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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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In vitro transcription showing the effect of varying salt concentrations of potassium acetate (Panel A), and sodium chloride (panel B). Transcription of the p28-Omp19 promoter region in pRG198 plasmid was assessed using the purified E. chaffeensis RNAP.
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Figure 5: In vitro transcription showing the effect of varying salt concentrations of potassium acetate (Panel A), and sodium chloride (panel B). Transcription of the p28-Omp19 promoter region in pRG198 plasmid was assessed using the purified E. chaffeensis RNAP.

Mentions: In support of testing the functionality of p28-Omp14 and p28-Omp19 gene promoters, we constructed in vitro transcription templates, pRG147 and pRG198, by cloning the promoter regions of the genes into the pMT504 plasmid (Figure 3). The plasmid pMT504 is a G-less cassette containing two transcription templates cloned in opposite directions to aid in driving transcription from promoters introduced upstream of the G-less cassette sequences [26]. (The promoter segments were amplified from E. chaffeensis genomic DNA using the primers listed in Table 1.) The functionality of the promoters of p28-Omp14 and p28-Omp19 in correct orientation, in plasmids pRG147 and pRG198, was initially confirmed using E. coli holoenzyme containing its σ70 polypeptide (Figure 4). Subsequently, transcriptional activity of the heparin-agarose purified RNAP fractions was evaluated. E. chaffeensis RNAP activity was detected in purified pooled fractions (data shown for pRG198 in Figure 4). The purified enzyme is completely inhibited in the presence of anti-σ70 monoclonal antibody, 2G10, or in the presence of rifampicin (Figure 4). Further characterization using varying salt concentrations showed that the enzyme was active in presence of potassium acetate up to 200 mM concentration and was inhibited at 400 mM (Figure 5A), and the optimum concentration for activity of the enzyme for sodium chloride was observed at 80 mM (Figure 5B).


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)

In vitro transcription showing the effect of varying salt concentrations of potassium acetate (Panel A), and sodium chloride (panel B). Transcription of the p28-Omp19 promoter region in pRG198 plasmid was assessed using the purified E. chaffeensis RNAP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: In vitro transcription showing the effect of varying salt concentrations of potassium acetate (Panel A), and sodium chloride (panel B). Transcription of the p28-Omp19 promoter region in pRG198 plasmid was assessed using the purified E. chaffeensis RNAP.
Mentions: In support of testing the functionality of p28-Omp14 and p28-Omp19 gene promoters, we constructed in vitro transcription templates, pRG147 and pRG198, by cloning the promoter regions of the genes into the pMT504 plasmid (Figure 3). The plasmid pMT504 is a G-less cassette containing two transcription templates cloned in opposite directions to aid in driving transcription from promoters introduced upstream of the G-less cassette sequences [26]. (The promoter segments were amplified from E. chaffeensis genomic DNA using the primers listed in Table 1.) The functionality of the promoters of p28-Omp14 and p28-Omp19 in correct orientation, in plasmids pRG147 and pRG198, was initially confirmed using E. coli holoenzyme containing its σ70 polypeptide (Figure 4). Subsequently, transcriptional activity of the heparin-agarose purified RNAP fractions was evaluated. E. chaffeensis RNAP activity was detected in purified pooled fractions (data shown for pRG198 in Figure 4). The purified enzyme is completely inhibited in the presence of anti-σ70 monoclonal antibody, 2G10, or in the presence of rifampicin (Figure 4). Further characterization using varying salt concentrations showed that the enzyme was active in presence of potassium acetate up to 200 mM concentration and was inhibited at 400 mM (Figure 5A), and the optimum concentration for activity of the enzyme for sodium chloride was observed at 80 mM (Figure 5B).

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