Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation
Figure 2
A: Northern blot analysis of sRamA5.
15 μg of total RNA extracted from Ecl8/pGEMTpI+romA were loaded into wells. The blots were either incubated with the 32P-end labelled sRamA5 specific DNA probe or romA specific DNA probe. The bands pointed as 1 and 2 are primary transcripts for both the RNA codes for sRamA5 and romA. The band referred to as sRamA5 is specifically detected by the sRamA5 DNA probe, sized at around 60 nucleotides; the band referred to as romA was specifically detected by the romA DNA probe. B: EMSA of RamR-sRamA5 or RamR-PI interaction in the presence of sRamA5. (i). RamR-sRamA5 interaction. The concentrations of sRamA5 and RamR were 40 nM and 1 μM respectively. (ii). RamR-pI interaction in the absence/presence of sRamA5. Radioactive labeled pI was 2 nM from lane 1 to 4. RamR’s concentrations from lane 1 to 4 were: 0, 2, 0, 2 μM. Cold sRamA5’s concentrations from lane 1 to 4 were: 0, 0, 1, 1 μM. FR = free RNA, CR = RNA-protein complex, FD = free DNA, C = RNA-DNA-protein complex. C: qPCR for the level of romA and sRamA5’s transcriptions in Ecl8ΔramR. qPCR using LNA probe for determining the levels of sRamA5 transcription in Ecl8 and Ecl8ΔramR. Despite sharing the same TSS, the transcript levels of sRamA5 are not linked to romA levels, thereby reducing the likelihood of sRamA5 being a 5’ untranslated region of romA. The log2 fold changes in Ecl8ΔramR displayed in the bar chart are relative to their transcript levels in Ecl8. One-way ANOVA analyses (P<0.001) were performed to demonstrate statistical significance. D: qPCR assay for the level of ramR, romA and sRamA5 in Ecl8ΔramR pACYCramR and Ecl8ΔramR pACYC177. The log2 fold changes in the two strains displayed in the bar chart are relative to their transcript levels in Ecl8ΔramR. sRamA5 levels are elevated in the presence of ramR, implying that RamR could stabilise the sRamA5 transcript. One-way ANOVA analyses (P<0.001) were performed to demonstrate statistical significance.