## 3 Discussion
Interestingly, up to now, the sole dosage-dependent suppressor of _lapC_ mutant bacteria expressing only the periplasmic domain has been the _acpT_ gene, whose product encodes a phosphopantetheinyl transferase [54]. In this work, we also isolated the _acpT_ gene as a multicopy suppressor; however, this suppressing phenotype was much weaker than the others. Surprisingly, it has been shown that AcpT-mediated suppression of _lapC_ (_yejM_) mutant bacteria for the Ts phenotype does not require its enzymatic activity [54]. The global transcriptomes of MarA and DksA are known [55, 56, 57], but only sketchy information about StrA is available. Thus, in this study, we systematically attempted to identify genes whose presence is required for multicopy suppression by MarA and StrA. In _E. coli_, MarA acts as a transcriptional activator and is known to activate the expression of _acrAB-tolC_-encoded efflux pump, thereby contributing to resistance against antimicrobial compounds [58, 59, 60]. More recently, approximately 30 transcriptional units have been identified that could be regulated by MarA [61]. Here, we used saturated transposon mutagenesis to identify genes whose inactivation abrogates the _marA_- and _srrA_-mediated restoration of the growth of _lapC190_ mutant bacteria at high temperatures. The vast majority of transposon mutants that prevented MarA-mediated multicopy suppression mapped to the _mla_ operon, whose products are implicated in lipid trafficking, leading to the removal of the mislocalized phospholipids from the OM [29]. Consistent with our results, a MarA-binding motif has been identified in the promoter region of the _mla_ operon [61]. Thus, our results provide a rationale for the identification of the _marA_ gene as a multicopy suppressor of _lapC190_ mutant bacteria, and this suppression requires the functionality of _mla_ genes. In accordance with these findings, the _pldA_ gene was isolated as a multicopy suppressor since both PldA and Mla pathways sense OM disturbance due to the accumulation of phospholipids in the OM when LPS is limiting. In a similar manner, we addressed the pathway of StrA-mediated suppression. Our results show that the overexpression of the _srrA_ gene efficiently restores the growth at high temperatures, LpxC levels as well as permeability defects. This suppression was found to be contingent on the presence of the functional copy of the _clsA_ gene encoding cardiolipin synthase A and the envelope-responsive two-component system CpxA/R. The CpxA/R system, along with the RpoE sigma factor, responds to severe defects in LPS assembly and biosynthesis as well as protein misfolding in the periplasm [13, 19, 62, 39]. We have previously shown that ClAs is required for the viability of strains synthesizing either penta or tetraacylated lipid A, or when LapD assembly protein is missing [15; 28]. Since LPS is presumably sensed in the inner membrane by LapC, the absence of ClsA could further accentuate this defect as it is required to assist MsbA-mediated LPS translocation. However, this requires further studies because the _clsA_ gene is per se essential in _lapC190_ bacteria. Our gene expression studies did not show that SrrA regulates transcription of the _clsA_ gene based on q-RT-PCR results. Thus, we are initiating a global transcriptome study to identify genes that are regulated by SrrA, and initial results, which require further in-depth experimentation, suggest that SrrA acts as a transcriptional repressor of the envelope stress response and fatty acid metabolism in _E. coli._  
Another important finding from this study is the partial overlap between LapD and LapC functions in the regulation of LpxC and LPS levels. This is based on the following results: (i) Some of the multicopy suppressors identified in this work that relieve the Ts phenotype of _lapC190_ bacteria were earlier also identified as suppressors of \(\Delta\)_lapD_. These include _dksA_, _srrA_, _acpP_, _accB_ and _yfgM_[28].