## 2 Results and Discussion
### Role of Leptospiral ECF\(\sigma\) Factors in ClpB\({}_{Li}\) Gene Expression  
It has been demonstrated that \(\sigma^{\rm E}\) contributes to heat shock and oxidative stress response in a number of bacteria and is also involved in the regulation of virulence genes and virulence-associated genes in many bacterial pathogens as well [26]. Since _L. interrogans rpoE_ expression was reported to be up-regulated in _Leptospira_ cells exposed to elevated temperatures, it is possible that leptospiral \(\sigma^{\rm E}\) factors also regulate expression of genes required for virulence or pathogenesis [13]. This correlates well with a specific function of ClpB in _L. interrogans_ and ClpB's importance during infections [15; 17; 25]. Of the ten genes encoding ECF \(\sigma\) (\(\sigma^{\rm E}\)) factors listed in Table 2, _LIC_12757_ (_LA0876_), and _LIC_10559_ (_LA3652_) were found to be 1.5- and 2-fold up-regulated at higher temperatures, respectively [13]. Therefore, we considered their products as potential candidates ECF (\(\sigma^{\rm E}\)) that transcribe _clpB\({}_{Li}\)_ and we evaluated their effect on the transcriptional activity of _clpB\({}_{Li}\)P1_. To this end, we replaced the _E. coli rpoE_ gene in the previously employed two-plasmid system with either _LIC_12757_ or _LIC_10559_ genes. Additionally, _LIC_10144_, which also encodes ECF \(\sigma\) factor (see Table 2) but is not up-regulated under heat shock, was also cloned and used in our assay. As shown in Figure 5, products of the _LIC_12757_ and _LIC_10144_ genes elevated the transcriptional activity of _clpB\({}_{Li}\)P1_ in both the exponential and stationary phase of bacterial growth, supporting our previous observations obtained for \(\sigma^{\rm E}\) from _E. coli_ and indicating that an ECF \(\sigma^{\rm E}\) factor is indeed involved in _clpB\({}_{Li}\)_ regulation (see Figure 3). Of note, _clpB\({}_{Li}\)P1_ displays a much higher activity in the presence of _LIC_12757_. This result suggests that its product may play a key role in _clpB\({}_{Li}\)_ expression, especially under stressful conditions like high temperature. To our surprise, _LIC_10559_, whose expression level in response to thermal stress was reported to be more increased than that of _LIC_12757_[13], did not significantly affect the transcriptional activity of _clpB\({}_{Li}\)P1_. Figure 4: _ClpB\({}_{Li}\)_ expression in _E. coli_ MC4100\(\Delta\)_clpB_[pcDpB\({}_{Li}\)] cells both under heat shock and \(\sigma^{\rm E}\) excess conditions. (**A**) Immunodetection of ClpB\({}_{Li}\) with specific antibodies in bacterial lysates of the _E. coli_\(\Delta\)_clpB_ cells carrying pClpB\({}_{Li}\) (marked as p\(P\)_TclpB\({}_{Li}\)) and grown at 30 \({}^{\circ}\)C, and after 1 h of heat shock at 42 \({}^{\circ}\)C. Cells carrying p\(\sigma^{32}\)-_clpB\({}_{Li}\)_(pGB2-ClpB\({}_{Li}\); [25]) were used as the control for the heat-inducible expression, while the \(\Delta\)_clpB_ mutant cells without a plasmid were the negative control. (**B**) Immunodetection of ClpB\({}_{Li}\) in the lysates of _E. coli_\(\Delta\)_clpB_ [pcDpB\({}_{Li}\), pAC-_rpoE\(4\)] cells grown at 30 \({}^{\circ}\)C both, in the absence and presence of 0.02% arabinose. An asterisk indicates ClpB\({}_{Li}\) (96 kDa), while a short form of ClpB\({}_{Li}\) (80 kDa) is marked by a square. The positions of protein size markers (M), the PageRank prestained protein ladder (Thermo Scientific), are also shown. Interestingly, comparison of promoter transcriptional activity in cells harboring \(pclpB_{Li}P1\)-\(luxAB\) alone or together with \(LIC\_10144\) or \(LIC\_12757\) genes, as well as with _E. coli_ rpoE, revealed that \(clpB_{Li}P1\) is more active in the presence of excess \(\sigma^{\text{E}}\) in the stationary growth phase than in the mid-exponential phase when compared to basal \(\sigma^{\text{E}}\) level (see Figures 3 and 5). Similar effect has been previously observed and carefully investigated for the _E. coli_ rpoH promoter [27; 28] and also for other \(\sigma^{\text{E}}\)-controlled promoters [29].