# Regulated Assembly of LPS, Its Structural Alterations and Cellular Response to LPS Defects
## 1 Introduction  
The cell envelope of Gram-negative bacteria, including _Escherichia coli_, contains two distinct membranes, an inner (IM) and an outer (OM) membrane separated by the periplasm, a hydrophilic compartment that includes a layer of peptidoglycan. The OM is an asymmetric bilayer with phospholipids forming the inner leaflet and lipopolysaccharide-ascharide (LPS) forming the outer leaflet. LPS is essential for bacterial viability. Because of the strong lateral chemical interactions, LPS provides the essential permeability barrier function. It is a complex glycolipid, a major component of the OM and highly heterogeneous in composition. Bacteria, like _E. coli_ and _Salmonella_, contain approximately \(2\)-\(3\times 10^{6}\) molecules of LPS that cover more than 75% of the OM [1]. The biosynthesis, translocation and various modifications of LPS requires the function of more than 100 genes. Several of them are essential and unique to bacteria and hence they are excellent targets for the identification of their inhibitors for the development of new antibiotics. The biosynthesis of LPS begins with the acylation of UDP-GlcNAc with \(R\)-3-hydroxyristate derived from \(R\)-3-hydroxyristoyl-ACP by LpxA [2]. \(R\)-3-Hydroxymyristoyl-ACP also serves as a precursor for the synthesis of phospholipids. The second reaction of the lipid A biosynthesis is catalysed by LpxC [UDP-3-O-(\(R\)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase] a Zn\({}^{2+}\)-dependent deacetylase, constituting the first committed step in the LPS synthesis, as the equilibrium constantfor the first reaction catalysed by LpxA is unfavourable. Following deacetylation, a second \(R\)-3-hydroxymyristate chain is added by LpxD leading to the synthesis of UDP-2,3-diacyl-GlcNAc. This serves as a substrate for LpxH to generate 2,3-diacyl-GlcNAc-1-phosphate, also called lipid X [3]. The next steps involve a condensation reaction generating the \(\beta\),1\({}^{\prime}\)-6-linked disaccharide by LpxB, followed by its phosphorylation at 4\({}^{\prime}\) position by LpxH. This generates the lipid IV\({}_{\text{A}}\) precursor, which serves as an acceptor for the WaaA-mediated incorporation of two 3-deoxy-\(\alpha\)-d-_manno_-oct-2-ulsonic acid (Kdo) residues. Up to the synthesis of Kdo\({}_{2}\)-lipid IV\({}_{\text{A}}\), all the required seven enzymes are essential for the bacterial viability [4]. Kdo\({}_{2}\)-lipid IV\({}_{\text{A}}\) comprises a key intermediate in LPS biosynthesis that acts 2-fold as a specific substrate: (_i_) for acyltransferases that generate Kdo\({}_{2}\)-lipid A moiety by the transfer of two additional fatty acids to the (\(R\))-3-hydroxyl groups of both acyl chains, which are directly bound to position 2\({}^{\prime}\) and 3\({}^{\prime}\) of the non-reducing GlcN residue and (_ii_) for glycosyltransferases catalyzing further steps of the core oligosaccharide biosynthesis. The _E. coli_ K-12 genome encodes three paralogous acyltransferases (LpxL, LpxM and LpxP), which catalyze acylation reactions using acyl carrier protein-activated fatty acids as co-substrates [5]. At ambient temperatures, a lauroyl residue is first transferred by LpxL to the OH group of the amide-bound (\(R\))-3-hydroxymyristate residue at position 2\({}^{\prime}\). This catalytic step is partially replaced at low temperature (12 \({}^{\circ}\)C) by LpxP, which transfers palmitoleate to the same position in approximately 80% of LPS molecules [6]. The free OH group of the ester-bound (\(R\))-3-hydroxymyristate residue at position 3\({}^{\prime}\) within both pentaacylated intermediates is then myristoylated by LpxM to give a hexaacylated lipid A moiety. However, it is noteworthy that under slow growth conditions and at low temperatures, the lipid IV\({}_{\text{A}}\) precursor without the Kdo incorporation can be the substrate for a secondary acylation by LpxP, LpxL, and LpxM, as observed in \(\Delta\)_waaA_ grown at 21-23 \({}^{\circ}\)C [7]. In _Enterobacteriaceae_ members, the core region of LPS is always attached to lipid A via a Kdo residue.