Journal: International Journal of Molecular Sciences

Article Title: Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation

doi: 10.3390/ijms232112719

Figure Lengend Snippet: SouthWestern blotting for the promoters of E. chaffeensis with recombinant DNA-binding proteins. Recombinant DNA-binding proteins were purified and resolved in a 12% SDS-PAGE, transferred to a nitrocellulose membrane and then incubated with 32 P-labeled probes to visualize signals by autoradiography. BSA was used as the control non-specific protein control. ( A ) South-Western blot analysis to assess the interaction of Tr1, EcxR, CtrA, HU and MerR proteins with the full gene promoter segments of p28-Omp19 and p28-Omp14 . ( B ) Full promoters of E. chaffeensis genes, tr1 , clpB , groES/L and dnaK , were similarly subjected to the interactions with DNA-binding protein EcxR and Tr1.

Article Snippet: E. chaffeensis Arkansas isolate (ATCC # CRL-10389, Manassa, VA, USA) was cultivated in DH82 macrophage cells [ ].

Techniques: Southwestern Blot, Recombinant, DNA Binding Assay, Purification, SDS Page, Membrane, Incubation, Labeling, Autoradiography, Control, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation

doi: 10.3390/ijms232112719

Figure Lengend Snippet: E. chaffeensis EcxR secondary structure and tertiary structure predictions. ( A ) The filled boxes with different colors indicate alpha regions (alpha helical), beta regions (beta strand), turn regions and coli regions, as identified using the Garnier-Osguthorpe-Robson algorithm. (The scale indicates 5-amino-acid intervals.) In the alpha amphipathic and beta amphipathic diagram, the filled boxes indicate regions that are predicted to form alpha helices and beta regions, respectively, and are comprised of amphipathic amino acids, as identified by the Eisenberg algorithm. The Kyte-Doolittle algorithm was used to identify hydrophobic (histogram above the x axis) and hydrophilic (histogram below the axis) regions described at the Hydrophobicity diagram. All of the analyses were carried out using Protean, as a part of the Lasergene software package. ( B ) Tertiary structure prediction of EcxR. EcxR forming as a homodimer was predicted by using SEWISS-MODEL algorithms.

Article Snippet: E. chaffeensis Arkansas isolate (ATCC # CRL-10389, Manassa, VA, USA) was cultivated in DH82 macrophage cells [ ].

Techniques: Software

Journal: International Journal of Molecular Sciences

Article Title: Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation

doi: 10.3390/ijms232112719

Figure Lengend Snippet: Electrophoretic Mobility Shift Assays (EMSAs) to assess the interaction of EcxR with different E. chaffeensis gene promoters. ( A ) From left to right, biotin-labeled probes of clpB, dnaK, groES/L and hup were incubated with recombinant EcxR. High excess cold competitor DNA (unlabeled) was added to show the specificity for protein-promoter interactions. Two DNA segments containing the coding region of dnaK (dnaK-ORF) and ecxR (ecxR-ORF) were used as additional controls to define the specific interactions of EcxR (far right data panels). ( B ) Biotin-labeled probes of different p28-Omp14 promoter segments (as described in ), were used in the EMSA analysis to identify specific regions of these two gene promoters with EcxR. ( C ) Similarly, biotin-labeled probes of different p28-Omp19 promoter segments were used in the EMSA analysis.

Article Snippet: E. chaffeensis Arkansas isolate (ATCC # CRL-10389, Manassa, VA, USA) was cultivated in DH82 macrophage cells [ ].

Techniques: Electrophoretic Mobility Shift Assay, Labeling, Incubation, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation

doi: 10.3390/ijms232112719

Figure Lengend Snippet: Yeast one-hybrid assay to independently confirm the binding of EcxR to three E. chaffeensis promoters; p28-Omp14 (P14), p28-Omp19 (P19) and tr1 (Ptr1). The β-galactosidase assays were used to quantitate the interaction strength of EcxR binding to three promoters, respectively. Eenzyme activities relaive to the negative control (the empty vector pDEST22) were shown for the three promoters. The values are the means ± standard deviations for three independent biological replicates. Ctrl refers to the negative control (the empty vector pDEST22), while EcxR represents to the expression of AD-EcxR chimeric protein in pDEST22-ecxR. Significant differences from the values for samples lacking the expression of protein EcxR were determined by the t test (* p < 0.05).

Article Snippet: E. chaffeensis Arkansas isolate (ATCC # CRL-10389, Manassa, VA, USA) was cultivated in DH82 macrophage cells [ ].

Techniques: Y1H Assay, Binding Assay, Negative Control, Plasmid Preparation, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation

doi: 10.3390/ijms232112719

Figure Lengend Snippet: Total RNA recovered from synchronously cultured E. chaffeensis in DH82 cells at different time points were used to perform real-time RT-PCR analysis for ecxR gene and normalized against bacterial 16S rRNA. Relative values to the amount at 0 h p.i. are shown. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test (* p < 0.05; ** p < 0.01).

Article Snippet: E. chaffeensis Arkansas isolate (ATCC # CRL-10389, Manassa, VA, USA) was cultivated in DH82 macrophage cells [ ].

Techniques: Cell Culture, Quantitative RT-PCR, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation

doi: 10.3390/ijms232112719

Figure Lengend Snippet: Assess the role of EcxR on the transcription of E. chaffeensis promoter-lacZ reporter constructs. β-galactosidase assays were performed to measure the transcriptional activities of lacZ reporter constructs. ( A , C ) Western blot analyses of samples from β-galactosidase assays were carried out using an anti-His tag antibody to verify the expression of RpoH (σ 32 ) or RpoD (σ 70 ) alone or with EcxR expression. The positions of RpoH, RpoD and EcxR are indicated by arrowheads. ( B ) The β-galactosidase expression driven by E. chaffeensis promoters constructs encoding clpB, groES/L and dnaK were quantitated in the CAG57101 strain of E. coli expressing E. chaffeensis EcxR and σ 32 (EcxR-RpoH) or only E. chaffeensis σ 32 (RpoH), which were grown at 30 °C. ( D ) The β-galactosidase expression driven by E. chaffeensis promoters constructs encoding p28-Omp14, p28-Omp19 and hup were quantitated in the CAG20177 strain of E. coli expressing E. chaffeensis EcxR and σ 70 (EcxR-RpoD) or only E. chaffeensis σ 70 (RpoD), which were grown at 37 °C. The values are the means ± standard deviations for three independent biological replicates. Significant differences from the values for samples lacking the expression of protein EcxR were determined by the t test (**, p < 0.01; ***, p < 0.001; ns, not significant).

Article Snippet: E. chaffeensis Arkansas isolate (ATCC # CRL-10389, Manassa, VA, USA) was cultivated in DH82 macrophage cells [ ].

Techniques: Construct, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation

doi: 10.3390/ijms232112719

Figure Lengend Snippet: In vitro transcription analysis of the role of protein EcxR on the transcription of the E. chaffeensis promoters, including clpB, groES/L, dnaK , p28-Omp14 and p28-Omp19 . The promoter segments of E. chaffeensis genes, clpB , groES/L, dnaK , p28-Omp14 and p28-Omp19 were cloned upstream to the G-less cassette in pMT504 plasmid vector in the correct orientation and used in the assays with reconstituted RNAP containing E. chaffeensis recombinant σ 32 or σ 70 . In vitro transcription analysis was performed using E. coli RNAP holoenzyme containing E. chaffeensis recombinant σ 32 for clpB, groES/L and dnaK , and E. chaffeensis recombinant σ 70 for p28-Omp14 and p28-Omp19. Lane 1, template DNA with RNAP holoenzyme with no EcxR added. Lanes 2–3, template DNA with RNAP holoenzyme and with 10 or 20 nM recombinant EcxR, respectively. The abundance of the transcripts for the template in the presence of σ 32 or σ 70 alone or with recombinant EcxR is captured from the Biotin-14-CTP incorporation in the RNA. The upper panel indicate the transcription products, which were resolved on a 6% denatured polyacrylamide gel containing 7 m urea in 1 × Tris-borate-EDTA buffer. The lower panel indicated the intensity of a band signals in a gel for in vitro transcriptions, as determined using the software ImageJ. The bars show the relative change of transcription products with different concentrations of EcxR as the percentage of transcripts compared to the controls lacking EcxR.

Article Snippet: E. chaffeensis Arkansas isolate (ATCC # CRL-10389, Manassa, VA, USA) was cultivated in DH82 macrophage cells [ ].

Techniques: In Vitro, Clone Assay, Plasmid Preparation, Recombinant, Software