## 3 Discussion  
In this study, we identified new additional factors that regulate LpxC levels. Regulation of LpxC is critical for our understanding of the mechanism of balancing biosynthesis of LPS and phospholipids, since they both share the same (\(R\))-3-hydroxymyristate-ACP as the common metabolic precursor. Recent studies have shown that LpxC stability is regulated by the FtsH-LapB complex, wherein FtsH degrades LpxC, which requires LpB function for its proteolytic activity towards LpxC [19; 22]. However, this activity is counteracted by the essential LapC (YejM) protein [6; 23; 25]. In addition to FtsH-LapB and LapC, many additional factors participate in the regulation of LpxC amounts and, hence, the overall LPS content. Mutations in either the _ftsH_ or _lapB_ genes lead to an increased abundance of LPS due to the stabilization of LpxC, whereas loss-of-function mutations in the _lapC_ gene exhibit enhanced LpxC degradation with a concomitant reduction in the overall LPS content. During earlier work, we isolated temperature-sensitive _lapC190_, _lapC377s_ and _lapC_ F349S mutant bacteria [6]. Because _lapC190_ mutant bacteria exhibit a tight Ts phenotype, we exploited such a property to isolate multicopy suppressors that restore growth at elevated  
Figure 10: Amino acid residues that are essential for DksA’s transcriptional activity and those required for its peptidyl-prolyl _cis/trans_ isomerase activity are required for its multicopy suppressing ability of _lapC190_ bacteria. Competent cells of _lapC190_ bacteria were transformed by plasmid DNA carrying either the wild-type _dksA_ gene or with specific single-amino amino acid replacement in the tip domain of the coiled-coil domain of DksA. Cultures of such isogenic cultures of _lap190_ bacteria were grown at 30 \({}^{\circ}\)C in LB medium and bacterial growth was quantified by serial spot dilution assay on LA 30 \({}^{\circ}\)C and 43.7 \({}^{\circ}\)C. The relevant genotype and temperature of incubation are indicated. temperatures to identify additional factors that regulate LpxC amounts. Such an approach should also reveal what are the limiting factors that confer the Ts phenotype to _lapC190_ mutant bacteria. Using such a procedure, thirteen genes were identified whose mild induction overcomes the Ts phenotype. As _lapC190_ bacteria exhibit permeability defects and reduced LpxC and LPS levels, we examined whether overexpression of any of these genes also suppress such defects. Quite satisfactorily, a major proportion of such genes can be implicated either directly or indirectly in functions related to LPS, phospholipid/fatty acid biosynthesis and regulation of LpxC protease FtsH amount (_lapD_, _pldA_, _acpP_, _acpT_, _accB_, _gnSA_, _ygM_, _marA_) or envelope stress response (_strA_, _dksA_, _ymgG_). These thirteen genes were thus grouped into different categories based on their known or predicted functions. Three out of them (_marA_, _dksA_ and _srrA_) act as transcriptional factors and should contribute by their regulation of downstream target gene(s), whose gene products either directly regulate LPS or phospholipid biosynthesis. Another group of suppressors include genes encoding the LPS assembly protein LapD, the essential AcpP protein that shuttles fatty acid chains in the lipid A and fatty acid/phospholipid biosynthesis, the outer membrane phospholipase A encoded by the _pldA_ gene. Isolation of these genes is interesting since this shows that _lapC_ mutant bacteria have imbalanced phospholipid and LPS biosynthesis. Importantly, PldA degrades phospholipids that have been mislocalized to the outer leaflet in the OM, which signal stabilization of LpxC and an increased production of LPS [29], explaining its isolation as a multicopy suppressor. However, it should be noted that overexpression of the _pldA_ gene does not overcome the permeability defects of _lapC190_ bacteria, as reflected by an inability to restore growth on bile-salt-containing MacConkey agar.