ArticleApplied and environmental microbiology2005
Analysis of fluorescent protein expression in transformants of Rickettsia monacensis, an obligate intracellular tick symbiont.
Article in Applied and environmental microbiology, 2005. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 42 papers.
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Who cites it
42 citing papers in PubMed, 60 citations in OpenAlex.
- Recent advances in genetic systems in obligate intracellular human-pathogenic bacteria.Frontiers in cellular and infection microbiology · 2023Review
- Examination of Rickettsial Host Range for Shuttle Vectors Based onApplied and environmental microbiology · 2022Article
- Analysis of Orientia tsutsugamushi promoter activity.Pathogens and disease · 2021Article
- Cells within cells: Rickettsiales and the obligate intracellular bacterial lifestyle.Nature reviews. Microbiology · 2021Review
- Growing Ungrowable Bacteria: Overview and Perspectives on Insect Symbiont Culturability.Microbiology and molecular biology reviews : MMBR · 2020Review
- The neglected challenge: Vaccination against rickettsiae.PLoS neglected tropical diseases · 2020Review
- Optimized fixation of actin filaments for improved indirect immunofluorescence staining of rickettsiae.BMC research notes · 2019Article
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- Stable Expression of Modified Green Fluorescent Protein in Group B Streptococci To Enable Visualization in Experimental Systems.Applied and environmental microbiology · 2018Article
- GFPuv-Expressing Recombinant Rickettsia typhi: a Useful Tool for the Study of Pathogenesis and CD8Infection and immunity · 2017Article
- Actin-based motility of bacterial pathogens: mechanistic diversity and its impact on virulence.Pathogens and disease · 2016Article
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- A Coming of Age Story: Chlamydia in the Post-Genetic Era.Infection and immunity · 2015Review
- Electrotransformation and Clonal Isolation of Rickettsia Species.Current protocols in microbiology · 2015Article
- Disrupting protein expression with Peptide Nucleic Acids reduces infection by obligate intracellular Rickettsia.PloS one · 2015Article
- An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum.PLoS pathogens · 2015Article
- Recent molecular insights into rickettsial pathogenesis and immunity.Future microbiology · 2013Review
- Transcriptional pathways associated with the slow growth phenotype of transformed Anaplasma marginale.BMC genomics · 2013Article
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Corrections and comments
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
Abstract
We developed and applied transposon-based transformation vectors for molecular manipulation and analysis of spotted fever group rickettsiae, which are obligate intracellular bacteria that infect ticks and, in some cases, mammals. Using the Epicentre EZ::TN transposon system, we designed transposons for simultaneous expression of a reporter gene and a chloramphenicol acetyltransferase (CAT) resistance marker. Transposomes (transposon-transposase complexes) were electroporated into Rickettsia monacensis, a rickettsial symbiont isolated from the tick Ixodes ricinus. Each transposon contained an expression cassette consisting of the rickettsial ompA promoter and a green fluorescent protein (GFP) reporter gene (GFPuv) or the ompB promoter and a red fluorescent protein reporter gene (DsRed2), followed by the ompA transcription terminator and a second ompA promoter CAT gene cassette. Selection with chloramphenicol gave rise to rickettsial populations with chromosomally integrated single-copy transposons as determined by PCR, Southern blotting, and sequence analysis. Reverse transcription-PCR and Northern blots demonstrated transcription of all three genes. GFPuv transformant rickettsiae exhibited strong fluorescence in individual cells, but DsRed2 transformants did not. Western blots confirmed expression of GFPuv in R. monacensis and in Escherichia coli, but DsRed2 was expressed only in E. coli. The DsRed2 gene, but not the GFPuv gene, contains many GC-rich amino acid codons that are rare in the preferred codon suite of rickettsiae, possibly explaining the failure to express DsRed2 protein in R. monacensis. We demonstrated that our vectors provide a means to study rickettsia-host cell interactions by visualizing GFPuv-fluorescent R. monacensis associated with actin tails in tick host cells.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.