Bacterial cells were negatively stained with 2% phosphotungstic acid. Quantitative RT-PCR analysis Total RNA from LB5010, Δstm0551,
Δstm0551 (pSTM0551), and Δstm0551 (pACYC184) strains were prepared and analyzed for the fimbrial subunit GDC941 gene, fimA, and the regulatory genes, fimZ, fimY, and fimW, by quantitative RT-PCR. 16 S ribosomal (r) RNA expression was used as a control. Individual gene expression profiles were first normalized against the 16 S rRNA gene and then compared to the expression level of fimA, fimZ, fimY, and fimW obtained from agar. As for the parental LB5010 strain, fimA expression obtained from static LB broth was about 150-fold higher than the value obtained from LB agar. The fimA expression of the Δstm0551 strain grown on agar was significantly higher than that of LB5010 grown on agar. Transformation of Δstm0551 with a plasmid possessing the stm0551 coding sequence repressed fimA expression whether this strain was cultured on agar or in static broth, whereas transformation of the same bacterial strain with the plasmid cloning vector pACYC184 de-repressed fimA expression in both culture conditions (Figure 5, panel A). The fimZ expression levels from different strains demonstrated a check details similar profile to that observed
for fimA. The parental LB5010 strain exhibited significant elevated level of click here fimZ when grown in broth than on agar. The fimZ expression of Δstm0551 was higher than that of the parental strain grown on agar. Transforming Δstm0551 with pSTM0551 repressed fimZ expression on both culture conditions, while transforming Δstm0551 with pACYC184 cloning vector de-repressed fimZ expression, leading to comparable level of expression as seen in the Δstm0551 strain (Figure 5, panel B).
Selleck Alectinib However, the expression levels of fimY were not significantly different between strains under both growth conditions (Figure 5, panel C). Δstm0551(pACYC184) had higher fimW expression than Δstm0551(pSTM0551) did when both strains were culture on agar medium (Figure 5, panel D). Figure 5 Detection of the relative transcript levels of fimA , fimZ , fimY , and fimW genes using quantitative RT-PCR. The mRNA transcript levels of the major fimbrial subunit gene fimA (panel A), fimZ (panel B), fimY (panel C), and fimW (panel D) in the parental LB5010, Δstm0551, Δstm0551 (pSTM0551), and Δstm0551 (pACYC184) strains were detected by quantitative RT-PCR. The mRNA transcript levels were obtained by delta-delta Ct (ΔΔCt) method, and the expression levels (2-ΔΔCt) of the parental LB5010 strain cultured on LB agar were set to 1 fold for each gene tested. The asterisk indicated that the differences in transcript levels were statistically significantly (p<0.05).