Rickettsial ompB promoter regulated expression of GFPuv in transformed Rickettsia montanensis

Gerald D. Baldridge, Nicole Y. Burkhardt, Adela S. Oliva, Timothy J Kurtti, Ulrike G Munderloh

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Background: Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, α-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. Methodology/Principal Findings: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/Significance: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.

Original languageEnglish (US)
Article numbere8965
JournalPLoS One
Volume5
Issue number1
DOIs
StatePublished - Jan 29 2010

Fingerprint

Rickettsia montanensis
Rickettsia
transposons
Chloramphenicol O-Acetyltransferase
Genes
promoter regions
chloramphenicol acetyltransferase
Acetyl-CoA C-Acetyltransferase
arthropods
acetyl-CoA acetyltransferase
typhus
Rickettsiaceae
Arthropods
Rickettsia rickettsii
Chloramphenicol
Rickettsiales
Chromosomes
Green Fluorescent Proteins
Escherichia coli
electroporation

Cite this

Rickettsial ompB promoter regulated expression of GFPuv in transformed Rickettsia montanensis. / Baldridge, Gerald D.; Burkhardt, Nicole Y.; Oliva, Adela S.; Kurtti, Timothy J; Munderloh, Ulrike G.

In: PLoS One, Vol. 5, No. 1, e8965, 29.01.2010.

Research output: Contribution to journalArticle

@article{d831bb4f4f6542d4847e7e1fc8907d25,
title = "Rickettsial ompB promoter regulated expression of GFPuv in transformed Rickettsia montanensis",
abstract = "Background: Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, α-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. Methodology/Principal Findings: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/Significance: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.",
author = "Baldridge, {Gerald D.} and Burkhardt, {Nicole Y.} and Oliva, {Adela S.} and Kurtti, {Timothy J} and Munderloh, {Ulrike G}",
year = "2010",
month = "1",
day = "29",
doi = "10.1371/journal.pone.0008965",
language = "English (US)",
volume = "5",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "1",

}

TY - JOUR

T1 - Rickettsial ompB promoter regulated expression of GFPuv in transformed Rickettsia montanensis

AU - Baldridge, Gerald D.

AU - Burkhardt, Nicole Y.

AU - Oliva, Adela S.

AU - Kurtti, Timothy J

AU - Munderloh, Ulrike G

PY - 2010/1/29

Y1 - 2010/1/29

N2 - Background: Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, α-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. Methodology/Principal Findings: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/Significance: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.

AB - Background: Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, α-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. Methodology/Principal Findings: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/Significance: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.

UR - http://www.scopus.com/inward/record.url?scp=77952475789&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77952475789&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0008965

DO - 10.1371/journal.pone.0008965

M3 - Article

VL - 5

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 1

M1 - e8965

ER -