Figure 6. Glucose and cAMP-related proteins affect rcsA transcription.

(A) Diagrammatic representation of rcsA loci. The large arrows represent the open reading frames. The relative positions of the primer sets used in PCR amplification of the DNA probes are indicated, and the numbers denote the positions relative to the translational start site. Names of the DNA probes are shown on the left. The dashed boxes indicate the predicted CRP binding sites and the alignment is shown below. (B) qRT-PCR analysis of rcsA expression was measured in WT, ΔcyaA, ΔcpdA, and Δcrp strains in LB or indicated LB medium. The results shown are an average from triplicate measurements in one single experiment representative of three independent experiments. Error bars indicate standard deviations. *P<0.05 and **P<0.01 compared with WT. (C) The β-galactosidase activities of K. pneumoniae CG43S3ΔlacZ and the isogenic strain (ΔlacZΔcrp) carrying the reporter plasmid prcsAZ15 (P_rcsA::lacZ) were determined using log-phased cultures grown in LB medium. **P<0.01 compared with ΔlacZ. (D) CRP binds directly to P_rcsA. Different concentrations of purified His₆-CRP were incubated with 10 ng of various truncated DNA fragments of the upstream region of rcsA. Following incubation at room temperature for 30 min, the mixtures were analyzed on a 5% non-denaturing polyacrylamide gel containing 200 µM cAMP. The gel was stained with SYBR Green I dye and photographed.