## 2 Results and Discussion
### ppGpp Is Required for the Thermogulation but Not for the Osmoregulation of _hlyCABDII_ Expression  
The expression of the \(\alpha\)-hemolysin in the conjugative plasmid pHy152 is affected by environmental parameters such as temperature and osmolarity of the culture media [31]. Experiments were performed to determine whether the expression of the _hlyCABDII_ operon was equally thermo- and osmo-regulated, and to describe a possible role of ppGpp in the environmental regulation. As seen in Figure 5, \(\alpha\)-hemolysin expression, monitored as the amount of HlyA present in the external fraction, is regulated by both temperature and external osmolarity. The toxin was barely detectable in cultures grown at low temperature (22 \({}^{\circ}\)C), a significant increase was detected when growing at 30 \({}^{\circ}\)C, and the maximal expression was detected at 37 \({}^{\circ}\)C (Figure 5, left panel). The ppGpp-deficient strain did not grow at 22 \({}^{\circ}\)C, even after a prolonged incubation period. At 30 \({}^{\circ}\)C, the amount of \(\alpha\)-hemolysin was significantly lower compared to the Wt strain, and no further induction was observed when the incubation temperature was raised to 37 \({}^{\circ}\)C (Figure 5, left panel). Regarding the osmolarity, the \(\alpha\)-hemolysin expression is induced at low osmolarity (LB with 0 M NaCl) in both ppGpp-proficient and -deficient strains (Figure 5, right panel). These data indicate that ppGpp is required for the thermoregulation of \(\alpha\)-hemolysin expression, but not for its osmoregulation. ppGpp-Deficiency Causes a Drastic Drop in the \(\alpha\)-Hemolysin Production in the Uropathogenic Isolate J96  
As previously indicated, the _hlyCABD\({}_{II}\)_ operon is located in a pathogenicity island of the pyelonephritis isolate J96 [17; 18]. Experiments were conducted to confirm that the ppGpp regulation described also occurs in the natural genetic background of the _hlyCABD\({}_{II}\)_ operon. Cytotoxicity assays on T24 cells were performed with LB cultures from J96 and its ppGpp-deficient derivative and cell-free supernatants from these cultures (Figure 6A). The data obtained indicate that the cytotoxicity is severely impaired in the ppGpp\({}^{0}\) mutant derivative compared to Wt. Moreover, the amount of secreted \(\alpha\)-hemolysin was about 10-fold lower in ppGpp\({}^{0}\) compared to Wt in cell-free supernatants, as seen by Coomassieie staining, after SDS-PAGE (Figure 6B). The J96 strain carries two _hlyCABD_ operons, the extensively studied _hlyCABD\({}_{II}\)_ operon, and the _hlyCABD\({}_{I}\)_ operon, which has been poorly characterized. The coding sequences from both operons are almost identical, whereas the sequences upstream of _hlyC_ carrying the promoter and regulatory motifs clearly diverge between both operons [28]. The two operons respond differently to specific environmental inputs, suggesting a specific role at distinct stages during the infection process [18; 28]. The expression from the two _hlyCABD_ operons present in the strain J96 was studied, using deletion mutants of the _hlyA\({}_{I}\)_ and _hlyA\({}_{II}\)_ genes, and monitoring the amount of external toxin produced in both Wt and ppGpp\({}^{0}\) strains (Figure 6). ppGpp is required for the appropriate expression of the _hlyA_ gene from both _hlyCABD_ operons of the J96 strain. A reduction in the production of \(\alpha\)-hemolysin from both hemolytic operons was detected in the corresponding ppGpp\({}^{0}\) derivatives. Despite the disparity of the regulatory regions, the expression of the \(\alpha\)-hemolysin from both operons is stimulated by ppGpp, indicating a central role for this second messenger in the expression of this toxin. Interestingly, under the culture conditions used, there is apparently higher production of \(\alpha\)-hemolysin from the _hlyCABD\({}_{II}\)_ operon. The downregulation of the hemolysis production from the _hlyCABD\({}_{II}\)_ operon in both commensal and  
Figure 5: Thermo- and osmo-regulation of the _hlyCABD\({}_{II}\)_ operon.