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Rickettsial ompB promoter regulated expression of GFPuv in transformed Rickettsia montanensis.

Baldridge GD, Burkhardt NY, Oliva AS, Kurtti TJ, Munderloh UG - PLoS ONE (2010)

Bottom Line: Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, alpha-proteobacteria that have historically been associated with blood-feeding arthropods.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, University of Minnesota, St Paul, Minnesota, United States of America. baldr001@umn.edu

ABSTRACT

Background: Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, alpha-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.

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Detection of GFPuv and CAT mRNAs in Rmontanensis700 RNA extracts by Northern blotting.(A) Rickettsial RNA extracts electrophoresed on 1% agarose formaldehyde gels stained with EtBr. Lanes 1 and 4: 0.5 to 4.0 kb RNA marker ladder with sizes indicated at left. Lanes 2 and 5: untransformed R. montanensis RNA (5 µg). Lanes 3 and 6: Rmontanensis700 RNA (5 µg). (B) Northern blots of gels shown in panel A. Lanes 1–3 were hybridized with a GFPuv gene probe and lanes 4–6 with a CAT gene probe.
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pone-0008965-g005: Detection of GFPuv and CAT mRNAs in Rmontanensis700 RNA extracts by Northern blotting.(A) Rickettsial RNA extracts electrophoresed on 1% agarose formaldehyde gels stained with EtBr. Lanes 1 and 4: 0.5 to 4.0 kb RNA marker ladder with sizes indicated at left. Lanes 2 and 5: untransformed R. montanensis RNA (5 µg). Lanes 3 and 6: Rmontanensis700 RNA (5 µg). (B) Northern blots of gels shown in panel A. Lanes 1–3 were hybridized with a GFPuv gene probe and lanes 4–6 with a CAT gene probe.

Mentions: We used Northern blots to further characterize the expression profiles of the GFPuv and CAT genes in Rmontanensis700. Rickettsial RNA extracts were electrophoresed in 1% agarose formaldehyde gels and stained with EtBr. Intact ribosomal RNA migrated as bands at approximately 1.6 and 3.8 kb relative to RNA markers (Fig. 5A, lane 1) in RNA extracts of untransformed R. montanensis (lanes 2 and 5) and Rmontanensis700 (lanes 3 and 6). The RNAs were transferred from the gel onto nylon membranes for hybridization with GFPuv and CAT gene probes. We predicted a minimum length 920 nucleotide GFPuv mRNA transcript based on the predicted ompB promoter transcription start site and untranslated mRNA sequence [45] as well as transcription of 70 nucleotides of the ompA terminator to form mRNA hairpin structures and stop transcription [46]. Similarly, we predicted an 860 nucleotide CAT mRNA transcript. The Northern blots showed that the GFPuv and CAT probes did not hybridize to untransformed R. montanensis RNA (lanes 2 and 5, respectively). The GFPuv probe hybridized to unexpected 1,200 and 600 nucleotide transcripts in Rmontanensis700 RNA extracts (lane 3). Similarly, the CAT probe hybridized to unexpected 1,400 and less abundant 700 nucleotide transcripts in Rmontanensis700 RNA extracts (lane 6). The results showed that the GFPuv and CAT genes in the Rmontanensis700 transformant were both expressed under regulation of the ompB promoter as two transcripts that were longer and shorter than the predicted lengths.


Rickettsial ompB promoter regulated expression of GFPuv in transformed Rickettsia montanensis.

Baldridge GD, Burkhardt NY, Oliva AS, Kurtti TJ, Munderloh UG - PLoS ONE (2010)

Detection of GFPuv and CAT mRNAs in Rmontanensis700 RNA extracts by Northern blotting.(A) Rickettsial RNA extracts electrophoresed on 1% agarose formaldehyde gels stained with EtBr. Lanes 1 and 4: 0.5 to 4.0 kb RNA marker ladder with sizes indicated at left. Lanes 2 and 5: untransformed R. montanensis RNA (5 µg). Lanes 3 and 6: Rmontanensis700 RNA (5 µg). (B) Northern blots of gels shown in panel A. Lanes 1–3 were hybridized with a GFPuv gene probe and lanes 4–6 with a CAT gene probe.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2813287&req=5

pone-0008965-g005: Detection of GFPuv and CAT mRNAs in Rmontanensis700 RNA extracts by Northern blotting.(A) Rickettsial RNA extracts electrophoresed on 1% agarose formaldehyde gels stained with EtBr. Lanes 1 and 4: 0.5 to 4.0 kb RNA marker ladder with sizes indicated at left. Lanes 2 and 5: untransformed R. montanensis RNA (5 µg). Lanes 3 and 6: Rmontanensis700 RNA (5 µg). (B) Northern blots of gels shown in panel A. Lanes 1–3 were hybridized with a GFPuv gene probe and lanes 4–6 with a CAT gene probe.
Mentions: We used Northern blots to further characterize the expression profiles of the GFPuv and CAT genes in Rmontanensis700. Rickettsial RNA extracts were electrophoresed in 1% agarose formaldehyde gels and stained with EtBr. Intact ribosomal RNA migrated as bands at approximately 1.6 and 3.8 kb relative to RNA markers (Fig. 5A, lane 1) in RNA extracts of untransformed R. montanensis (lanes 2 and 5) and Rmontanensis700 (lanes 3 and 6). The RNAs were transferred from the gel onto nylon membranes for hybridization with GFPuv and CAT gene probes. We predicted a minimum length 920 nucleotide GFPuv mRNA transcript based on the predicted ompB promoter transcription start site and untranslated mRNA sequence [45] as well as transcription of 70 nucleotides of the ompA terminator to form mRNA hairpin structures and stop transcription [46]. Similarly, we predicted an 860 nucleotide CAT mRNA transcript. The Northern blots showed that the GFPuv and CAT probes did not hybridize to untransformed R. montanensis RNA (lanes 2 and 5, respectively). The GFPuv probe hybridized to unexpected 1,200 and 600 nucleotide transcripts in Rmontanensis700 RNA extracts (lane 3). Similarly, the CAT probe hybridized to unexpected 1,400 and less abundant 700 nucleotide transcripts in Rmontanensis700 RNA extracts (lane 6). The results showed that the GFPuv and CAT genes in the Rmontanensis700 transformant were both expressed under regulation of the ompB promoter as two transcripts that were longer and shorter than the predicted lengths.

Bottom Line: Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, alpha-proteobacteria that have historically been associated with blood-feeding arthropods.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, University of Minnesota, St Paul, Minnesota, United States of America. baldr001@umn.edu

ABSTRACT

Background: Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, alpha-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.

Show MeSH
Related in: MedlinePlus