# Lipopolysaccharide: Recent Advances in Its Biosynthesis and Controlling Cell Envelope Homeostasis
###### Abstract
The suitability of various techniques, including visualization of differences in various chemotypes by SDS-PAGE analysis, structural analysis by MALDI-MS, and chemical methods for quantification, was evaluated. The authors describe in detail the structural variability of LPS with examples of two _Yersinia_ strains grown at different temperatures and LPSs from other genera, particularly variations in the acylation of the lipid A part of LPS. Furthermore, the impact of different LPS structures obtained under different bacterial growth conditions on selective LPS detection methods, such as Limulus Amoebocyte Lysate (LAL), HEK-blueTLR-4, LC-MS\({}^{2}\), and MALDI-MS, is described. As expected, LPSs responded differently in these assays. This was attributed to the properties of lipid A structures, including the length and number of acyl chains, the presence of phosphate groups, and their possible substitutions. The impact of structural differences on the toxicity of LPS was shown by cytokine induction and MAT or HEK-Blue hTLR4 signalization. However, the LAL assay did not respond to LPS modifications and did not differentiate between toxic and non-toxic LPS forms, although it exhibited the highest sensitivity. Additionally, HEK-Blue hTLR4 was shown to be more specific for measuring potential LPS toxicity, and its use along with the LAL assay is suggested. In continuation of the theme of diversity of LPS structure, another manuscript describes the structural properties of the LPS from the rhizosphere bacterium _Oclroductrum quorumocens_ T1 Kr02 [68]. Analysis of the fatty acid composition of LPS revealed the presence of 3-hydroxytetradacanoic, hexadecenoic, octadecenoic, and 27-hydroxyoctacosaoic acids. The authors analyzed the structure of its \(O\)-polysaccharide (PS-1). The remarkable feature of this polysaccharide is that it contains d-Fuc\(p\) and d-Fuc\(f\) residues, with 50% \(O\)-acetylation of d-Fuc. These findings are significant because d-Fuc monosaccharides are relatively rare components of bacterial polysaccharides. This study also examined the physicochemical properties of its LPS in aqueous solution using the dynamic light-scattering method. The results showed that LPS particles had a diameter of 72.2 \(\pm\) 3.6 nm and a zeta-potential of \(-\)21.5 \(\pm\) 0.7 mV, which can be a useful marker for future comparison of strains from related bacteria. Regarding biological activity, it was shown that LPS from _O. quorumocens_ strain T1 Kr02 improves the development of microplants in vitro and hence could be used to increase the efficiency of microclonal propagation of potato. In the manuscript by [PERSON] et al., the authors evaluated the impact of variants of antimicrobial peptides cathelicidins (As-CATHs) obtained from _Alligator sinensis_ on the membranes of Gram-negative bacteria [69]. The authors used As-CATH4, AS4-1, AS4-5, and AS4-9 peptides with decreasing charges but increasing hydrophobicity and evaluated them by performing molecular dynamics simulations. All four variant peptides disrupted the structures of the IM of bacteria, with AS4-9 exhibiting the highest antibacterial activity. As the AS4-9 peptide has moderate charges and hydrophobicity, it significantly perturbs the membrane structure by squeezing the lipids in the proximal leaflet and can even extract them from the membrane at sufficiently high peptide concentrations. The authors postulate that the binding of AS4-9 promotes PL flipping from the inner leaflet to the outer leaflet, changing OM asymmetry. In this manner, the AS4-9 peptide could bind to PL and extract it from the membrane. This mode of action could promote their antibacterial activity and allow the future construction of novel peptide-based antibiotics. In an extension of previous work on LapC- and LapD-mediated regulation of LpxC, [PERSON] et al. used new strategies to identify additional players that could mediate LpxC regulation [70].