# Regulated Assembly of LPS, Its Structural Alterations and Cellular Response to LPS Defects
## 10 Mechanism of Signal Transduction  
Here, we will discuss the activation of RpoE regulon in response to LPS defects with an overlap in the activation of the Rcs phosphorelay system, which jointly respond to LPS defects (Figure 3). We do not know how the Cpx system senses LPS defects as it can respond to several envelope stress signals in the IM, periplasm and OM. One of the main observations has been that only those structural LPS changes/defects that induce the Rcs system also induce RpoE. Thus, induction of PhoB/R-dependent GlcUA incorporation, BasS/R-dependent lipid A modifications, lack of either WaaY, or WaaZ or WaaS or truncation of outer core neither induce the Rcs system nor the RpoE-regulated stress response [11; 55]. In a comprehensive study, a systematic approach of dissection of the transcriptional regulation of the _rpoE_ gene and stimuli that induce or repress was taken based on previously observed RpoE induction in \(\Delta\)_xwaA_ or strains synthesizing Kdo\({}_{2}\)-lipid IV\({}_{A}\) derivatives [7; 11]. When the _rpoE_ gene was initially identified, two complementary studies showed that transcription of the _rpoE_ gene is positively autoregulated from one of its promoters since it is recognized by E\({}^{E}\) polymerase and its activity is induced when OMP maturation is dysfunctional or due to an imbalance in OMP expression [81; 82]. However, the regulation of distal promoter or promoter region (previously called the _rpoE_P1 promoter) remained elusive. Besides positive transcriptional regulation, RpoE is negatively regulated by ResA and ResB proteins, whose encoding genes are co-transcribed with the _rpoE_ gene [83]. The ResA inner membrane protein associates via its cytoplasmic N-terminal domain with RpoE, thereby sequestering RpoE in the IM under non-stress conditions [83]. This inhibitory effect of ResA is relieved upon OMP misfolding or overexpression of some OMPs by sequential degradation of ResA initiated by DegS in the periplasm, followed by the second site inner membrane proteolysis by ResP (EcfE) and subsequent degradation of the N-terminal ResA in the cytoplasm [84]. The periplasmic C-terminal domain of ResA associates with ResB and prevents RseA degradation. Since the autoregulated _rpoE_ promoter (P6 promoter), previously called _rpoE_P2 promoter, responds to effective concentration of available RpoE that complexes with core RNA polymerase, its activity reflects both transcriptional increase in the _rpoE_ expression and its release from ResA and hence its activity or activity of reporter promoters of its regulon members (_rpoH_P3) does not directly reflect response to LPS defects. During the studies aimed to understand the mechanism of _rpoE_ induction in response to LPS defects and examination of transcriptional regulation of upstream element, it turned out that the _rpoE_ gene is transcribed from six promoters and, among these, activity of _rpoE_P2 and _rpoE_P3 promoters is specifically induced when LPS is defective (Figure 3). Among these, transcription from the _rpoE_P3 promoter is significantly induced when either the LPS core biogenesis is defective or when LPS assembly is compromised or by challenge with LPS-binding antibiotic like polymyxin B or treatment with agents like AMV that induce non-specifically different TCS [11]. Global mutational screen identified insertion/deletion mutations mapping to genes involved in the early steps of LPS core biosynthesis such as _waaC_, _waaF_, _waaG_, _waaO_ and _waaP_. Among these, maximal 7-fold induction of the _rpoE_P3 promoter was observed in \(\Delta\)_xwaaC_ mutants synthesizing Kdo\({}_{2}\)-lipid IV\({}_{A}\)[11]. This activation was nearly abrogated in \(\Delta\)(_xwaC rcsB_) mutants suggesting that the signal of activation due to defective LPS requires the RcsB response regulator [11]. Interestingly, mutations in either _waaC_ or _waaF_ or _waaP_ exhibit deep-rough phenotype accompanied by the induction of Rcs-regulated exopolysaccharideacharide synthesis.