Figure 1.

cAMP receptor protein influence on the regulatory network of early-stage virulence genes of Y. pseudotuberculosis. (A) The carbon storage regulator (Csr) system is composed of the RNA-binding protein CsrA and the two regulatory RNAs CsrB and CsrC. Expression and activity of the system components are tightly autoregulated and controlled by the two-component system BarA/UvrY. Upregulation of one or both of the regulatory RNAs lead to the sequestration of CsrA, whereby RovM synthesis is repressed and allows production of RovA, the activator of inv transcription. Arrows display direct activation of gene expression or protein synthesis, where as dashed arrows label indirect regulation. T represents repression or inactivation. (B) Expression of the CsrB and CsrC RNA, RovM, and RovA in the presence and absence of Crp. Whole cell extracts from overnight cultures of Y. pseudotuberculosis wildtype strain YPIII and the mutant strain YP89 (Δcrp) harboring the empty vector pAKH85 (V) or the crp-encoding plasmid pAKH37 (pcrp⁺) grown at 25°C were prepared, and analyzed by northern blotting with a CsrC- or CsrB-specific probe (two upper panels), or by western blotting with polyclonal antibodies directed against RovM or RovA (two lower panels). The 23S rRNAs are shown as RNA loading control, the sizes of RNA or protein markers are given on the left. The respective negative controls are loaded in the last lanes: YP79 (ΔcsrBC, two upper panels), YP3 (ΔrovA) thrid panel, YP72 (ΔrovM) lowest panel. (C) To investigate Crp-dependent flhDC expression in the absence of csrA, the empty vector pAKH85 (V) or its crp⁺ derivative pAKH37 (pcrp⁺) were transformed into Y. pseudotuberculosis strains YPIII, YP89 (Δcrp) or YP53 (ΔcsrA) harboring a flhDC’-’lacZ fusion (pAKH58). The bacteria were grown overnight in LB medium at 25°C. β-Galactosidase activity from overnight cultures was determined and is given in μmol min⁻¹ mg⁻¹ for comparison. The data represent the average ±SD from at least three different experiments each done in duplicate. (D) Analysis of Crp-dependent RovM protein levels in the absence of csrA. Whole cell extracts from overnight cultures of the Y. pseudotuberculosis wildtype strain YPIII and YP53 (ΔcsrA) harboring the empty vector pAKH85 (V) or the crp-encoding plasmid pAKH37 (pcrp⁺) grown at 25°C were prepared, and analyzed by western blotting with polyclonal antibodies directed against RovM. The negative control YP72 (ΔrovM) is loaded in the last lane. The loading controls for the western blots are marked with c.