## 2 Results and Discussion  
### Identification of the \(clpB\) Transcriptional Start Sites by Primer Extension  
In order to determine \(clpB\) transcriptional start sites and identify functional promoters responsible for its expression in _Leptospira_, primer extension assays were performed. Three synthetic oligonucleotides, p51-72, p290-321, p565-591 (see Table 3) were hybridized to RNA extracted from both, _Leptospira interrogans_ and _E. coli_ MC4100\(\Delta clpB\) cells carrying the _L. interrogans clpB_ gene (\(clpB_{Li}\)) cloned into a low-copy pGB2 plasmid together with a 500-bp DNA fragment containing sequence located upstream of the gene (pClpB\({}_{\rm Li}\)). A BLAST analysis did not show a potential cross-hybridization of the designed primers with regions of the _E. coli_ genome, suggesting high specificity of the designed primers. To prepare RNA, the _L. interrogans_ strain was grown at 28 \({}^{\circ}\)C for optimal in vitro growth and also under thermal stresses at 37 and 42 \({}^{\circ}\)C for 4 and 2 h, respectively, whereas _E. coli_ cells were grown at 30 or 42 \({}^{\circ}\)C for 30 min (heat shock). Following reverse transcription and analysis on a sequencing gel, one major product was observed when the p51-72 primer was used in reactions with RNA isolated from _Leptospira_ cells grown at all tested temperatures (Figure 1). This product corresponds to a transcriptional start site at a cytidine residue located 41 bp upstream of the first \(clpB\) ATG codon (\(-\)41 relative to ATG). In addition, a lesser band is observed at an adenosine residue located at the \(-\)39 position relative to ATG. It is interesting to note that this alternative start point of transcription (A-39) coincides with the primary transcriptional start site (tctAaac) previously predicted by [10] using differential RNA-sequencing (dRNA-seq). It is also worth mentioning that the secondary transcriptional start site (G-526 of the GenBank \(clpB\) gene sequence, accession number M28364) was found to be close to the primary transcriptional start site (A-525) in primer extension analysis of the _E. coli \(clpB\)_ transcripts [18]. It is also known that _Mycoplasma pneumoniae_ reference strain M129 uses two different transcriptional start sites (G-10 and A-7 relative to the ATG initiation codon) to produce ClpB [19]. Thus, the presence of the primary and secondary transcriptional start site in the \(clpB_{Li}\) promoter region is not surprising. Further, the amount of both _Leptospira \(clpB\)_ transcripts is increased after exposure of cells to a temperature of 42 \({}^{\circ}\)C for 2 h. This result indicates that \(clpB_{Li}\) \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Gene ID a** & **Protein Accession** & **Number of Amino** & **Identity/Similarity b** \\  & **Number** & **Acids** & **(\%)** \\ \hline _LLC\_10144_ & AAS68777 & 174 & 26.0/50.3 \\ _LLC\_10225_ & AAS68853 & 301 & 20.8/51.6 \\ _LLC\_10386_ & AAS69009 & 182 & 20.1/55.6 \\ _LLC\_10559_ & AAS69180 & 181 & 28.3/58.7 \\ _LLC\_10644_ & AAS69265 & 174 & 26.8/61 \\ _LLC\_11817_ & AAS70405 & 184 & 26.1/55.7.9 \\ _LLC\_12490_ & AAS71055 & 206 & 32.7/70.8 \\ _LLC\_12757_ & AAS71314 & 180 & 24.0/55.0 \\ _LLC\_13266_ & AAS71810 & 192 & 31.1/63.3 \\ _LLC\_13285_ & AAS71829 & 169 & 27.6/62.9 \\ \hline \hline \end{tabular}  
* Gene ID was based on ORFs of the genome sequence of _L. interrogans_ serovar Copenhagen deposited in GenBank under accession numbers AE016823 (chromosome I) and AE016824 (chromosome II) [12]. b Identity/similarity scores were determined from sequence alignment of the ECF \(\sigma\) factors from _L. interrogans_ serovar Copenhagen and _E. coli_ of c\({}^{\rm E}\) (in GenBank under accession number CDJ72918) using Clustal software. Genes in bold were found to be up-regulated at elevated temperatures [13].  
\end{table} 
Table 2: ECF \(\sigma\) factors (\(\sigma^{\rm E}\)) from _Leptospira interrogans_.