## 2 Results
### MarA-Mediated Suppression Requires the Presence of mla Genes  
As the induction of the _marA_ gene expression restored the growth of _lapC190_ mutant bacteria at an elevated temperature, we further investigated its possible mechanism of suppression. Thus, strain SR23660 (_lapC190_ + _pmarA_+) served as a host to generate a saturated Tn_10_ Kan transposon library at 30 \({}^{\circ}\)C. Nearly 50,000 such transposon mutants were screened for the growth on LA medium at 43 \({}^{\circ}\)C in the presence of 75 uM IPTG and as a control at 30 \({}^{\circ}\)C without the presence of an inducer. This is the strategy that we earlier successfully employed to identify genes whose products are required for the multicopy suppressor phenotype of the _dksA_ gene [36]. Thus, we retained those Tn_10_ insertion mutants that grew at 30 \({}^{\circ}\)C but were unable to propagate at 43 \({}^{\circ}\)C, even with the induction of the _marA_ gene expression. Such transposon mutations were backcrossed into SR23660 and verified for lack of suppression of _lapC190_ mutant bacteria in the presence of the inducible _marA_ gene. This resulted in the identification of fifty-four independent Tn_10_ mutants. Next, such Tn_10_ Kan transposon insertions were introduced into the wild type by bacteriophage P1-mediated transductions to eliminate mutations that conferred a Ts phenotype at 43 \({}^{\circ}\)C, even in the wild-type background. Such experiments identified 22 Tn_10_ insertions that were found to confer the Ts phenotype in the wild type, while the remaining were specific to the _lapC190_ strain. Most of the Ts mutants were found to have the Tn_10_ insertion in the _degP_ gene, whose product is known to be required for bacterial growth at temperatures above 42 \({}^{\circ}\)C [37; 38]. This is consistent with a similar spectrum of Ts insertions obtained in an identical strategy described to unravel the requirement of specific genes for DksA-mediated multicopy suppression [36]. The mapping and characterization of the remaining Tn_10_ insertions that prevent the multicopy suppression by _marA_ identified 12 out of 15 mapping to genes whose products are part of the Mla pathway. To facilitate the mapping of Tn_10_ insertions, we used a previously described linked _arpON_[39] to find how many can cross out the Tn_10_ insertion mutation. Such transductions revealed that the majority of Tn_10_ insertion mutants had a disruption of the _mla_ operon. DNA sequence analysis of the Tn_10_ junction from chromosomal DNA of _mla_ Tn10 insertions was obtained by inverse PCR, which identified four insertions each in _mlaC_ and _mlaD_, three insertions in _mlaF_ and one insertion in the _mlaA_ gene. The Mla system is known to be required for the maintenance of outer membrane lipid asymmetry and the inactivation of _mla_ genes confers sensitivity to SDS+EDTA [40]. The _mla_ operon contains _mlaF_, _mlaE_, _mlaD_, _mlaC_ and _mlaB_ genes, out of which the _mlaF_ gene encodes the ATP-binding protein of an inner membrane complex (MlaFDB). The Mla pathway functions as part of the retrograde and/or anterograde intermembrane phospholipid trafficking system [40; 41]. The Mla system also contains MlaA, whose encoded gene is located outside the _mla_ operon. The Mla system can function by removing mislocalized outer leaflet phospholipids and transporting them back to the inner membrane. Thus, the isolation of Tn_10_ insertion in genes encoding the Mla pathway that prevent MarA-mediated suppression of _lapC190_ bacteria is consistent with LapC function in regulating LPS amounts and a balance with phospholipid amounts. Thus, to further support these results, we used previously constructed in-frame deletion derivatives of _mlaC_ and _mlaD_[42] and a new construct \(\Delta\)(_mlaC__mlaD_) to verify if such mutations abolish _marA_ multicopy suppression. This was required because Tn_10_ insertions are expected to be polar on the expression of downstream genes.