## 3 Discussion
(ii) Of significance is the isolation of the _lapD_ gene as a multicopy suppressor of _lapC_ mutant bacteria for the Ts phenotype. We recently proposed that LapD could also act upstream of LapB-FtsH in a manner similar to LapC, since suppressors mapping to _lapB_, _ftsH_ and _lpxC_ that stabilize LpxC, can suppress \(\Delta\)_lapD_ and _lapC190_ bacteria. (iii) However, some suppressors, like the _marA_ gene and the _acpT_ gene cannot relieve the Ts phenotype of \(\Delta\)_lapD_ bacteria upon their overexpression and are specific to _lapC_ mutant bacteria. Consistent with these results, the overexpression of the _lapC_ gene does not relieve the Ts phenotype of \(\Delta\)_lapD_ bacteria. These results are not surprising because, at the biochemical level, despite the LapC and LapD co-purification with LapB, several other interacting partners of LapD are unique to it and were not found to be part of the LapC complex [6; 24; 28]. To further understand LapD-mediated multicopy suppression of _lapC_ mutant bacteria, we constructed a series of mutations in the _lapD_ gene to identify the essential domains of the encoded protein. This allowed us to express such mutant versions in a controlled manner from a regulated inducible promoter in _lapC_ mutant bacteria. This revealed that the N-terminal membrane anchor and the last 32 conserved C-terminal amino residues are critical for LapD function. Furthermore, several residues that were predicted for LapD to interact with its partners [50] were also found to be essential for its function since their replacement by Ala amino acid residues abolished LapD function (inability to suppress the Ts phenotype of either \(\Delta\)_lapD_ or _lapC190_ mutant bacteria). This includes a motif constituted by the N93 P94 F96 triplet, which we show is essential for the LapD function and has been predicted to interact with LapA. This conserved NFP motif is located after the long cytoplasmic helical domain. Regarding the mechanism of suppression by overproduction of the global transcriptional regulator DksA, we show that, most critically, its PPIase activity is required for this activity. However, the replacement of conserved amino acid residues in the tip region of the coiled-coil domain of DksA which are known to be essential for its role in interaction with RNA polymerase and hence the transcriptional regulation caused a highly attenuated ability to confer suppressing ability of _lapC190_ bacteria. However, the most severe phenotype was the replacement of F82 by Y, which resulted in the total abolishment of the multicopy-suppressing ability of DksA. This amino acid residue is important for conferring the PPIase activity of DksA but is not essential for transcriptional function [36]. Hence, the PPIase activity of DksA is critical for its multicopy-suppressing ability, and its transcriptional activity also plays an important role. Further studies are required to determine the mechanism(s) of the restoration of growth at elevated temperatures by other multicopy suppressors that were identified in this study. It would be interesting to determine whether the suppression by _gnsA_ overexpression is due to an increase in the unsaturated fatty acid synthesis that could lead to the stabilization of LpxC. The _gnsA_ gene was initially identified as a multicopy suppressor of the _secG_ null mutant and was shown to increase the acidic phospholipid content and inhibit phosphatidylcholine accumulation via an unknown mechanism [63]. Furthermore,oveexpression of the \(gnsA\) gene also suppresses the Ts phenotype of _fabA6_ mutation and causes an increase in the unsaturated fatty acid content, such as _cis_-vaccenic acid (18:1), at the expense of palmitic acid (16:0), particularly at low temperatures [64]. At present, the molecular basis of the suppression of either _lapC190_ bacteria or the _fabA6_ Ts mutation or the ascribed increase in the unsaturated fatty acid content is not known.