## 2 Results
### Lauroyl and Myristoyl Transfers Are Required for the Viability of lapC190 Mutant Bacteria  
Next, we examined if acyltransferases that mediate the transfer of Lauroyl (LpxL) and myristoyl (LpxM) acyl chains to Kdo\({}_{2}\)-lipid IV\({}_{A}\) are essential for the growth of _lapC190_ mutant bacteria. Bacteriophage P1-mediated transfer of _lapC190_ or an introduction of _\(\Delta\)lpxL_ or \(\Delta\)lpxM_ revealed that \(\Delta\)lpxL _lapC190_ and \(\Delta\)lpxM _lapC190_ mutational combinations are only possible in the ectopic presence of covering plasmid containing the wild-type gene (Tables 2 and 3). However, at a very low frequency, viable suppressors could be obtained after an incubation of more than 48 h when \(\Delta\)lpxL _lapC190_ and \(\Delta\)lpxM _lapC190_ transductants were plated. Further characterization of two suppressors of each such mutational combinations identified a single amino acid exchange, L412P, in the _msbA_ gene. Interestingly, the same _msbA_ suppressor mutation was found to overcome the lethality of \(\Delta\)(_lpxL _lapD_) and \(\Delta\)(_lpxM _lapD_) combinations [28]. Thus, we can conclude that truncation of the _lapC_ gene, resulting in the expression of only the transmembrane anchor, requires complete acylation of the lipid A. Since underacylated LPS (lack of LpxL or LpxM) is poorly translocated by MsbA as compared to hexaacylated lipid A, this could lead to a further reduction in the amount of LPS in the OM in _lapC190_ bacteria and hence the lethality. Taken  
\begin{table}
\begin{tabular}{c c c} \hline \hline  & **Numbers of Transductants on LA 30 \({}^{\circ}\)C** \\ \hline
**Donor** & **Wild Type** & _lapC190_ \\ \hline \(\Delta\)_marA_ & 1274 & 92 \({}^{\dagger}\) \\ \(\Delta\)_srrA_ & 650 & 2 \\ \(\Delta\)_dksA_ & \textgreater{}2000 & 163 \({}^{\ddagger}\) \\ \(\Delta\)_pldA_ & 1460 & 34 tiny \\ \(\Delta\)_lpxL_ & 742 & 6 \\ \(\Delta\)_lpxM_ & 960 & 12 \\ \(\Delta\)_lapD_ & 733 & 25 \\ \hline \hline \end{tabular} \({}^{\dagger}\) not viable. \({}^{2}\) viable but heterogenous.  
\end{table}
Table 2: Synthetic lethal combinations reveal essentiality of _marA_, _srrA_ and _lapD_ genes in _lapC190_ bacteria.  
\begin{table}
\begin{tabular}{c c c} \hline \hline  & **Numbers of Transductants on LA 30 \({}^{\circ}\)C** \\ \hline
**Donor** & **Wild Type** & _lapC190_ \\ \hline P1 \(\Delta\)_lpxL_ & wild type + p/_pxL_ & _lapC190_ + p/_pxL_ \\ \hline P1 \(\Delta\)_lpxM_ & 1234 & 1164 \\ \hline P1 \(\Delta\)_lpxM_ & wild type + p/_pxM_ & _lapC190_ + p/_pxM_ \\ \hline P1 \(\Delta\)_lpxM_ & 968 & 1005 \\ \hline P1 \(\Delta\)_lpxD_ & wild type + p/_lapD_ & _lapC190_ + p/_lapD_ \\ P1 \(\Delta\)_lapD_ & 811 & 840 \\ \hline  & wild type + p/_srrA_ & _lapC190_ + p/_srrA_ \\ P1 \(\Delta\)_srrA_ & 794 & 736 \\ \hline \hline \end{tabular}
\end{table}
Table 3: Synthetic lethal combinations reveal essentiality of _lpxL_ and _lpxM_ genes in _lapC190_ bacteria, which can be overcome in the presence of ectopic plasmid containing a wild-type copy of either of the genes.  
together, _lapC190_ derivatives synthesizing either tetraacylated or pentaacylated lipid A, cannot be constructed unless a suppressor mutation in the _msbA_ gene, which encodes lipid A flippase, is present.