## 2 Results and Discussion
### Identification of the \(clpB\) Transcriptional Start Sites by Primer Extension
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Oligonucleotide** & **Sequence (5’ to 3’)** & **Purpose** \\ \hline P51-72 & ATITTCCGACAGCCGCTTGTGC & primer extension \\ P290-321 & AAGATACTCGCTTTTAAATTCTTCTTACT & primer extension \\ P565-591 & ACAGCATCAATTTCCTTGTTGGGC & primer extension \\ _luxANdel_ & CATATGAAATTTGAAAAACTCTTCTC & cloning of luciferase reporter genes \\ _luxBrintIII_ & CGACACAAACTTCAGGTATTTGACGATG & cloning of luciferase reporter genes \\ _SD\({}_{Li}\)LuxCAM_ & CCCGGACACATTATTTAGAAAAAGACT & cloning of luciferase reporter genes \\ _clpB\({}_{Li}\)Ndel_ & CATATAGAAATTGAAAAACTACAAATT & cloning of _clpB\({}_{Li}\)_ \\ _clpB\({}_{Li}\)HindIII_ & AAGCTTTTTAAACTACAACAACTACTCTTCCCT & cloning of _clpB\({}_{Li}\)_ \\ _prLiXmI_ & CCCGGATAAAATTCCGACTCCGATT & cloning of _clpB\({}_{Li}\)_ promoter region \\ prLIC\({}_{1014}\)_Ndel_ & CATATGTTCAATCTGATTCTC & cloning of LLC\({}_{10144}\) \\ prLIC\({}_{1014}\)_AtIn_ & AAGCTTAGATGAATTGAATTCGTTAG & cloning of LLC\({}_{10144}\) \\ prLIC\({}_{10559}\)_Ndel_ & CATATGATGCCAATTCC & cloning of LLC\({}_{10559}\) \\ prLIC\({}_{10559}\)_HindIII_ & AAGCTTTTTCATTATAAAATTTTCTC & cloning of LLC\({}_{10559}\) \\ prLIC\({}_{12757}\)_Ndel_ & CATATGACCAAAATTCCGAAAC & cloning of LLC\({}_{12757}\) \\ prLIC\({}_{12757}\)_HindIII_ & AAGCTCTCTATATATACTCAAAGCTG & cloning of LLC\({}_{12757}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Oligonucleotides used in this study. Presence of a Promoter Dependent on \(\sigma^{E}\) Upstream of the ClpB\({}_{Li}\) Gene and its Activity in E. coli Cells  
After the mapping of the two \(clpB_{Li}\) transcriptional start sites (both probably originating from the same promoter) in _Leptospira_ (see Figure 1), the nucleotide sequence upstream of \(clpB_{Li}\) was carefully investigated. A putative \(\sigma^{\rm E}\) promoter element was successfully identified on the basis of sequence similarity to the well-characterized _E. coli_\(\sigma^{\rm E}\) core promoters. As shown in Figure 2, sequences of the \(-10\) and \(-35\) motifs of the potential \(clpB\) promoter (\(clpB_{Li}P1\)) exhibit a considerable homology to the known promoters controlled by _E. coli_\(\sigma^{\rm E}\). Figure 1: Determination of \(clpB_{Li}\) transcriptional start sites by primer extension assays. (**A**) Primer extension results for the P\({}_{565-591}\) (left) and P\({}_{51-72}\) (right) primers, and the corresponding sequencing ladders. Arrows indicate mapped transcriptional start sites; closed arrowheads—_L. interrogans_, opened arrowheads—_E. coli_. The temperature at which bacteria were grown prior to RNA isolation is indicated (28, 30, 37, or 42 \({}^{\circ}\)C). (**B**) \(clpB_{Li}\) promoter and coding region with the \(clpB_{Li}P1\) promoter region underlined; transcriptional start sites are indicated by arrows as in (A), primer annealing sites and putative _rbs_ sequences are also annotated; \(clpBLi\)-FL: start codon yielding ClpB95; \(clpBLi\)-short: internal ATG site producing ClpB80. To investigate whether the \(\sigma^{\rm E}\) factor may be really involved in the \(clpB_{Li}\) transcriptional regulation, promoter activity assay was carried out under \(\sigma^{\rm E}\)-limiting (basal) and \(\sigma^{\rm E}\) excess (overproduction) conditions. It is worth to emphasize that under normal growth conditions, the expression level of _rpoE_ in _E. coli_ cells is low. Therefore, it was of great interest to compare the activity of the potential \(clpB_{Li}\) promoter (\(clpB_{Li}P1\)) under basal and increased levels of \(\sigma^{\rm E}\). To our knowledge, there has been only one report to date, describing the construction and utility of the _gfp_ reporter plasmid for assessing promoter activity in _L. interrogans_[22]. Therefore, due to the still existing limitations of genetic tools that could be easily utilized for examining gene expression in _L. interrogans_, experiments were performed in _E. coli_ cells.