## 2 Results and Discussion
### Identification of the \(clpB\) Transcriptional Start Sites by Primer Extension
A modified two-plasmid system has been employed that has been previously successfully used for the identification of many \(\sigma^{\rm E}\)-cognate promoters from _E. coli_ and also from other bacteria in studies that demonstrated the suitability of this system for assessing promoter activity in _E. coli_[23, 24]. Briefly, this system uses two compatible plasmids. One of them is pAC-_rpoE4_ carrying the _E. coli rpoE_ gene under the control of an arabinose inducible pbad promoter, and the second plasmid carries a reporter gene fused to a potential \(\sigma^{\rm E}\) promoter. We used the _V. harveyi luxAB_ gene, encoding luciferase, as a reporter cloned into a low-copy pGB2 plasmid under control of the tested \(clpB_{Li}P1\) promoter (\(pclpB_{Li}P1\)-\(luxAB\)). The method used assumes that shortly after arabinose addition \(\sigma^{\rm E}\) factor overproduced from pAC-_rpoE4_ interacts with the _E. coli_ RNA polymerase core enzyme forming functional RNA polymerase holoenzyme that is capable of recognizing \(\sigma^{\rm E}\) -controlled promoters located upstream of a reporter gene carried on the second plasmid; in our study, it was the \(pclpB_{Li}P1\)-\(luxAB\) plasmid carrying the \(luxAB\) reporter genes. As shown in Figure 3, the activity of luciferase in both, the exponential and stationary phase of bacterial growth was indeed significantly higher in the presence of the pAC-_rpoE4_ plasmid expressing the _E. coli rpoE_ (i.e., under \(\sigma^{\rm E}\) excess conditions) than that in cells transformed only with \(pclpB_{Li}P1\)- \(luxAB\) and producing a basal level of \(\sigma^{\rm E}\). Figure 2: Nucleotide sequence alignment of the well-characterized _E. coli_\(\sigma^{\rm E}\)-dependent promoters and the potential \(clpB_{Li}\) promoter (\(clpB_{Li}P1\)). The two transcriptional start sites are marked in bold and underlined, and the major transcriptional start site is denoted by +1. The consensus sequence for the \(\sigma^{\rm E}\)-regulated promoter element is shown below the alignment. Importantly, no significant luminescent signal was detected for cells carrying a construct with promoter-less _luxAB_ genes. These results strongly suggested that the activity of _clpBLiP1_ is dependent on \(\sigma^{\rm E}\). Furthermore, we examined _clpBLi_ expression driven by the _clpBLiP1_ promoter in _E. coli_ cells under heat shock exposure at 42 \({}^{\circ}\)C and also under \(\sigma^{\rm E}\) excess conditions (in the presence of PAC-_rpoE4_). Thus, the main aim of this experiment was to compare the _clpBLiP1_ promoter behavior under thermal stress and \(\sigma^{\rm E}\) overproduction, by monitoring the amount of ClpBLi that was produced. Such an approach was impossible in case of experiments with _pclpBLiP1-luxAB_ because luciferase is thermolabile and would not withstand the heat shock conditions. To this end, Western blotting analysis of whole-cell lysates of the _E. coli_ MC4100\(\Delta\)_clpB_ strain carrying pClpBLi alone or together with pAC-_rpoE4_ was carried out using anti-ClpBLi158-334 serum [15]. Expression of _E. coli rpoE_ was induced by the addition of 0.02% arabinose for 30-90 min, while the expression of pClpBLi was induced by exposure to 42 \({}^{\circ}\)C for 1 h. As shown in Figure 4A, thermal stress was not sufficient to induce the ClpBLi production (96-kDa protein) in the cells carrying the _clpBLi_ gene under the control of the _clpBLiP1_ promoter (see lanes 5 and 6). This is in agreement with our results of primer extension analysis (see Figure 1A) that did not show the presence of the _clpBLi_ transcripts in _E. coli_ cells after heat shock stress. Consistent with our previous results [25], the use of pGB2-ClpBLi [25], which carries the heat-inducible promoter \(\sigma^{\rm 32}\) from _E. coli_ located upstream of _clpBLi_, resulted in increased synthesis of ClpBLi after heat treatment (see lanes 3 and 4). The most interesting observation pertains to the _clpBLi_ expression under \(\sigma^{\rm E}\) excess conditions (Figure 4B), as the appearance of the 96-kDa ClpBLi in the _E.