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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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E. chaffeensis RNA polymerase purification by employing heparin agarose column purification method. A) Silver-stained SDS-PAGE gel profile of heparin agarose purified fractions of E. chaffeensis RNA polymerase. M, protein standards (kDa); C, E. chaffeensis crude lysate; W1, first wash fraction from the column; W2, second column wash; E1, first elution fraction; E2, second elution fraction; P, pooled dialyzed fractions of eluted fractions 3 to 6; Ec, E. coli holoenzyme from Epicenter® B) Western blot analysis of the proteins resolved in panel A with E. coli anti-sigma70 monoclonal antibody, 2G10.
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Figure 1: E. chaffeensis RNA polymerase purification by employing heparin agarose column purification method. A) Silver-stained SDS-PAGE gel profile of heparin agarose purified fractions of E. chaffeensis RNA polymerase. M, protein standards (kDa); C, E. chaffeensis crude lysate; W1, first wash fraction from the column; W2, second column wash; E1, first elution fraction; E2, second elution fraction; P, pooled dialyzed fractions of eluted fractions 3 to 6; Ec, E. coli holoenzyme from Epicenter® B) Western blot analysis of the proteins resolved in panel A with E. coli anti-sigma70 monoclonal antibody, 2G10.

Mentions: E. chaffeensis DNA-dependent RNA polymerase (E. chaffeensis RNAP) was partially purified from the organisms grown in macrophage cultures by adapting heparin-agarose column purification method described earlier for other bacterial systems [27]. To determine the purity and polypeptide composition of the E. chaffeensis RNAP, several eluted fractions were electrophoresed on a polyacrylamide gel that was stained using silver nitrate (Figure 1A). The gel pattern revealed that the E. chaffeensis RNAP had a subunit structure similar to E. coli RNAP (that is also typical of other eubacteria) with five major subunits (α2, β, β', σ). Western blot analysis confirmed the presence of E. chaffeensis σ70 polypeptide when assessed using a heterologous E. coli anti-σ70 monoclonal antibody, 2G10 (Figure 1B). Amino acid alignment of the sequence of E. chaffeensis σ70 polypeptide with E. coli σ70 polypeptide revealed significant homology which also spanned to the putative binding site sequence of 2G10 antibody to E. coli σ70 polypeptide [28,29] (Figure 2). The homology between amino acid residues of σ70 polypeptides recognised by 2G10 antibody [28] is considerably higher between E. chaffeensis and E. coli than between E. chaffeensis and Chlamydia trachomatis . Protein BLAST search (at National Center for Biotechnology Information Bethesda, MD, USA) of the putative amino acid binding site sequence of 2G10 in E. coli [28,29] against E. chaffeensis (Arkansas isolate) genome identified only one significant match (E-value of 1e-11 and having 69% identity) with E. chaffeensis RNAP σ70 polypeptide, RpoD.


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)

E. chaffeensis RNA polymerase purification by employing heparin agarose column purification method. A) Silver-stained SDS-PAGE gel profile of heparin agarose purified fractions of E. chaffeensis RNA polymerase. M, protein standards (kDa); C, E. chaffeensis crude lysate; W1, first wash fraction from the column; W2, second column wash; E1, first elution fraction; E2, second elution fraction; P, pooled dialyzed fractions of eluted fractions 3 to 6; Ec, E. coli holoenzyme from Epicenter® B) Western blot analysis of the proteins resolved in panel A with E. coli anti-sigma70 monoclonal antibody, 2G10.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: E. chaffeensis RNA polymerase purification by employing heparin agarose column purification method. A) Silver-stained SDS-PAGE gel profile of heparin agarose purified fractions of E. chaffeensis RNA polymerase. M, protein standards (kDa); C, E. chaffeensis crude lysate; W1, first wash fraction from the column; W2, second column wash; E1, first elution fraction; E2, second elution fraction; P, pooled dialyzed fractions of eluted fractions 3 to 6; Ec, E. coli holoenzyme from Epicenter® B) Western blot analysis of the proteins resolved in panel A with E. coli anti-sigma70 monoclonal antibody, 2G10.
Mentions: E. chaffeensis DNA-dependent RNA polymerase (E. chaffeensis RNAP) was partially purified from the organisms grown in macrophage cultures by adapting heparin-agarose column purification method described earlier for other bacterial systems [27]. To determine the purity and polypeptide composition of the E. chaffeensis RNAP, several eluted fractions were electrophoresed on a polyacrylamide gel that was stained using silver nitrate (Figure 1A). The gel pattern revealed that the E. chaffeensis RNAP had a subunit structure similar to E. coli RNAP (that is also typical of other eubacteria) with five major subunits (α2, β, β', σ). Western blot analysis confirmed the presence of E. chaffeensis σ70 polypeptide when assessed using a heterologous E. coli anti-σ70 monoclonal antibody, 2G10 (Figure 1B). Amino acid alignment of the sequence of E. chaffeensis σ70 polypeptide with E. coli σ70 polypeptide revealed significant homology which also spanned to the putative binding site sequence of 2G10 antibody to E. coli σ70 polypeptide [28,29] (Figure 2). The homology between amino acid residues of σ70 polypeptides recognised by 2G10 antibody [28] is considerably higher between E. chaffeensis and E. coli than between E. chaffeensis and Chlamydia trachomatis . Protein BLAST search (at National Center for Biotechnology Information Bethesda, MD, USA) of the putative amino acid binding site sequence of 2G10 in E. coli [28,29] against E. chaffeensis (Arkansas isolate) genome identified only one significant match (E-value of 1e-11 and having 69% identity) with E. chaffeensis RNAP σ70 polypeptide, RpoD.

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