## 2 Overview of OM Structure and Functions
### Overview of LPS Biosynthesis  
The biosynthesis of LPS is a complex process that occurs in three different cellular compartments, cytoplasm, IM and periplasm, thus, requiring spatial and temporal coordination of several independent biosynthetic pathways (lipid A, Kdo, and oligosaccharidesacharide core) that converge in an ordered assembly line to give the mature molecule (Figure 1) [23, 33, 34]. Nine enzymes are required to for the synthesis of the Kdo\({}_{2}\)-lipid A moiety. The first reaction in the synthesis of lipid A is the fatty acylation of UDP-\(N\)-acetylglucosamine (UDP-GlcNAc) by LpxA to UDP-3-\(O\)-acyl-GlcNAc that is deacetylated by the LpxC enzyme [35]. Further acylation is then carried out by LpxD the third enzyme of the biosynthetic machinery. In both acylation steps catalyzed by LpxA and LpxD the fatty acyl donor is R-3-hydroxymethyl ACP that also serves as an acyl donor for the synthesis of membrane phospholipids and therefore represents a crucial metabolic branch point in the biosynthesis of membrane components [36, 37]. Next, reactions catalyzed by LpxH, LpxB, and LpxK result in the synthesis of the tetra-acylated lipid IV\({}_{\rm A}\) that, in _E. coli_, is the substrate of WaaA the CMP-Kdo dependent transferase that catalyzes the sequential incorporation of two Kdo residues synthesized in a separate pathway [23]. Finally, the late acyltransferases LpxL and LpxM catalyze the addition of secondary acyl chains to the distal glucosamine [38, 39] to give the hexa-acylated Kdo\({}_{2}\)-lipid A moiety. The additional sugars composing the oligosaccharidesacharide core are added to Kdo\({}_{2}\)-lipid A by specific glycosyltransferases to generate the core-lipid A structure. The core-lipid A, which is anchored to the IM with its hydrophilic moiety exposed to the cytoplasm, is then flipped over the IM by the ABC transporter MsbA and becomes exposed in the periplasm [40, 41]. \(O\)-antigen repeat units are synthesized in the cytoplasm and then flipped at the periplasmic face of the IM attached to the lipid carrier undecaprenyl diphosphate. Mature LPS is then formed at the periplasmic side of the IM by the WaaL ligase that catalyzes the ligation of _O_-antigen to core-lipid A [42]. The _O_-antigen moiety is not essential and is missing in common laboratory _E. coli_ K12 strains due to inactivation of the _wobL_ gene [43, 44] but is present in most wild type strains and clinical isolates where it contributes to virulence by protecting the bacteria from phagocytosis and complement-mediated killing [45]. The enzymes for lipid A and Kdo biosynthesis are constitutively expressed [23, 46]. However, in _E. coli_ the production of Kdo\({}_{2}\)-lipid A is post-transcriptionally regulated by FtsH, an essential membrane protease belonging to the AAA family (\(\Delta\)TPase associated with various cellular activities) that controls the turnover of LpxC [47] the enzyme that catalyzes the first committed step of the lipid A biosynthetic pathway [48]. Mutations in _ftsH_ lead to increased cellular levels of LpxC and are lethal [47]. This can be explained by the fact that both lipid A and phospholipid biosynthetic pathways  
Figure 1: LPS biosynthesis in _E. coli_ K12 strains. Cytoplasm and inner leaflet of IM: lipid IV\({}_{\rm A}\) is synthesized from two molecules of the sugar nucleotide UDP-GlcNAc. Sequential addition of two Kdo molecules to lipid IV\({}_{\rm A}\) produces the Kdo\({}_{2}\)-lipid IV\({}_{\rm A}\) moiety which undergoes two late acylation reactions to give Kdo\({}_{2}\)-lipid A. Core oligosaccharides include is assembled on Kdo\({}_{2}\)-lipid A via sequential glycosyl transfer from nucleotide sugar precursors. Outer leaflet of IM: core-lipid A is translocated across the IM by MsbA transporter. Schematic representation of core lipid A: violet rectangle, Kdo; yellow heptagon, heptose; red hexagon, glucose; orange hexagon, galactose. In the inset the chemical structure of _E. coli_ K12 LPS is shown. largely depend on the same precursor molecule, R-3-hydroxymyristoyl ACP.