# Lipopolysaccharide: Recent Advances in Its Biosynthesis and Controlling Cell Envelope Homeostasis
###### Abstract
Although the main regulation of LpxC occurs post-transcriptionally up to a 20-fold level [35], at the transcriptional level, SoxS induction can increase _lpxC_ gene expression, which causes cell lysis [36]. Consistent with these findings, the accumulation of SulA in the absence of Lon protease in nitrofurantoin-treated bacteria increased LPS synthesis and transporter-related gene expression, resulting in increased LPS production [37]. Another post-transcriptional regulator that requires further study is the GcvB sRNA-mediated control of LpxC. Overexpression of GcvB sRNA represses the accumulation of LpxC and suppresses the lethality of LpAB absence [38]. Interestingly, LpxC can also be subjected to proteolysis independent of FtsH-LapB by the HsIVU protease in _E. coli_[39], HsIVU-mediated proteolysis of LpxC may be particularly important for regulating LPS synthesis at high temperatures, as the activity of this protease is enhanced under these growth conditions. (v) New insights in LPS transport machinery: It is well established that the essential inner membrane protein MsbA plays a pivotal role in flipping the LPS precursor from the cytoplasmic to the periplasmic leaflet of the IM. LPS flipping involves the binding of LPS to the interior binding site and a conformational change from an inward- to an outward-facing conformation [40; 41]. These studies further revealed that MsbA has two LPS-binding sites: one located in the central cavity and the other at a membrane-facing exterior site [40; 41]. More recent studies have further probed these MsbA-LPS interactions using native mass spectrometry with Kdo\({}_{2}\)-LA precursor as a substrate [42]. Kdo\({}_{2}\)-lipid A can tune the selectivity of MsbA for ATP over ADP [42]. Another interesting recent study addressed the interplay between TMDs and NBDs of MsbA by examining the involvement of two intracellular loops, coupling helix 1 and 2 (CH1, CH2) [43]. The authors observed significant chemical shift changes within both CH1 and CH2 upon substrate binding, in the ATP hydrolysis transition state, and upon MsbA inhibitor binding [43]. Once LPS is flipped to the periplasmic leaflet of the IM, it is further extracted by the Lpt protein machinery, which comprises seven essential Lpt proteins (LptB\({}_{2}\)FGCADE) that function together as a transenvelope bridge. Four of the proteins, LptB\({}_{2}\)FGC, form an ABC transporter that extracts LPS from the IM [44]. After extraction from the IM, LPS is transferred from the periplasmic domain of the IM-anchored LptC to LptA in the periplasm and subsequently delivered to the OM protein complex LptDE [45]. In the last decade, several studies have shown that the proper functioning of the Lpt bridge is the assembly and maturation of LptD, which requires disulfide bond-forming proteins (Dsbs) [46]. Recently, another lipoprotein, LptM, was shown to facilitate the rearrangement of disulfide bonds in LptD, adding a new layer of regulation [47]. Latest structural studies of LptDE-LptM suggest that LptM translocates to the OM via LptDE, in a manner similar to LPS transport [48]. (vi) Targeting LPS biosynthesis and transport for antibiotic discovery: The emergence of multidrug resistance in ESKAPE pathogens that include Gram-negative bacteria, such as _Acinetobacter_ and _Pseudomonas_, poses a new challenge for human health. As LPS is essential in the majority of Gram-negative bacteria and several proteins involved in LPS biogenesis, assembly, and transport are unique and required for bacterial growth, they have become important targets for the discovery of new antibiotics. Thus, the discovery of inhibitors such as CHIR09, which targets LpxC, mediating the first committed step in LPS biosynthesis, proved vital in this direction. However, many LpxC inhibitors exhibit unexpected cardiovascular toxicity, hampering their use.