Figure 5
From: Methylomic and phenotypic analysis of the ModH5 phasevarion of Helicobacter pylori
ModH5-mediated methylation of the flaA promoter region modulates downstream gene expression. (a) pTM117-flaA reporter construct design. DNA containing GACC sites upstream of, and at the start of, the P12 flaA ORF was inserted upstream of a promoterless gfpmut3 gene within plasmid pTM11733. (b) Transformation of H. pylori modH5 strain 7.13 resulted in both GFP-fluorescent and non-fluorescent kanamycin-resistant transformants as measured by flow cytometric analysis of transformants (green peak) compared to parental 7.13 (grey peak); x-axis denotes GFP fluorescence intensity and y-axis denotes number of cells. Inset chromatograms of the polyG-tract in modH5 gene of each transformant showing the GFP-fluorescent clones were modH5-ON and the non-fluorescent clones were modH5-OFF. (c) gfp mRNA levels in GFP-fluorescent versus non-fluorescent transformants as determined by qPCR using 16S-specific and gfp-specific qPCR of random primed cDNA generated from bacterial RNA. Three independent transformants were assessed per fluorescence phenotype; each symbol type (circle, triangle or square) represents an individual clone; open symbol = 16S qRT-PCR, filled symbol = gfp qRT-PCR; each point represents mean (±SD) of technical triplicates.
