Fig 2. Effects of the absence of YfcJ, YhjE, and YdiM on the transcription of bo₃-Qox.

One step RT-PCR was performed on total RNA extract from bo₃⁺ strain as well as ∆bo₃ (cyoB), ∆yfcJ, ∆yhjE, and ∆ydiM mutants using the cyoB primers to amplify a 322 bp DNA fragment, and the rrsA primers to amplify a 100 bp region of the 16S ribosomal mRNA as a control (Materials and Methods). (A) Early stage of growth. The cells were grown under aerobic conditions at OD₆₀₀ of 0.05 (top panel) and 0.1 (bottom panel) where cell division continues. The transcription of cyoB was readily detected and showed no difference at both OD₆₀₀. (B) Late stage of growth. The cells were grown under aerobic conditions at OD₆₀₀ of 0.15 (maximum OD₆₀₀ reached). The transcripts of cyoB gene were barely detectable when the bo₃-Qox or the MFS-type transporters YfcJ, YhjE, and YdiM are absent. The numbers below each panel indicate the intensities of the corresponding bands, normalized to that of rrsA then compared to that seen with the bo₃⁺ strain (taken as 100%). These intensities were determined using ImageJ software (NIH). A control PCR where the reverse transcriptase enzyme was inactivated at 95°C was performed for each total RNA extract to check for DNA contamination. Each experiment is repeated at least three times, and a representative sample is shown for each case.