Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation
Figure 5
A. Gene Expression analyses of lpx genes.
All qPCR experiments were performed as outlined in materials and methods. Expression levels were normalized to 16S levels, and fold change values were generated by calibrating against Ecl8∆ramA. Two-way ANOVA analyses (P<0.05) were performed to demonstrate statistical significance. B. Regulation of lpx genes. (i) EMSA using lpxC and lpxK promoter regions. Purified RamA (200nM) and the different labelled DNA probes (2 nM) were incubated on ice. All reactions were performed on ice prior to electrophoresis on 7.5% native gel. (ii) Transcription in vitro of lpxC promoter region. Relative fold increase was determined using densitometric analysis as described previously [55]. Fold increases in the presence of RamA were determined by first normalising to the control promoter (gnd) prior to comparison to the no protein control. C: Lipid A analysis from K. pneumoniae Ecl8 (WT), Ecl8ΔramA, Ecl8ΔramR and Ecl8ΔramRA. Lipid A analysis was undertaken as described before [31]. Negative ion MALDI-TOF mass spectrometry of lipid A isolated from K. pneumoniae Ecl8 and its derivatives. Peaks in bold correspond to LpxO dependent 2’ secondary chain modifications.