## 2 Results and Discussion  
### The Production of the \(\alpha\)-Hemolysin Is Impaired in ppGpp-Deficient Strains  
The effect of ppGpp on the expression of \(\alpha\)-hemolysin produced by the _hlyCABD\({}_{II}\)_ of J96 was initially characterized using the plasmid pSF4000, a PACYC184-based plasmid carrying the _hlyCABD\({}_{II}\)_ operon. The pSF4000 plasmid was transformed in the _E. coli_ K12 strain AAG1 (MG1655 \(\Delta\)lac) and its ppGpp-deficient derivative (ppGpp\({}^{0}\)) counterpart (JFV2). An increase in cytotoxicity by the presence of plasmid pSF4000 in a non-hemolytic strain has been previously described [22]. Infection challenges with both Wt and its ppGpp\({}^{0}\) derivative carrying the pSF4000 plasmid were performed on T24 cells. Cytotoxicity, monitored as lactate dehydrogenase activity released from the T24 cells, was significantly higher in the Wt strain as compared to its isogenic ppGpp\({}^{0}\) derivative (Figure 1A, black bars). A similar pattern was detected when T24 cells monolayers were incubated with cell-free supernatants from cultures of both strains (Figure 1A, white bars), confirming the involvement of a secreted product in the observed cytotoxic effect. The activity of the \(\alpha\)-hemolysin can be easily characterized by its ability to lyse erythrocytes. Comparison of the hemolytic haloes surrounding the colonies on blood agar plates also substantiates the impaired ability to produce the toxin by the ppGpp-deficient strain (Figure 1B). To further corroborate these results, the amount of secreted toxin was monitored in cell-free supernatants from cultures of both Wt and ppGpp\({}^{0}\) strains. The \(\alpha\)-hemolysin, with an estimated molecular mass of 110 kDa, was detected as a major band in cell-free supernatant of Wt cultures after SDS-PAGE and Coomassie staining (Figure 1C, labelled with an arrowhead). A similar band with much lower intensity was detected in the supernatant of the ppGpp\({}^{0}\) strain. The amount of secreted \(\alpha\)-hemolysin was about 8-fold lower in ppGpp\({}^{0}\) as compared to Wt. Our data show that ppGpp is required for optimal production of external \(\alpha\)-hemolysin. This conclusion can be draw either by ppGpp acting as a positive regulator of the \(\alpha\)-hemolysin expression or by ppGpp promoting its secretion to the external medium. If ppGpp is involved in the secretion of the \(\alpha\)-hemolysin, one would expect accumulation of the toxin in the cytoplasm of the ppGpp\({}^{0}\) strains. The intracellular \(\alpha\)-hemolysin in whole cell extracts of Wt and ppGpp-deficient strains was monitored by immunoblot analyses (Figure 1C). The amount of intracellular \(\alpha\)-hemolysin was higher (10-fold) in the Wt strain than in its ppGpp\({}^{0}\) derivative, clearly indicating that ppGpp stimulates the production of \(\alpha\)-hemolysin. Suppressor Mutants of the Auxotrophic Phenotype Depicted by the ppGpp\({}^{0}\) Restore \(\alpha\)-Hemolysin Expression from the hlyCABD\({}_{\Pi}\) Operon  
The ppGpp\({}^{0}\)_E. coli_ strains are impaired to induce the expression of several amino acid biosynthetic pathways and, consequently, these strains are unable to grow on minimal media [23]. Spontaneous suppressor mutants of ppGpp\({}^{0}\) strains restoring prototrophy can be selected by plating cultures on minimal media plates. Consistent with the fact that ppGpp binds to the RNA polymerase, mutations associated to prototrophy restoration are frequently located within the _rpoB_ and _rpoC_ genes that encode the \(\beta\) and \(\beta^{\prime}\) subunits of the  
Figure 1: Effect of ppGpp on the expression of the _hlyCABD\({}_{\Pi}\)_ operon of J96. (**A**) Bacterial-induced cytotoxicity on T24 cells was monitored as lactate dehydrogenase (LDH) activity released (OD\({}_{490\text{nm}}\)) after 3 h incubation with bacterial cell suspensions (black bars) or cell-free supernatants (white bars).