Figure 3.

RydC acts on one of the two isoforms of cfa mRNA. (A) Sequence of the Salmonella cfa upstream region. Transcription initiates from two start sites indicated by arrows at −210 bp (σ⁷⁰-dependent; cfa1 mRNA) or −33 bp (σ^S-dependent; cfa2 mRNA) relative to the translational start site, respectively. Promoter elements are highlighted in grey, and the start codon is boxed. (B) RydC specifically acts on the longer cfa mRNA isoform. Salmonella cfa::3xFLAG ΔrydC cells or an isogenic ΔrpoS mutant were transformed with the pBAD control plasmid (−) or the pBAD-RydC overexpression plasmid (+); Salmonella Δcfa ΔrydC served as a negative control. All strains were grown to an OD₆₀₀ of 2, and total RNA samples withdrawn prior to and 15′ after arabinose addition were used as templates for primer extension. Transcripts originating from either TSS1 or TSS2 were identified using gene-specific sequencing ladders. (C) Schematic representation of the cfa gene including the upstream promoter region. Translational cfa::gfp fusions (under control of the constitutive P_LTetO-1 promoter) were constructed comprising the 5′ upstream region from the distal (cfa1::gfp) or the proximal start site (cfa2::gfp) plus the first 45 nucleotides of the cfa CDS. (D) Regulation of reporter fusions was monitored by western blot analysis. At an OD₆₀₀ of 1, total protein samples were prepared from Salmonella ΔrydC ΔrpoS mutants carrying plasmids to express cfa2::gfp or cfa1::gfp in combination with a control plasmid (−) or pP_LRydC (+). GroEL served as a loading control. Expression of RydC was validated on a northern blot.

Source data for this figure is available on the online supplementary information page.

Source Data for Figure 3 ^{(2.4MB, pdf)}