## 4 Materials and Methods
### Bacterial Strains, Plasmids and Media
In this library, the expression of an individual gene is driven from the tightly regulated IPTG-inducible P\({}_{\text{T5}}\)-_lac_ promoter. The concentration of IPTG used in this study has been optimized to provide a mild induction of genes without any toxicity [19]. Cultures of such Ts\({}^{+}\) colonies were grown to obtain plasmid DNAs and used to retransform parental _lapC190_ mutant bacteria. Transformants were plated on growth medium in the presence of IPTG at 43 \({}^{\circ}\)C and 43.5 \({}^{\circ}\)C, and plasmids which allowed restoration of growth were retained. DNA insert of all relevant plasmids that restored the growth upon retransformation on LA-rich medium at 43.5 \({}^{\circ}\)C was sequenced. The Isolation of Chromosomal Transposon Insertion Mutations That Prevent the Suppression by Overexpression of Either the marA Gene or the _srrA_ Gene  
As overexpression of either the _marA_ gene or the _srrA_ gene restore the growth of _lapC190_ bacteria, chromosomal Tn_10_ transposon insertions were isolated to understand the mechanism of their suppression as previously described [36]. Thus, the strains SR23660 and SR23951 _lapC190_ derivatives, which can grow at 43.5 \({}^{\circ}\)C carrying either the _marA_ gene or the _srrA_ gene, respectively, were used as a host to isolate Tn_10_ insertions. In these strains, suppressing _marA_ and _srrA_ genes are cloned in pCA24N vector with expression regulated by P\({}_{\text{T5}}\)-_lac_ promoter. For the mutagenesis, SR23660 (_lapC190 + pmarA\({}^{+}\)_) and SR23951 (_lapC190 + psrrA\({}^{+}\)_) isogenic strains served as recipients using \(\uplambda\)Tn_10_ kan bacteriophage to obtain random saturated transposon insertions. More than 50,000 transposon insertion mutants were isolated in each strain background in LA medium at 30 \({}^{\circ}\)C (permissive growth conditions) as described previously [36]. Transductants were screened for a Ts phenotype on LA medium at 43.5 \({}^{\circ}\)C supplemented by 75 \(\upmu\)M IPTG to induce the expression \begin{table}
\begin{tabular}{l l l} \hline \hline
**Plasmids** & **Genotype** & **Reference** \\ \hline pCP20 & ts replicon with inducible FLP recombinase & [70] \\ pSR23599 & _lapD\({}^{+}\)_ in pDUCT & This study \\ pSR22189 & _dksA\({}^{+}\)_ in pBR322 _lacI\({}^{\text{q}}\)_ tet\({}^{\text{S}}\) amp\({}^{\text{R}}\) & [36] \\ pSR22505 & _dksA\({}^{+}\)_ D74N in pBR322 _lacI\({}^{\text{q}}\)_ tet\({}^{\text{S}}\) amp\({}^{\text{R}}\) & [36] \\ pSR22511 & _dksA\({}^{+}\)_ R82Y in pBR322 _lacI\({}^{\text{q}}\)_ tet\({}^{\text{S}}\) amp\({}^{\text{R}}\) & [36] \\ pSR22519 & _dksA\({}^{+}\)_ S83A in pBR322 _lacI\({}^{\text{q}}\)_ tet\({}^{\text{S}}\) amp\({}^{\text{R}}\) & [36] \\ pSR22498 & _dksA\({}^{+}\)_ L84A in pBR322 _lacI\({}^{\text{q}}\)_ tet\({}^{\text{S}}\) amp\({}^{\text{R}}\) & [36] \\ pSR22525 & _dksA\({}^{+}\)_ E85A in pBR322 _lacI\({}^{\text{q}}\)_ tet\({}^{\text{S}}\) amp\({}^{\text{R}}\) & [36] \\ JW0141 & _dksA\({}^{+}\)_ in pCA24N cm\({}^{\text{R}}\) & [33] \\ JW0844 & _artJ\({}^{+}\)_ in pCA24N cm\({}^{\text{R}}\) & [33] \\ JW0976 & _gnsA\({}^{+}\)_ in pCA24N cm\({}^{\text{R}}\) & [33] \\ JW1044 & _qcycl\({}^{+}\)_ in pCA24N cm\({}^{\text{R}}\) & [33] \\ JW1080 & _acpP\({}^{+}\)_ in pCA24N cm\({}^{\text{R}}\) & [33] \\ JW2497 & _yfgM\({}^{+}\)_ in pCA24N cm\({}^{\text{R}}\) & [33] \\ JW3223 & _accB\({}^{+}\)_ in pCA24N cm\({}^{\text{R}}\) & [33] \\ JW3440 & _acpT\({}^{+}\)_ in pCA24N cm\({}^{\text{R}}\) & [33] \\ JW3794 & _pldA\({}^{+}\)_ in pCA24N cm\({}^{\text{R}}\) & [33] \\ JW5178 & _ymgG\({}^{+}\)_ in pCA24N cm\({}^{\text{R}}\) & [33] \\ JW5249 & _marA\({}^{+}\)_ in pCA24N cm\({}^{\text{R}}\) & [33] \\ JW5539 & _lapD\({}^{+}\)_ in pCA24N cm\({}^{\text{R}}\) & [33] \\ pSR14857 & _srrA\({}^{+}\)_ in pCA24N cm\({}^{\text{R}}\) & This study \\ pEB797 & pBAD-CBP-ACP (S36T) & [71] \\ \hline \hline \end{tabular}
\end{table} 
Table 6: _Cont._of _marA_ and _srrA_ genes. Transductants that were unable to grow at 43.5 \({}^{\circ}\)C were retained and a bacteriophage P1 was grown on them individually.