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Isolation and characterization of Ehrlichia chaffeensis RNA polymerase and its use in evaluating p28 outer membrane protein gene promoters.

Faburay B, Liu H, Peddireddi L, Ganta RR - BMC Microbiol. (2011)

Bottom Line: In recent studies, we demonstrated significant host-specific differences in protein expression in E. chaffeensis originating from its tick and vertebrate host cells.Our experiments demonstrated that both the native and recombinant proteins are functional and have similar enzyme properties in driving the transcription from E. chaffeensis promoters.This study marks the beginning to broadly characterize the mechanisms controlling the transcription by Anaplasmataceae pathogens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA.

ABSTRACT

Background: Ehrlichia chaffeensis is a tick-transmitted rickettsial pathogen responsible for an important emerging disease, human monocytic ehrlichiosis. To date how E. chaffeensis and many related tick-borne rickettsial pathogens adapt and persist in vertebrate and tick hosts remain largely unknown. In recent studies, we demonstrated significant host-specific differences in protein expression in E. chaffeensis originating from its tick and vertebrate host cells. The adaptive response of the pathogen to different host environments entails switch of gene expression regulated at the level of transcription, possibly by altering RNA polymerase activity.

Results: In an effort to understand the molecular basis of pathogen gene expression differences, we isolated native E. chaffeensis RNA polymerase using a heparin-agarose purification method and developed an in vitro transcription system to map promoter regions of two differentially expressed genes of the p28 outer membrane protein locus, p28-Omp14 and p28-Omp19. We also prepared a recombinant protein of E. chaffeensis σ70 homologue and used it for in vitro promoter analysis studies. The possible role of one or more proteins presents in E. chaffeensis lysates in binding to the promoter segments and on the modulation of in vitro transcription was also assessed.

Conclusions: Our experiments demonstrated that both the native and recombinant proteins are functional and have similar enzyme properties in driving the transcription from E. chaffeensis promoters. This is the first report of the functional characterization of E. chaffeensis RNA polymerase and in vitro mapping of the pathogen promoters using the enzyme. This study marks the beginning to broadly characterize the mechanisms controlling the transcription by Anaplasmataceae pathogens.

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Transcription of pRG198 with varying potassium acetate concentrations showing transcription by E. chaffeensis RNAP saturated with the recombinant σ70 and by E. coli core RNAP reconstituted with recombinant σ70.
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Figure 7: Transcription of pRG198 with varying potassium acetate concentrations showing transcription by E. chaffeensis RNAP saturated with the recombinant σ70 and by E. coli core RNAP reconstituted with recombinant σ70.

Mentions: We reconstituted E. coli RNAP core enzyme with recombinantly expressed E. chaffeensis σ70 and the resulting holoenzyme effectively transcribed the promoters of p28-Omp14 and p29-Omp19 (data presented for p28-Omp 19 promoter in Figure 6), but the core enzyme alone or recombinant E. chaffeensis σ70 alone did not drive the transcription. Saturation of the purified enzyme with recombinant σ70 also resulted in enhanced transcriptional signals (Figure 6). General transcriptional profile of both the reconstituted enzymes in the presence of varying potassium acetate concentrations were similar (Figure 7), although a relatively stronger transcriptional signal at 400 mM salt concentration was detected for E. coli core enzyme saturated with E. chaffeensis recombinant σ70 subunit (Figure 7).


Isolation and characterization of Ehrlichia chaffeensis RNA polymerase and its use in evaluating p28 outer membrane protein gene promoters.

Faburay B, Liu H, Peddireddi L, Ganta RR - BMC Microbiol. (2011)

Transcription of pRG198 with varying potassium acetate concentrations showing transcription by E. chaffeensis RNAP saturated with the recombinant σ70 and by E. coli core RNAP reconstituted with recombinant σ70.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108270&req=5

Figure 7: Transcription of pRG198 with varying potassium acetate concentrations showing transcription by E. chaffeensis RNAP saturated with the recombinant σ70 and by E. coli core RNAP reconstituted with recombinant σ70.
Mentions: We reconstituted E. coli RNAP core enzyme with recombinantly expressed E. chaffeensis σ70 and the resulting holoenzyme effectively transcribed the promoters of p28-Omp14 and p29-Omp19 (data presented for p28-Omp 19 promoter in Figure 6), but the core enzyme alone or recombinant E. chaffeensis σ70 alone did not drive the transcription. Saturation of the purified enzyme with recombinant σ70 also resulted in enhanced transcriptional signals (Figure 6). General transcriptional profile of both the reconstituted enzymes in the presence of varying potassium acetate concentrations were similar (Figure 7), although a relatively stronger transcriptional signal at 400 mM salt concentration was detected for E. coli core enzyme saturated with E. chaffeensis recombinant σ70 subunit (Figure 7).

Bottom Line: In recent studies, we demonstrated significant host-specific differences in protein expression in E. chaffeensis originating from its tick and vertebrate host cells.Our experiments demonstrated that both the native and recombinant proteins are functional and have similar enzyme properties in driving the transcription from E. chaffeensis promoters.This study marks the beginning to broadly characterize the mechanisms controlling the transcription by Anaplasmataceae pathogens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA.

ABSTRACT

Background: Ehrlichia chaffeensis is a tick-transmitted rickettsial pathogen responsible for an important emerging disease, human monocytic ehrlichiosis. To date how E. chaffeensis and many related tick-borne rickettsial pathogens adapt and persist in vertebrate and tick hosts remain largely unknown. In recent studies, we demonstrated significant host-specific differences in protein expression in E. chaffeensis originating from its tick and vertebrate host cells. The adaptive response of the pathogen to different host environments entails switch of gene expression regulated at the level of transcription, possibly by altering RNA polymerase activity.

Results: In an effort to understand the molecular basis of pathogen gene expression differences, we isolated native E. chaffeensis RNA polymerase using a heparin-agarose purification method and developed an in vitro transcription system to map promoter regions of two differentially expressed genes of the p28 outer membrane protein locus, p28-Omp14 and p28-Omp19. We also prepared a recombinant protein of E. chaffeensis σ70 homologue and used it for in vitro promoter analysis studies. The possible role of one or more proteins presents in E. chaffeensis lysates in binding to the promoter segments and on the modulation of in vitro transcription was also assessed.

Conclusions: Our experiments demonstrated that both the native and recombinant proteins are functional and have similar enzyme properties in driving the transcription from E. chaffeensis promoters. This is the first report of the functional characterization of E. chaffeensis RNA polymerase and in vitro mapping of the pathogen promoters using the enzyme. This study marks the beginning to broadly characterize the mechanisms controlling the transcription by Anaplasmataceae pathogens.

Show MeSH
Related in: MedlinePlus