Fig. 5

Analysis of nrdA and nrdJ mRNA stability using transcriptional shut-off in plasmid-based and chromosomal systems. (a) Decay curves and retrotranscribed transcript levels of nrdA and nrdJ following rifampicin treatment at 0, 5 and 20 minutes in P. aeruginosa PAO1 WT carrying pETS130-GFP derivatives. Constructs include either the full 5’UTR (blue, WT + PnrdA) and truncated 5’ UTR (pink, WT + PnrdA- Δ5’ UTR). In this plasmid-based system, cat mRNA, located downstream of the promoter and 5’ UTR, was used as proxy to estimate nrdA transcript stability. This approach was necessary due to technical limitations in directly quantifying plasmid-derived nrdA. nrdJ levels were measured directly from the chromosomal copy and are shown in green and yellow (WT + PnrdA and WT + PnrdA-Δ5’ UTR, respectively). (b) Decay curves and retrotranscribed transcript levels of nrdA and nrdJ following rifampicin treatment at 0, 2-, 6-, 10- and 20-min in P. aeruginosa wild-type (WT) and PAO1 Δ5’ UTR (Δ5’ UTR). (c,d) One-phase exponential decay model fitted to the data to calculate best-fit mRNA half-life values (in minutes, min) and decay rate constant (k, in minutes, min⁻¹). In panel (c), nrdA values were derived from cat mRNA (proxy), while nrdJ was measured directly from the chromosome (endogenous). In panel (d), both nrdA and nrdJ were quantified from their endogenous chromosomal copies. All qRT-PCR data were normalized to the endogenous control 16S rRNA. The dashed line represents the 50% threshold used to determine mRNA half-life. Decay parameters were calculated using nonlinear regression with a one-phase exponential model, as described in Materials and Methods.