# Regulated Assembly of LPS, Its Structural Alterations and Cellular Response to LPS Defects
## 8 Regulated Structural Alterations in LPS
### Regulation of Lipid A Modifications  
Most prevalent non-stoichiometric modifications that occur in the lipid A part involve either reducing the net negative charges of lipid A by modifying the 1 and/or 4\({}^{\prime}\) ends of phosphate residues, or the addition or removal of acyl chains. Most commonly observed modifications of lipid A include the incorporation of P-EtN and l-Ara4N at 1 and/or 4\({}^{\prime}\) ends, respectively [57]. Such substitutions are known to confer resistance to cationic antimicrobial peptides like polymycin B. This non-stoichiometric incorporation of P-EtN and l-Ara4N residues requires IM-located EptA and ArnT transferases, respectively with active site facing the periplasm [57]. Genes encoding these transferases are part of operons, whose transcription is positively regulated by the BasS/R (PmrA/B) TCS with an overlap with the PhoP/Q system. These systems specifically respond to changes in Fe\({}^{3+}\) and divalent cationic concentrations, respectively. The PhoP/Q system in _Salmonella_ and in some pathogenic Gram-negative bacteria regulates the expression of virulence genes, including those encoded in pathogenic islands. Lipid A of strains synthesizing tetraacylated lipid A can have phosphate residues at 1 and/or 4\({}^{\prime}\) ends modified by P-EtN, since they do not incorporate l-Ara4N due to defects in translocation and such lipid A species could be poor substrates for ArnT. Such modifications occur after the translocation at the periplasmic side and often serve as markers for LPS translocation with l-Ara4N incorporation as a more stringent signature [7]. In _E. coli_ and _Salmonella_, the activation of PhoP/Q TCS upon depletion of Mg\({}^{2+}\) and Ca\({}^{2+}\) also leads to the BasS/R induction, which requires PmrD as the adaptor protein [58]. Such a cross talk between BasS/R and PhoP/Q TCSs allows integration of signals from different environmental cues and amplification of output response. The majority of lipid A in _E. coli_ K-12 contains monophosphate at positions 1 and 4\({}^{\prime}\). Approximately one-third of lipid A molecules in the _E. coli_ K-12 outer membrane contains a diphosphate unit at the 1 position. The enzyme LpxT is responsible for this phosphorylation at 1\({}^{\prime}\) position, generating hexaacylated lipid A with two phosphate residues under ambient growth conditions in the absence of induction of lipid A modification systems increasing the negative charge [59]. However, upon induction of the BasS/R system, P-EtN is incorporated at 1\({}^{\prime}\) position due to inhibition of the LpxT activity. This inhibition of LpxT activity upon BasS/R-inducing conditions is due to the expression of a short peptide PmrR, which directly bind to LpxT [60] and hence constitutes a post-translational control [13]. In _Salmonella_, PhoP activation can also positively regulate _lpxT_ transcription. The PhoP-dependent _lpxT_ expression induced in low Mg\({}^{2+}\) results in 1-PP lipid A, favors further modification of lipid Aphosphates with l-Ara4N and the exclusion of P-EtN. Thus, _Salmonella_ favors lipid A modified with l-Ara4N under low Mg\({}^{2+}\) and with both l-Ara4N and P-EtN when exposed to a mildly acidic pH [61]. As RpoE and LPS structural alterations are intricately linked, an interesting regulatory control of lipid A alteration by RpoE-regulated sRNAs has emerged. Structural analysis of LPS obtained from several different strains under simultaneous RpoE- and BasS/R-inducing conditions shows the absence of LpxT-dependent phosphorylation of lipid A, presumably due to the transcriptional induction of _micA_[62]. The RpoE-regulated McA sRNA can also exert an influence on lipid A composition by connecting regulation of PhoP/Q TCS as well as LpxT to RpoE. Analysis of McA targets revealed that besides known OMP-encoding genes, McA represses PhoP synthesis by base pairing in the translation initiation region of _phoP_ mRNA and inhibits its translation [63].