# Lipopolysaccharide: Recent Advances in Its Biosynthesis and Controlling Cell Envelope Homeostasis
###### Abstract
In this study, the authors exploited the temperature sensitivity (Ts) and membrane permeability defects of _lapC_ mutant bacteria expressing only the N-terminal domain of LapC. Isolation and characterization of multicopy suppressors that corrected the Ts phenotype identified specific factors predicted to be involved in maintaining homeostasis between LPS and PL biosynthesis. Thus, one group comprises genes encoding LPS assembly factor (LapD), acyl-carrier protein AcpP, known to shuttle fatty acids for lipid A and fatty acid biosynthesis, subunit of acetyl-CoA carboxylase enzyme AccB, and outer membrane phospholipase PldA, which removes mislocalized PL from the outer leaflet of the OM. Another set of genes encoded either transcription factors (_dksA_, _marA_, and _srrA_) or the _gtsA_ gene, whose product is predicted to regulate the amounts of saturated versus unsaturated fatty acids, or could act indirectly (_yfgM_, _ycef_). It was further shown that overexpression of _srrA_ and _marA_ genes fully restored LpxC levels, and DksA-mediated suppression was only to a milder extent. However, interestingly, overexpression of _acpP_ or _accB_ did not restore either LpxC or LPS levels in _lapC_ mutant bacteria and was proposed to act by balancing PL and LPS amounts. Overexpression of _acpP_ could inhibit PL synthesis at the PlsB level, and _accB_ could disrupt the assembly of the ACC complex, thereby reducing fatty acid synthesis and helping in balancing PL amounts to reduced amounts of LPS in _lapC_ mutant bacteria. It was further shown that MarA suppression requires the functionality of the Mla system involved in the maintenance of lipid asymmetry, providing a rationale for the requirement of MarA in the regulation of LPS. A similar analysis revealed that StrrA-mediated suppression was contingent on the presence of cardiolipin synthase A encoded by the _clsA_ gene. Confirming the important roles of these players, this study also showed that _srrA_, _marA_, and _pldA_ became essential when LapC was dysfunctional. As overexpression of the _lapD_ gene can overcome the Ts phenotype of _lapC_ mutant bacteria in an LpxC-independent manner, it was shown that both the N-terminal IM anchor and the last 32 C-terminal residues are critical for LapD function. Concerning DksA, which is a well-characterized transcriptional factor (TF) [71] that exhibits peptidyl _cis/trans_ isomerase activity (PPIase) [72], it was shown that both TF and PPIase activities of DksA are required for its role in regulating LpxC levels. Thus, in this study new players in LpxC regulation, such as MarA, StrrA, and GnsA, were identified, and the roles of the acyl-carrier protein AcpP and acetyl-CoA carboxylase complex in maintaining a balance between LPS and PL were proposed. The manuscript by [PERSON] et al. examined the effect of mutations that alter LPS structure on the activation of Toll-like receptor (TLR4) in the pathogenic bacteria _Bordetella pertussis_[73]. The authors showed that dephosphorylation of lipid A in _B. pertussis_ through the heterologous production of the phosphatase LpxE from _Francisella novizeta_ does not affect TLR4-stimulating activity. The authors rationalized these results due to the presence of phosphorylated Kdo in the inner core of _B. pertussis_ LPS, which could mediate the interaction with the TLR4-MD2 receptor complex. Thus, the Kdo region in the inner core was modified either mutationally or by the heterologous expression of bifunctional _E. coli_ Kdo transferase, as KdtA in _B. pertussis_ is essential. Interestingly, the presence of KdoI in _B. pertussis_ LPS impaired the substitution of the outer core with the terminal trisaccharide with a variable loss of the secondary C\({}_{14}\) chain at the 2\({}^{\prime}\) position of lipid A. The authors next identified the _eptB_ gene, whose product mediates the non-stoichiometric substitution of Kdo-bound phosphate with P-EtN, and furthermore inactivated this gene on the chromosome.