Figure 13. PhoB regulates kpnO in K. pneumoniae.

A. Promoter region analysis of kpnO. The numbers in brackets represent the distance from the transcription start site. The −35 and −10 region in the promoter is underlined. Putative PhoB binding site has been shown in bold. B. SDS-PAGE profile of pET-phoB ^KP. Lane 1: medium size marker, Lane 2: pET28C/BL21DE3 uninduced, Lane 3: pET28C/BL21DE3 induced, Lane 4: pET-phoB ^KP/BL21DE3 uninduced, Lane 5: pET- phoB ^KP/BL21DE3 induced, purified PhoB^KP-fractions E1 and E2 (lanes 6–7) respectively. Protein samples after induction were subjected to SDS/PAGE (15% gel) followed by coomassie brilliant blue staining. C. Gel shift assays demonstrating the binding of PhoB to promoter of outer membrane protein kpnO in K. pneumoniae in a concentration dependent manner. Lane 1 (shows free probe), lanes 2–7 with increasing concentrations of PhoB protein (50 nM to 500 nM) respectively. Slower moving bound complexes and free probe has been indicated by arrows respectively. The gels are representative of at least three independent experiments. D. Gel shift assays demonstrating the sequence-specific binding of PhoB to kpnO using different controls as in lane 1 (shows free probe), lanes 2–4 (labeled kpnO promoter with increasing amount (100 nM, 200 nM and 400 nM) of PhoB), lane 5 (labeled kpnO promoter and PhoB with specific competitive inhibitor: 10 fold excess of unlabeled kpnO promoter), lane 6 (labeled non-specific DNA: promoter of gyrA and PhoB, 100 nM), lane 7 (labeled kpnO promoter with non-specific protein: BSA, 100 nM) respectively. The gels are representative of at least three independent experiments. E. Relative transcriptional level of kpnO in ΔphoB ^KP and ΔphoB ^KPΩphoB ^KP strains determined using real time RT-PCR is showed in comparison with wild type. The wild type expression level is represented as one fold. Each bar represents the average value of three independent experiments. Error bars are standard deviations.