## 2 Results
### Absence of _mla_ Genes Does Not Alter LPS Composition  
In _E. coli_, up to now, no structural/compositional analysis of LPS has been reported when the Mla system is impaired. To investigate if the deletion derivatives of _mla_ genes cause any major alterations in the LPS composition or in the incorporation of non-stoichiometric modifications, purified LPS obtained from the wild type and its isogenic derivatives were examined using mass spectrometric analysis. For such experiments, LPS was extracted after growth in phosphate-limiting growth medium as described earlier, since such growth conditions are inductive for lipid A and LPS core modifications due to the induction of BasS/R and PhoB/R two-component systems [13; 43]. Examination of mass spectra of LPS obtained from the wild type and isogenic _mla_C and _mlaD_ derivatives shows similar mass peaks corresponding to the presence of complete glycoform I represented by mass peaks at 3936.7 Da and its derivatives with predicted incorporation of additional 1 or 2 P-EtN residues and also modification by L-Ara4N and GlcUA moieties as indicated. These mass peaks can be explained as \(\text{LA}_{\text{hexa}}\) (\(\text{Kdo}_{2}\text{Hep}_{4}\text{Hex}_{4}\text{P}_{2}\)) accompanied by additional substitutions with P-EtN and L-Ara4N, as shown (Figure 7). The mass peaks at 4489.9 Da and 4612.9 Da can be attributed to the addition of GlcNAc and GlcUA to glycoporm I (Figure 7). These spectra from LPS obtained from either the wild type or its _mla_C and _mlaD_ derivatives also reveal a prominent presence of mass peaks corresponding to glycoforms IV and V, which are represented by mass peaks at 3948.7, 4079.8, 4202 and 4298.9 Da. These mass peaks are explained by the incorporation of a third Kdo and Rha linked to the Kdo disaccharide with a concomitant truncation of the outer core with a predicted composition of \(\text{LA}_{\text{hexa}}\) (\(\text{Kdo}_{3}\text{RhaHep}_{3}\text{Hex}_{3}\text{P}_{2}\)). The mass differences in these derivatives of glycoforms with a third Kdo and Rha are explained by the additional incorporation of P-EtN, L-Ara4N and GlcUA, as shown (Figure 7). The main findings from mass spectrometry data are that a deletion of _mla_ genes does not alter LPS core and lipid. A structural properties under these defined growth conditions. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & & \multicolumn{5}{c}{**P1 Donor**} \\ \cline{2-7}
**Recipient** & & \multicolumn{2}{c}{**30 \({}^{\circ}\)C**} & \multicolumn{2}{c}{**43 \({}^{\circ}\)C**} & \\  & \(\Delta\)_mlaC_ & \(\Delta\)_mlaD_ & \(\Delta\)_mlaCD_ & \(\Delta\)_mlaC_ & \(\Delta\)_mlaD_ & \(\Delta\)_mlaCD_ \\ \hline wt BW25113 & 1260 & 1380 & 1429 & 1412 & 1274 & 980 \\ _lapC190_ (SR23583) & 623 & 710 & 690 & 12 & 14 & 9 \\ _lapC190_ + _pmarA_ & & & & & & \\ (SR23660) & 654 & 740 & 565 & 9 & 12 & 8 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: MarA-mediated multicopy suppression of _lapC190_ bacteria requires the presence of Mla system. SrrA-Mediated Suppression of lapC190 Mutant Bacteria Requires the Presence of Cardiolipin Synthase A and ClsA Is Essential for lapC190  
Among various multicopy suppressors that were identified, SrrA has a distinguishing property that the corresponding gene in high dosage can suppress the Ts phenotype of strains lacking six cytoplasmic peptidyl-prolyl _cis/trans_ isomerases, \(\Delta\)_lapD_ bacteria and is again reisolated as a suppressor of the Ts phenotype of _lapC190_ bacteria [28; 36]. Since a modest overproduction of SrrA was found to restore LpxC amounts (Figure 5), we further investigated a possible pathway that can explain this suppression. The same strategy of isolating transposon insertion mutants was used as shown above with the _marA_ gene.