Fig. 4
Analysis of nrdA expression using GFP reporter assay and quantitative reverse transcription PCR (qRT-PCR) under aerobic and anaerobic conditions during exponential and stationary growth phases. (a) nrdA expression measured as relative fluorescence units (RFU), normalized to OD600 (RFU/OD600) from GFP transcriptional reporter fusions (pETS130-GFP derivatives) containing either the full-length 5’ UTR (PnrdA) or truncated version of 5’ UTR (PnrdA- Δ5’ UTR), under aerobic (left) and anaerobic conditions (right), during exponential (EX) and stationary (ST) growth phases. (b) Fold change in nrdA transcript levels measured by qRT-PCR in P. aeruginosa CRISPR-Cas9-engineered chromosomal 5’ UTR mutant (PAO1 Δ5’ UTR) compared to the wild-type strain (PAO1 WT), under aerobic (left) and anaerobic (right) conditions during both exponential and stationary growth phases. Comparisons were made between genotypes within each growth phase, and data were normalized accordingly to the WT sample in each condition to emphasize intra-phase differences. Dashed lines indicate the separation between exponential and stationary phases. (c) Fold change in nrdA transcript levels measured by qRT-PCR in P. aeruginosa PAO1 WT and the isogenic Δ5′ UTR strains, comparing exponential (EX) and stationary (ST) under aerobic (left) and anaerobic (right) conditions. Data were normalized to the exponential (EX) phase sample of each genotype to highlight intra-genotype variation. Dashed lines indicate the separation between WT and Δ5′ UTR strains. Significance: p-value < 0.0001(****). Bars are grouped by condition and genotype to facilitate intra-group comparisons.
