## 2 Results
### Overexpression of _srrA_, _marA_, _yceJ_ and _dksA_ also _Restore LPS Amounts in lapC190 Bacteria  
We previously showed that _lapC190_ bacteria have reduced amounts of LPS due to the enhanced degradation of LpxC as compared to the wild type. Thus, to address the mechanism of suppression by overexpression of specific genes that provide a robust restoration of growth at elevated temperatures, we estimated levels of LPS from total cell extracts. For such experiments, we measured levels of LPS from cellular extracts obtained from exponentially grown cultures of the isogenic wild type, its _lapC190_ derivative with the vector alone and _lapC190_ bacteria carrying plasmid that expresses a specific suppressor encoding gene under an inducible P\({}_{\text{TS}}\)-_lac_ promoter. Bacterial cultures were grown under permissive growth conditions at 30 \({}^{\circ}\)C in LB medium, the gene expression was induced with the addition of 75 uM IPTG at OD\({}_{\text{595}}\) 0.1 and samples obtained under the same conditions when LpxC amounts were measured as described in Figure 5. Total cell extracts were also applied from isogenic _dhfsH ghTc21_ derivative as a control. Equivalent  
Figure 5: Restoration of LpxC amounts to different extents upon the overexpression of various multicopy suppressor encoding genes in _lap190_ bacteria. Immunohistochemistry of whole cell lysates obtained from isogenic strains grown at 30 \({}^{\circ}\)C, followed by the addition of 75 uM IPTG and a temperature shift for 2 h at 43 \({}^{\circ}\)C (**A**,**B**). For immunoblotting, LpxC-specific antibodies were used and the relevant genotype is indicated. An equivalent amount of total proteins was resolved by an SDS-PAGE prior to immunoblotting. As controls, lane 2 in panel (**A**) and panel (**B**) samples from _lapC190_ bacteria are loaded, which have reduced LpxC amounts. Additional control involves samples from _dhfsH ghTc21_ which expectedly reveal higher amounts of LpxC due to its stabilization lane 10 (panel (**A**)) and lane 9 (panel (**B**)). As loading control, the same samples were probed with anti-TrxA antibodies.  
amount of whole cell lysates was treated with Proteinase K. Samples were resolved on an SDS-Tricine gel and LPS was transferred by Western blotting. Amounts of LPS were revealed using an LPS-specific WN1 222-5 monoclonal antibody as described in the Methods section.  
The estimation of relative amounts of LPS in such experiments show that _lapC190_ bacteria have highly diminished amounts of LPS (Figure 6 lane 9) as compared to the wild type and the _AflsH sflc21_ derivative contains elevated amounts of LPS. Most importantly, results from such immunoblots reveal that extracts from the _lapC190_ derivative overexpressing either _marA_, or _ycej_, or _srrA_ or the _dksA_ gene show the restoration of LPS amounts to near wild-type levels explaining the mechanism of their suppression (Figure 6). As with the estimation of LpxC amounts, overexpression of the _acpP_ gene did not cause any increase in the amounts of LPS and remained low as observed with _lapC190_ bacteria. Similarly, overexpression of the _lapD_ gene only marginally enhances LPS amounts. These results allow us to conclude that some of the multicopy suppressors such as _marA_, _dksA_, _srrA_ and _ycej_ contribute via increasing LpxC and LPS amounts, providing a reasonable explanation for their identification. However, the mode of restoration of growth of _lapC_ mutant bacteria by an overexpression of the _acpP_ gene could involve the LpxC independent mode.