - host range

KW - plasmids

KW - shuttle plasmids

KW - shuttle vector

KW - transformation

UR - http://www.scopus.com/inward/record.url?scp=85128487333&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85128487333&partnerID=8YFLogxK

U2 - 10.1128/aem.00210-22

DO - 10.1128/aem.00210-22

M3 - Article

C2 - 35323021

AN - SCOPUS:85128487333

SN - 0099-2240

VL - 88

JO - Applied and environmental microbiology

JF - Applied and environmental microbiology

IS - 7

ER -

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

Abstract

Bibliographical note

Keywords

Publisher link

Find It

Other files and links

Fingerprint

Cite this