## 2 Results
### SrrA Does Not Regulate Transcription of the clsA Gene
qRT-PCR analysis of mRNA isolated from wild-type bacteria, its isogenic derivative _AsnrA_, grown up to an OD\({}_{595}\) of 0.2 in LB medium at 37 \({}^{\circ}\)C. For heat shock experiments isogenic cultures of wild type with empty vector and _lapC190_ bacteria carrying the inducible _srrA_ gene on a plasmid were grown at 30 \({}^{\circ}\)C in M9 minimal medium up to an OD\({}_{995}\) of 0.2 and supplemented with 75 uM IPTG prior to a 15-min shift to 43 \({}^{\circ}\)C. Data presented are from RNA isolated from three biological replicates and error bars are indicated. three Ala residues. Similarly, L84, L85 and P86 and N93, P94 and P95 were also substituted with three Ala residues in each case. All generated constructs with in-frame mutations were expressed under a tightly regulated _ptac_ promoter and, when required, in-frame with an N-terminal hexa-His tag, where the expression is driven from a controlled T7 promoter. Plasmids where the expression is driven from the _ptac_ promoter were transformed either into the \(\Delta\)_lapD_ strain or in _lapC190_ bacteria and evaluated for the ability to suppress their Ts phenotype. Results from such experiments show that while the control plasmid overexpressing the wild-type _lapD_ gene restores growth of _lapC190_ at elevated temperature, neither the N-terminal nor the C-terminal deletions were able to suppress the Ts phenotype when the expression was induced by the addition of 75 \(\upmu\)M IPTG (Figure 9). Similarly, three plasmid sets expressing replacement by Ala substitutions of amino acids (69-71), (84-86) and (93-95) were also unable to restore the growth of _lapC190_ bacteria at elevated temperatures (Figure 9). These results allow us to conclude that specific conserved residues in the cytoplasmic domain are essential for LapD function. Furthermore, the single N-terminal membrane anchor and last 32 amino acid residues of the cytoplasmic domain are required for the _lapD_ gene to act as a multicopy suppressor. Multicopy Suppression of _lapC190_ Bacterial Defects Requires DksA's PPIase and Transcriptional Activities  
The results presented in the above sections have shown that overexpression of the global transcriptional regulator encoded by the _dksA_ gene can restore the bacterial growth of _lapC190_ bacteria at elevated temperatures and also suppress LpxC degradation defects. We have earlier shown that DksA also suppresses the Ts phenotype of \(\Delta\)_6 ppi_ bacteria and \(\Delta\)_lapD_ bacteria [28; 36]. Characterization of the biochemical properties of DksA further revealed that besides acting as a global transcriptional regulator, it also exhibits the PPIase activity, which can be inhibited by the FK506 macrolide [36]. Several studies have led to the conclusion that the coiled-coil domain of DksA inserts into the secondary channel of RNAP and that residues at the tip of the coiled-coil domain of DksA are important for its activity [51; 52; 53]. More recent studies suggest that amino acid residues in this coiled-coil domain play an essential role for the transcriptional and PPIase activities of DksA [36]. Thus, to examine which activity of DksA is required for the suppression of growth defects of _lapC190_ bacteria, cloned DNA of D74N, F82Y, S83A, L84A and E85A variants of the  
Figure 9: The N-terminal membrane anchor of LapD and its conserved amino acid residues in the C-terminal domain are essential for the restoration of the growth of _lap190_ bacteria at 43.7 \({}^{\circ}\)C. In-frame deletion of N-terminal 20 amino acid residues, \(\Delta\)N20, deletion of last 32 amino acid residues \(\Delta\)100-132 and a triple Ala replacement of conserved amino acid were constructed by gene synthesis and cloned in expression vector. Plasmid DNA with the wild-type _lapD_ gene and plasmids DNAs with various substitutions or deletions in the _lapD_ gene were used to transform _lapC190_ bacteria.