Figure 5. Self-quorum quenching regulates virB genes expression.

(A) B. melitensis wt and ▵aibP strains both carrying the pBBR PvirB-gfp(ASV) plasmid were grown in 2YT and GFP(ASV) fluorescence intensity was measured at indicated phases of growth by flow cytometry (5×10⁴ events acquired). Results are representative of two independent experiments. (B) The relative abundance of virB1, virB2, and virB8 mRNAs was determined by qRT-PCR on RNA isolated from bacteria harvested at the early exponential phase of growth in 2YT supplemented or not with exogenous C12-HSL (5 µM). Deletion of aibP results in significant upregulation of virB genes (P<0.001 in Student’s t test), whereas exogenous C12-HSL significantly downregulates their expression (P<0.001 in Student’s t test). ▵aibP PaibP on the right panel is the complemented strain. Results are representative of two independent experiments. Error bars represent standard deviation from biological triplicates. (C) (Left panel) Western Blot analysis of VirB8 production performed on whole protein lysates of bacteria harvested at the indicated phases of growth in 2YT. ▵aibP PaibP is the complemented strain. (Right panel) Bacteria were harvested in the log phase of growth in 2YT in the absence (–) or in the presence of C12-HSL. The VirB8 protein was detected at its expected size (26,5 kDa). Detection of PrlR or Omp89 proteins was used to normalize total protein content.