## 2 Results and Discussion
### Identification of the \(clpB\) Transcriptional Start Sites by Primer Extension
expression in _Leptospira_ is induced by temperature stress, which is consistent with the findings of [15], who demonstrated that _clcpB\({}_{Li}\)_ transcription in _Leptospira_ had increased by 3-folds after 2 h exposure to heat stress. One possible candidate regulating this expression could be RopoH (\(\alpha\)[32]; heat shock sigma factor); however, comparative genomics and genome-wide in silico analyses had shown no presence of a _rpoH_ gene in the _Leptospira_ genome [9; 10]. Thus, _Leptospira_ spp. does not possess \(\alpha\)[32] that is required for heat shock gene expression and heat shock response in _E. coli_. On the other hand, as shown in Figure 1, for RNA extracted from the _E. coli_ cells, no extension product was detectable with the p51-72 primer, indicating that _E. coli_ either does not possess an appropriate factor for _clpB\({}_{Li}\)_ expression or this factor is not induced in _E. coli_ by temperature stress. Additional analysis has been performed with two other primers. Primer extension assay utilizing the P290-321 primer did not detect any transcripts either with _Leptospira_ or with _E. coli_ RNA (not shown), while the use of the p565-591 primer revealed a few weak heat-inducible transcripts for _Leptospira_ and one transcript for _E. coli_ RNA that was not induced by heat shock stress and did not coincide with any leptospiral transcription start sites (Figure 1). All of these transcripts initiate within the region that is 20-96 nt upstream of a potential internal translation initiation site of _clpB\({}_{Li}\)_ with a good Shine-Dalgarno sequence (AGGGAA) and the ATG codon (see Figure 1). It is known that _E. coli_ uses two translational start sites to produce two isoforms of ClpB, the full-length ClpB95 and shorter ClpB80, which does not contain the substrate-interacting N-terminal domain [18; 20; 21]. It has been demonstrated that the two isoforms of ClpB from _E. coli_ cooperate in reactivation of aggregated proteins to form a highly efficient chaperone system [21]. It is very likely that the functional cooperation between ClpB isoforms arises from interactions between them because it was found that ClpB95 and ClpB80 associate into hetero-oligomers, which boost the aggregate-reactivation potential of the ClpB chaperone [21]. Thus, it was postulated that _E. coli_ produces two isoforms of ClpB to optimize its disagregase activity. The presence of two forms of ClpB, a 96-kDa and an 80-kDa protein, was also demonstrated in _L. interrogans_[15]. Our results point to the occurrence of an additional transcriptional start site located within the _clpB\({}_{Li}\)_ coding region in both _E. coli_ and _Leptospira_. Still, it is intriguing that even though the internal _clpB\({}_{Li}\)_ transcriptional start sites were detected in both instances, they differ depending on the host. It is also interesting to note the similarity between one of the internal transcriptional start sites (see Figure 1, gagfItt) that we mapped by primer extension analysis and one of the sixteen internal start points (gagfItt) previously predicted by [10]. Thus, it is highly possible that _clpB\({}_{Li}\)_ has an additional promoter. On the other hand, it cannot be excluded that the mapped minor transcripts result from the processing of the major transcript produced. Still, further studies are needed to examine the nature of those minor transcripts in _Leptospira_.