Figure 1.
Mapping Transcription Start Sites (TSS) of E. chaffeensis genes by primer extension (PE) and 5’ RACE.
A) The primer extended products resolved on sequencing gels with TSS identified for the genes dnaK, hup, DNAbp (DNA binding protein gene), clpA, clpB, operons of groE* (groES and groEL genes), and hsIV* (hslV and hslU genes). The location of the TSS for each gene was identified by comparing the Sanger’s DNA sequencing runs generated with the same primers used for the PE reactions, but using plasmid DNAs containing the respective gene segments. All genes had one TSS with the exception of hup, which contained two TSS. [The location of the TSS established from the PE results for all 7 genes (bold and underlined text) relative to the initiation codon of each gene were presented under gel data.] B) 5’RACE data identifying the TSS of the genes dksA and grpE, and the operon glyQ* (glyQ, glyS and dnaJ genes). Sequences generated from the 5’RACE products are compared with the sequences generated with DNA templates and are shown for each gene relative to the initiation codon. TSS are identified with bold and underlined text. The underlined G rich tails are added upstream to TSS during the 5’RACE reaction.
Figure 2.
RNAP binding motifs -35 and -10 of E. chaffeensis genes.
RNAP binding motifs, -35 and -10, are identified for the 12 E. chaffeensis genes for which TSS were mapped (listed in Tables 2). The upper panel has the consensus motifs for the σ32 dependent gene promoters; the middle panel includes the σ70-dependent promoters and the lower panel includes the consensus motifs for all 12 genes assessed.
Figure 3.
In vitro transcription analysis of E. chaffeensis genes dnaK, groE, hup, p28-Omp14 and p28-Omp19 promoters.
In vitro transcription analysis was performed using RNAP holoenzyme containing E. chaffeensis recombinant σ32 or σ70. The promoter segments of E. chaffeensis genes dnaK, groE, hup, p28-Omp14 and p28-Omp19 cloned upstream to the G-less cassette in pMT504 plasmid vector in the correct or reverse orientation were used in the assays with reconstituted RNAP containing E. chaffeensis recombinant σ32 or σ70. (Reverse orientation constructs were identified in the Figure as dnaK-R, groE-R, hup-R, p28-Omp14-R and p28-Omp19-R.) The p28-Omp14 and p28-Omp19 promoter constructs having -35 motif deletions (p28-Omp14-35 and p28-Omp19-35) were also prepared and used in the in vitro transcription assays. The abundance of the transcripts for each gene in the presence of σ32 or σ70 is captured from the 32P incorporation in the RNA. As reported earlier [37], assays performed with RNAP core enzyme alone or with purified σ32 or σ70 did not yield any transcripts (not shown).
Figure 4.
Promoter sequences and the plasmids used for the in vivo assay system for defining the promoter activities of E. chaffeensis genes in E. coli.
A) Sequences of promoter segments of genes hup, dnaK and groE (wild-type; hup, dnaK and groE or with -35 deletions; hup-35del, dnaK-35del and groE-35del) used for the in vivo assays were presented. The groE-35updel segment had a deletion lacking both the -35 motif and the entire sequence upstream from it. The transcription start sites (identified as +1), -35 and -10 motifs of the promoters were identified in the sequences as the bold and underlined text. The second predicted -35 sequence for groE promoter was identified as the bold, underlined and italics text. B) Illustrations of the two plasmids with distinct origins of replication used in the in vivo promoter mapping assays. Plasmid pSAKT32-Ech_rpoH contained either a wild-type E. chaffeensis rpoH gene sequence or mutant forms with mutations engineered in the region 4.2 of E. chaffeensis σ32. The pQF50K-Ech_promoter plasmids with promoter sequences described in panel A were cloned upstream to the lacZ gene coding sequence to drive the expression of lacZ gene.
Figure 5.
E. chaffeensis promoter activities (A, dnaK; B, groE; and C, hup) assessed in E. coli by measuring the β-galactosidase expression.
The β-galactosidase expression driven by E. chaffeensis promoters from the wild-type promoters (dnaK, groE or hup), promoters containing -35 motif deletion (dnaK-35del, groE-35del or hup-35del) or groE promoter having complete deletion from -35 to the entire upstream sequence (groE-35updel) were measured in the CAG57101strain of E. coli before or after the induced expression of E. chaffeensis rpoH. The CAG57101 strain contained either promoterless pQF50K (control) or one of the pQF50K-Ech_promoter plasmids together with the pSAKT32-Ech_rpoH plasmid (described in Figure 4). Three independent experiments were performed; the error bars indicate standard deviation. Significant changes in the β-galactosidase activity were identified with double asterisks where the P values were <0.01.
Figure 6.
E. chaffeensis σ32 or σ70 binding to dnaK, groE, hup, p28-Omp14, p28-Omp19 promoters assessed by EMSA analysis.
A) Biotin-labeled probes of dnaK, groE, hup, p28-Omp14 and p28-Omp19 promoter segments were used in the EMSA analysis in the presence or absence of the RNAP holoenzyme containing either the recombinant E. chaffeensis σ32 (Eσ32) or σ70 (Eσ70). Specificity of RNAP binding was determined by the inclusion of 100 fold molar excess of cold competitors. B) A DNA segment containing the coding region of dnaK (dnaK-ORF) was used as a control. C) Gel shift assays were performed with dnaK promoter segments by incubating with E. coli core RNAP or purified σ32 or σ70 to serve as additional controls.
Figure 7.
Mutational analysis of E. chaffeensis rpoH gene spanning the conserved region 4.2.
A) Protein sequence homology of σ32 and σ70 of E. chaffeensis and E. coli was assessed by Clustal X (version 2.0) for the entire sequence. The homology spanning region 4 was presented here. Numbers on the left indicate the amino acid position relative to the start codon of each protein. The four amino acids in E. coli, which are identified as critical for binding to the -35 motifs of σ32 and σ70, are also conserved in E. chaffeensis (highlighted with an underlined text). Amino acids that are conserved in all four protein primary sequences are identified with asterisks; homology found only in two or three proteins was identified with a dot or colon, respectively. B) Mutational analysis of the four conserved amino acid residues predicted to be involved in the binding of σ32 to the -35 motif. The amino acids at positions E266, R267, R269 and Q270 of E. chaffeensis σ32 were individually mutated to change the amino acids in the encoded proteins each to alanine. The mutant plasmids were used to assess the E. chaffeensis σ32 in driving the promoter activity of the wild-type dnaK gene (β-galactosidase expression measured relative to the wild-type E. chaffeensis σ32). The experiment was performed three times, and average values were presented with error bars to show the standard deviation.
Figure 8.
Transcripts of σ32 and σ70 assessed in E. chaffeensis infected macrophages.
Total RNA recovered from E. chaffeensis infected macrophages at different times post-infection was assessed by quantitative RT-PCR after normalizing the RNA levels used for the analysis relative to 16S rRNA and the data were presented as the fold change at each time point post-infection relative to zero time point. The experiment was performed three times, and the average values were used for plotting the graph.