## 2 Results
### SrrA Does Not Regulate Transcription of the clsA Gene  
SrrA is predicted to act as a transcriptional regulator since it contains an N-terminal signal recognition Per-Arnt-Sim (PAS) domain and a C-terminal helix-turn-helix motif [36]. However, the genes whose expression is regulated by SrrA are unknown. As described above, the overexpression of the _srrA_ gene efficiently suppresses the Ts phenotype of _lapC190_ bacteria and also either of the strains collectively lacking six cytoplasmic peptidyl-prolyl _cis/trans_ isomerases or of \(\Delta\)_lapD_ bacteria [28; 36]. Since SrrA-mediated suppression \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Recipient**} & \multicolumn{2}{c}{**P1 Donor \(\Delta\)_clsA_**} \\ \cline{2-3}  & **30 \({}^{\circ}\)C** & **43 \({}^{\circ}\)C** \\ \hline wt & 840 & 759 \\ _lapC190_ + vector alone & 3 & - \\ _lapC190_ + psrrrA\({}^{+}\) with IPTG & 331 small colonies & 38 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: The _srrA_-mediated multicopy suppression of _lapC190_ bacteria requires the presence of cardiolipin synthase A encoded by the _clsA_ gene. requires the presence of a functional _clsA_ gene, we undertook studies to address if SrrA acts by regulating transcription of the encoding gene. Thus, total RNA was extracted from the wild type and its isogenic derivative _AsnrA_ from cultures grown at 37 \({}^{\circ}\)C. In parallel, total RNA was also extracted from wild-type bacteria carrying either an empty vector or expressing the _srrA_ gene from an inducible promoter. For such experiments, cultures were grown at 30 \({}^{\circ}\)C. Prior to heat shock, 75 uM IPTG was added to induce transcription of the _srrA_ gene, and cultures were shifted to 43 \({}^{\circ}\)C for 15 min. The equivalent amount of total RNA from these four sets of RNAs was next subjected to q-RT-PCR analysis using _clsA_-specific oligonucleotides for the synthesis and quantification of cDNA. The quantification of the _clsA_ transcription pattern showed nearly similar levels of _clsA_ transcripts between the wild-type and _AsnrA_ bacteria at 37 \({}^{\circ}\)C (Figure 8). Similarly, when the _srrA_ gene expression was induced and when RNA was extracted from 43 \({}^{\circ}\)C and the relative abundance of transcripts were estimated by q-RT-PCR, no significant differences were observed in the _clsA_ expression with or without _srrA_ induction (Figure 8). Although a minor increase in _clsA_ mRNA can be observed upon shift to 43 \({}^{\circ}\)C as compared to the relative abundance at 37 \({}^{\circ}\)C, such an increase is not comparable to that of heat shock genes. Thus, results from q-RT-PCR allow us to conclude that the _clsA_ transcription is not regulated by SrrA either at 43 \({}^{\circ}\)C or at 37 \({}^{\circ}\)C, and overexpression of the _srrA_ gene or its absence does not alter the abundance of _clsA_ transcripts. LapD-Mediated Suppression of a lapC190 Mutant Bacteria Requires LapD N- and C-Terminal Domains and Identification of Some of the Critical Amino Acid Residues Required for LapD Function  
As overexpression of the _lapD_ gene restored the growth of _lapC190_ mutant bacteria at a high temperature, we sought to address if specific domains of LapD are required for its function. LapD has a single N-terminal transmembrane anchor and a highly conserved C-terminal domain. The crystal structure of the LapD counterpart in _Haemophilus ducreyi_ shows a unique tetrameric \(\alpha\)-helical coiled-coil structure in the cytosolic domain, which is predicted to mediate protein-protein interactions [50]. Thus, a series of point mutations (substitution of conserved residues by Ala), deletion of the N-terminal membrane anchor (1-20 amino acids) and deletion of the C-terminal last 32 amino acids were constructed. Among the Ala substitutions, we replaced conserved amino acids D69, Y70 and R71 with  
Figure 8: Transcription of the _clsA_ gene is not regulated by SrrA.