qRT-PCR verification of S. flexneri virulence plasmid genes repressed under anaerobic growth conditions and the role of FNR in the repression. Strand specific qRT-PCR analysis of S. flexneri M90T virulence plasmid genes mRNA levels shown to be repressed in RNA-seq analysis. Data were calculated as the n-fold difference relative to polA (2-ΔCt, where ΔCt represents the difference in threshold cycle between the target and control genes). Results are shown in relation to the wild-type 2-ΔCt levels under aerobic conditions (referred to as 1). Values greater than 1 indicate increased transcription under anaerobiosis, while lower than 1 indicate the opposite. Significant differences were detected with the wild-type strain 2-ΔCt levels under aerobic and anaerobic conditions, or wild-type vs. Δfnr 2-ΔCt levels under anaerobiosis were compared. P <0.01, **; n = 4; Mann–Whitney test. Error bars show Standard Deviation (SD).