RNA-Seq transcriptional analysis of Herbaspirillum seropedicae fnr1 and fnr3 deletion mutants in comparison with the wild type under two different oxygen regimes

To systematically address the role of different Fnr proteins in the transcription regulation in response to varying oxygen levels, we have established an oxygen switch protocol to study the transcriptional changes directly or indirectly associated with these oxygen-sensitive regulatory proteins. To ensure an efficient oxygen switch, cells were grown to the mid log phase (O.D600nm = 0.36±0.02) at high aeration rates (350 rpm) to ensure truly aerobic conditions and then switched to oxygen-limiting conditions (120 rpm) for 30 minutes prior to RNA extraction. Under these conditions, the switch in oxygen availability has minimal impact on growth rates of either the wild-type or the single fnr mutant strains, whilst allowing the detection of Fnr-dependent promoter activation. This enabled the comparison of the transcript profiles among the different strains under “high O2” (350 rpm or control) and “low O2” (120 rpm or switch) conditions.