## 3 Materials and Methods
### Cloning and PCR Methods  
To generate a transcriptional fusion between the _clpBLi_ promoter region (500 bp upstream of the first ATG codon) and luciferase reporter genes, the _V. harveyi luxAB_ genes were amplified from pLucVh [21] by PCR using Pfu Turbo DNA polymerase (Agilent Technologies/Perlan Technologies, Warsaw, Poland) with appropriate primers (see Table 3). First, the PCR product was cloned into a pJET1.2 blunt vector, then digested with appropriate restriction enzymes and ligated with the linearized pGB2 _Ndel-Hind_III vector and the _clpBLi_ promoter region to produce the _clpBLiP1-luxAB_ fusion. The sequence of the resulting construct was confirmed by DNA sequencing (Genomed S.A., Warsaw, Poland). To construct a negative control reporter plasmid (pSD\({}_{\lambda 1}\)_luxAB_; promoter-less _luxAB_), _V. harveyi luxAB_ genes were amplified together with the Shine-Dalgarno sequence of _clpBLi_ from  
Figure 6: Transcriptional activity of the _clpBLi_ gene promoter is dependent on ppGpp during the entry of bacteria into the stationary phase. _E. coli_ wild-type (MG1655; ppGpp\({}^{+}\)) and _NrelAAspoT_ mutant (ppGpp\({}^{0}\)) cultures carrying p_clpBLiP1-luxAB_ along with _LIC_10144_ or _LIC_12757_ genes were grow in LB at 30 \({}^{\circ}\)C to OD\({}_{\rm 600\,nm}\) of 0.45–0.5 and expression of the _LIC_ genes encoding ECF \({}^{\rm E}\) was induced by addition of 0.02% arabinose for 1 h (for mid-exponential-phase culture) or 6 h (for early-stationary-phase culture), and then the luciferase reporter assay was carried out. The luciferase activity (relative luminescence units/OD600 nm) in the _E. coli_ ppGpp\({}^{+}\) cells during exponential phase is taken as 100%; bars represent the percentage of luciferase activity normalized to that of _E. coli_ ppGpp\({}^{+}\) cells during exponential phase; results are presented as the average of three independent experiments, each performed with duplicate cultures, with standard deviations indicated. The paired _t_-test result: \({}^{**}\)\(p<0.01\) and \({}^{***}\)\(p<0.001\) calculated with GraphPad Prism software.  
pclpB\({}_{Li}\)P1-luxAB by PCR using Pfu Turbo DNA polymerase (Agilent Technologies) with appropriate primers (see Table 3). Next, the same cloning procedure was used as described above.  
To study the _clpB\({}_{Li}\)_ expression in _E. coli_ MC4100\(\Delta\)clpB cells, _clpB\({}_{Li}\)_ (2583 bp) and its promoter region (500 bp) were amplified by PCR from genomic DNA of _L. interrogans_ (extracted with a QIAamp DNA mini kit (Qiagen, Germantown, MD, USA), by using Pfu Turbo DNA polymerase (Agilent Technologies) with appropriate primers (see Table 3). Subsequently, the same cloning procedure was used as described above for the construction of transcriptional fusion.  
To clone leptospiral genes encoding ECF \(\sigma\) factors, _LIC_10144_, _LIC_10559_, and _LIC_12757_, into pAC7 under control of a pBAD promoter, the appropriate fragments of genomic DNA isolated from _L. interrogans_ were amplified by PCR using Pfu Turbo polymerase and appropriate primers (see Table 3). Then, the same cloning procedure was used as described above for the construction of transcriptional fusion.