## 2 Results and Discussion
### ppGpp Stimulates the Transcriptional Expression of the hly_CABD_\({}_{II}\)_Open_  
Transcription from the _hly_\(CABD\) operons generates two transcripts, _hlyCA_ and _hly-CABD_, through transcriptional termination occurring between _hlyA_ and _hlyB_[25]. Transcriptional studies by qPCR were performed to determine the levels of _hlyA_ and _hlyD_ transcripts from the _hlyCABD_\({}_{II}\) operon (Figure 3). An obvious reduction for both transcripts was detected in the ppGpp\({}^{0}\) strain as compared to Wt, being more pronounced for the _hlyA_ transcript. These results indicate that ppGpp stimulates the transcriptional expression of the _hlyCABD_\({}_{II}\) operon. The differential effect of ppGpp on the expression of _hlyA_ and _hlyD_ suggest that ppGpp affects somehow the termination occurring in the internal terminator. The ppGpp-Mediated Regulation of the hlyCABD\({}_{II}\) Operon Is Independent of RpoS, RfaH, Zur, and H-NS  
ppGpp promotes the expression of RpoS-dependent genes by stimulating both _rpoS_ expression and the interaction of RpoS with the RNA polymerase holoenzyme [2]. RpoS-deficiency did not cause any significant reduction in the \(\alpha\)-hemolysin expression from the  
Figure 2: Analysis of the hemolytic phenotype of the ppGpp\({}^{0}\) suppressor mutant _rpoB3370_. (**A**) Hemolytic phenotype on blood agar plates of Wt (AAGI), ppGpp\({}^{0}\) (IFY2), _rpoB3370_ (JFV19), and ppGpp\({}^{0}\)_rpoB3370_ (JFV20) carrying the plasmid pSF4000. (**B**) Electrophoretic analyses (Coomassieie blue stained 10% SDS-PAGE) of the cell-free supernatants from bacterial cultures grown in LB at 37 \({}^{\circ}\)C up to late exponential phase (OD\({}_{600\text{nm}}\) of 0.8) of the same strains as in (**A**). Relative intensities of the protein bands are indicated at the bottom of the panel. Lane M: molecular mass markers (size in kDa indicated along the left side). _hlyCABDII_ operon as compared to the Wt strain. Moreover, the absence of ppGpp causes a reduction in the production of \(\alpha\)-hemolysin in both _rpoS_ and _rpoS\({}^{+}\)_ strains (Figure 4A), indicating that _hlyCABDII_ expression is RpoS-independent. Figure 4: ppGpp-mediated stimulation of _hlyCABDII_ expression is independent of RpoS, RfaH, and H-NS. Electrophoretic analyses (Coomassie blue stained 10% SDS-PAGE) of cell-free bacterial culture supernatants of ppGpp-proficient and ppGpp-deficient derivatives of _rpoS_ (**A**), _rfaH_ (**B**), _zur_ (**C**), and _lns_ (**D**) and its corresponding Wt counterparts (AAG1/pSF4000 for _rpoS_ and _lns_ analyses and AAG1/pMGP-1 for _rfaH_ and _zur_ analyses). Cultures were grown in LB at 37 \({}^{\circ}\)C up to late exponential phase (OD600 nm of 0.8). Lane M: molecular mass markers (size in kDa indicated along the left side). The relative average intensity of the bands is indicated at the bottom of the panel. Figure 3: ppGpp stimulates the transcriptional expression of the _hlyCABDII_ operon from J96. Relative _hlyA_ and _hlyD_ mRNA quantification by qPCR in Wt (AAG1) and ppGpp\({}^{0}\) (JFV23) strains carrying plasmid pMGP-1 (pBR322 based plasmid carrying the _hlyCABDII_ operon of J96). RNA samples were extracted from cultures grown in LB at 37 \({}^{\circ}\)C up to late exponential phase (OD600 nm of 0.8). The results are mean and the standard deviation from three biological repeats. The transcriptional antiterminator RfafH regulates _hlyCABDII_ expression [26]. Hence, _rfuH_ mutation causes a dramatic reduction in the expression of the \(\alpha\)-hemolysin (Figure 4B). Nevertheless, the _rfaH_ strain was still responding to ppGpp since a significant reduction was observed in the ppGpp-deficient strain when _rfaH_ was removed. To corroborate the accumulative effect of ppGpp\({}^{0}\) and the _rfaH_ mutation, _hlyA_ mRNA levels were determined by qPCR. Consistent with previously published data [27], a clear reduction in the level of _hlyA_ transcript was observed in the _rfaH_ mutant strain (5-fold reduction).