Figure 8.

In vivo effect of CRP-cAMP complex on lipBA expression of S. oneidensis. (A) MacConkey agar plate-based visualization for effect of Escherichia coli CRP on Shewanella lipBA promoter-driven lacZ transcription. The two E. coli strains with the lipBA-lacZ transcriptional fusion include FYJ457 (WT) and FYJ458 (Δcrp). To assay lipBA-lacZ expression, we used MacConkey agar plate with 0.4% lactose as a sole carbon source. The bacteria were maintained at 37°C for around 36 h. Purple denotes high level of β-gal activity, whereas yellow indicates low level of β-gal activity. (B) β-gal analyses for CRP-mediated regulation of lipBA_she transcription in model organism E. coli. Mid-log phase cultures in RB media were collected to test β-gal activity. The data are expressed in average ± standard deviation (SD), and error bars indicate SD. No less than three independent experiments were performed. The two E. coli strains are FYJ457 (WT) and FYJ458 (Δcrp), respectively. (C) Real-time quantitative PCR (qPCR) assays for altered expression profile of lipA and lipB upon the removal of crp gene from Shewanella. The two strains of Shewanella grown in RB media are MR-1 (S. oneidensis MR-1, WT) and HG0624 (S. oneidensis MR-1, Δcrp). Mig-log phase bacteria were collected for isolation of total RNA. The data are expressed as averages ± standard deviations (SD), and error bars mean SD. Three independent experiments were performed here. Colony comparison (D) and β-gal activity (E) of the S. oneidensis reporter strains carrying the chromosomal lipBA-lacZ fusions grown on minimal medium plates. (F) Direct measurement of bacterial cAMP level. The intracellular (pelleted cells) and extracellular (supernatant) level of bacterial cAMP pools were assayed after centrifugation. A standard curve with cAMP by values of OD₄₅₀ was generated for each patch of samples. Relative levels were calculated by normalizing to the values of the wild-type, which was set to 1. Both CΔcrp and CΔcyaC strains were designed to express a single copy of the corresponding genes in trans. Error bars represent standard deviations from at least three independent experiments. CRP, cAMP-receptor protein.