# Regulated Assembly of LPS, Its Structural Alterations and Cellular Response to LPS Defects
## 10 Mechanism of Signal Transduction
Analysis of various other response regulators did not show any direct participation [11]. The activity of the same _rpoE_P3 promoter was found to be induced nearly 3-4 fold in \(\Delta\)_rfuH_ background and by the overexpression of _rirA_ sRNA [11]. The 73 nt RiraA sRNA contains _ops_ element, located in the 5' UTR of _waaQ_ operon and acts by decreasing the RfaH activity, by directly binding to RfaH in the presence of RNA polymerase. The overexpression of _rirA_ causes a reduction in the expression of operons that require the RfaH activity. When RirA is in excess, it causes a decrease in the expression of _waaQ_ operon members leading to truncation of the LPS core and abolishes the incorporation of _O_-antigen, explaining the specific induction of LPS responsive _rpoE_P3 promoter [11]. Additional evidence that the _rpoE_P3 promoter responds to LPS-specific alterations is based on an increase in transcription from this promoter when bacteria are challenged in early growth phase by either polymyxin B or AMV. The induction of RpoE regulon concomitant with Rcs pathway activation is also observed when any component of Lpt is depleted, since under such conditions LPS is modified by the Rcs-regulated colonic acid excopolysaccharide [85]. A direct proof that the _rpoE_P3 promoter is positively regulated by the Rcs system comes from in vitro DNA binding assays with purified RcsB, establishing the presence of conserved RcsB motif in the _rpoE_P3 promoter region [11]. This was supported by deletion and mutational analysis of RcsB-binding motif. Run-off assays established the authenticity of recognition by the house-keeping sigma factor and mutational analysis reinforced that RcsB acts as a positive regulator in response to LPS defects. Transcription from the _rpoE_P3 promoter is also activated in \(\Delta\)(_lapA-lapB_) which also requires RcsB. It should be noted that the RpoN-regulated _rpoE_P2 promoter also responds to LPS defects albeit to a much-reduced extent (only a 70% to 2-fold increase as compared to the 7-fold induction by \(\Delta\)_xuaaC_) and that too mainly upon entry into the stationary phase. Since the _rpo_EP2 promoter requires QseF as an activator [11], it will be interesting to know how the Qse TCS senses LPS. A role for sensing of LPS defects by the Rcs signalling pathway was proposed in earlier studies by examining a mutant strain carrying _rfa-1_ mutation. Such a strain synthesizes LPS composed of only two heptose residues lacking any glucose in the core region and exhibits induction of _cps_ genes leading to overexpression of excopolysaccharide [80]. Furthermore, it was shown that this LPS defect requires the outer membrane lipoprotein RcsF, since induction of cps transcription in _rfa-1_ mutant was abolished in \(\Delta\)_rcsF_ mutants [80]. Interestingly, overexpression of _cps_ genes due to LPS defects did not occur due to the increased _rcsF_ transcription, rather could be the consequence of other mechanisms of the RcsF activation. More recent studies on RcsF revealed that it exists in complex with many OMPs [86] and such RcsF complex uses its positively charged, surface-exposed N-terminal domain to detect alterations in lateral interactions between LPS molecules (changes in phosphorylation status of the LPS core), which lead to the induction of Rcs system. This activation of Rcs pathway in turn can activate RcsB to induce the _rpoE_P3 promoter when LPS defects are encountered (Figure 3). Another model postulates that the periplasmic RseB binds to LPS that might be released in an off pathway during translocation via the Lpt system [84]. Usually, RseB association with RseA inhibits its proteolysis. However, LPS binding to RseB relives inhibition of proteolysis by disassociation of the RseA-RseB complex (Figure 3).