## 2 Results  
Genes Whose Overexpression Overcomes the Temperature Sensitivity (Ts) of lapC Mutant Bacteria with a Truncation of LapC Periplasmic Domain Identify New Players That Can Regulate LpxC Amounts  
We previously reported the isolation of mutations in the essential gene encoding the LapC protein. Such mutations cause truncation in the periplasmic domain of LapC and confer a Ts phenotype, permeability defects and a reduced amount of LPS due to the hyperdegradation of LpxC [6]. Some of these phenotypic defects could be suppressed by chromosomal single-copy mutations mapping to _lpxC_, _ftsH_ and the _lapA/B_ operon [6]. This suppression was attributed to the stabilization of LpxC. However, since several  
Figure 2: Schematic depiction of various approaches that identify new regulators of LpxC. Multicopy suppressors of sensitivity to high temperature, growth on either MacConkey agar or erythromycincin of _lapC190_ identified several genes, some of which act by increasing LpxC and LPS amounts (bottom panel). Transposon mutagenesis showed that while as MarA-mediated suppression requires the functional presence of Mla system, SrrA-dependent suppression needs the presence of cardiolipin synthase A.  
factors can contribute towards the regulation of LpxC, here we asked if the overexpression of any gene can also suppress growth defects of _lapC_ mutant bacteria. Thus, we used _lapC190_ mutant bacteria, which lack the periplasmic domain of LapC on the chromosome, to isolate multicopy suppressors, using a complete genomic library. To achieve this goal, plasmid DNA pools from the ASKA collection of single ORFs expressed from the tightly regulated IPTG-inducible P\({}_{\text{TS}}\)-_lac_ promoter in the vector pCA24N [33] were used to transform competent cells of _lapC190_ mutant bacteria. Transformations were selected for the restoration of growth on LA medium supplemented with 75 \(\upmu\)M IPTG at 43.5 \({}^{\circ}\)C, a condition that is non-permissive for _lapC190_ mutant bacteria. This concentration of inducer of gene expression has been optimized as described earlier [19]. Cultures from identified Ts\({}^{+}\) colonies were used to extract plasmid DNA to retransform _lapC190_ bacteria. Plasmids that bred true were retained and their DNA was sequenced, which identified 13 unique genes, whose mild overexpression can rescue the Ts phenotype of _lapC190_ mutant bacteria (Table 1, Figure 3). Based on their known or predicted function, these genes can be grouped into various classes: those with a role in LPS, phospholipid/fatty acid biosynthesis or sensing (_lapD_, _acpP_, _acpT_, _accB_, _pldA_) (Figures 3 and 4), transcription factors (_marA_, _dksA_, _srrA_), an IM-anchored heat shock protein-encoding gene with an unknown function (_vecl_) and others whose products could either titrate the FtsH protease responsible for LpxC degradation (_yfgM_) or modulate saturated versus unsaturated fatty acid abundance (_gnsA_) or related with the stress response (_ymgG_), whose product is the part of toxin-antitoxin system. Next, we examined the suppression of permeability and growth defects. For such studies, we investigated the degree of restoration of bacterial growth/viability. Thus, spot dilution assays were performed on LA medium and on MacConkey agar supplemented with 75 \(\upmu\)M IPTG or when supplemented with 15 \(\upmu\)mL of erythromycincin. Data are presented for growth on MacConkey agar as a similar pattern was observed with resistance to erythromycincin. These experiments revealed that although most of the multicopy suppressors restored bacterial growth on LA agar at 43.5 \({}^{\circ}\)C, the permeability defect was suppressed to a variable extent (Figure 3). Even on LA medium, the colony-forming ability and the colony size indicated the degree of suppression of Ts phenotype was variable. For example, the overexpression of the _acpT_ gene conferred only a partial suppression (Figure 3). Thus, on MacConkey agar, only overexpression of _srrA_ and _yfgM_ genes restored growth to a near-wild-type level.