Fig 4. c3566-c3568 transcription and hlyCABD transcription are linked and controlled by c3564 and c3565.
(A) qPCR analysis indicates that c3564 and c3565 positively regulate the expression of c3566-c3568 expression. Bacteria were collected by scraping off the plates and immediately stabilized in RNAlater reagent. Total RNA was isolated and reverse transcribed to cDNA. The rpoB gene was used as an internal control, and the 2-ΔΔCt method was employed to determine fold-changes in CFT073 variants compared to the wild type. Dashed lines represent a 2-fold change cutoff, and a fold-change ≥2 was considered significant. Values are the mean ± SD of three replicates from 3 independent experiments. (B) RT-PCR analysis indicates that the c3566-c3568 operon and hlyCABD operon are cotranscribed. RNA and cDNA samples were prepared as described above. The black bars under the gene arrows denote the PCR amplicons generated during testing of cotranscription, and the number in front of each bar corresponds to the lane in the agarose gel. The #1 fragment was used as a negative control reaction, as c3565 and c3566 do not cotranscribe. RNA that was not reverse transcribed served as a negative control template, while genomic DNA served as a positive control template. 5’-RACE PCR was used to identify the TSS (+1) of the c3566-c3568 operon, and the -10 and -35 motifs were deduced afterward. Gray box, promoter region of c3566; underlined ATG, start codon; blue letters, ribosomal binding site; green letters, -10 box; red letters, -35 box; boxed letters, inverted repeats (IRs) likely bound by the C3565 protein. The genes are drawn to scale. (C) EMSA analysis of C3565 protein bound to the c3566 promoter region. C3565-His6 recombinant protein was purified to homogeneity, and the biotinylated c3566 promoter fragment was used as a probe. Concentrations of the proteins and the ratios of cold probe to the biotinylated probe were indicated above the image. (D) Hemolysis is regulated by the c3566 promoter (Pc3566). Hemolysis assays with liquid blood and blood agar were performed on various CFT073 strains. Strain names represent: WT, wild-type CFT073; ΔPc3566, c3566 promoter deletion mutant; Δc3565, c3565 deletion mutant; Pc3566::PCmR, a mutant in which c3566 promoter is replaced by PCmR; Δc3565-Pc3566::PCmR, a mutant in which c3565 is deleted and c3566 promoter is replaced by PCmR. All images are representative of at least two independent experiments, each containing 3 replicates.