Regulated Assembly of LPS, Its Structural Alterations and Cellular Response to LPS Defects

Abstract

Distinguishing feature of the outer membrane (OM) of Gram-negative bacteria is its asymmetry due to the presence of lipopolysaccharide (LPS) in the outer leaflet of the OM and phospholipids in the inner leaflet. Recent studies have revealed the existence of regulatory controls that ensure a balanced biosynthesis of LPS and phospholipids, both of which are essential for bacterial viability. LPS provides the essential permeability barrier function and act as a major virulence determinant. In Escherichia coli, more than 100 genes are required for LPS synthesis, its assembly at inner leaflet of the inner membrane (IM), extraction from the IM, translocation to the OM, and in its structural alterations in response to various environmental and stress signals. Although LPS are highly heterogeneous, they share common structural elements defining their most conserved hydrophobic lipid A part to which a core polysaccharide is attached, which is further extended in smooth bacteria by O-antigen. Defects or any imbalance in LPS biosynthesis cause major cellular defects, which elicit envelope responsive signal transduction controlled by RpoE sigma factor and two-component systems (TCS). RpoE regulon members and specific TCSs, including their non-coding arm, regulate incorporation of non-stoichiometric modifications of LPS, contributing to LPS heterogeneity and impacting antibiotic resistance.

Keywords: LapB; Lpt transport system; LpxC; Rcs two-component system; RpoE sigma factor; lipid A modifications; lipid IVA; noncoding small regulatory RNA.

Figure 1

Networks of regulatory pathways that control the lipopolysaccharide (LPS) assembly and its non-stoichiometric modifications. The RpoE sigma factor responds to severe defects in LPS biosynthesis and is also required for transcription of genes involved in LPS biosynthesis/translocation and modifications via sRNAs like rybB and micA. RpoE also transcribes the slrA (micL) sRNA, which represses the Lpp synthesis and acts in a feedback manner to repress RpoE. Other regulatory controls involve two-component systems like BasS/R, PhoP/Q, PhoB/R and Rcs system, which are required for transcription of genes whose products are involved in lipid A and inner core modifications. The RpoH heat shock sigma factor transcribes lapB and ftsH genes, whose products control balanced biosynthesis of LPS and phospholipid by regulating LpxC levels. The unique transcriptional factor RfaH is required for overcoming antitermination, enhance transcriptional elongation and couple transcription/translation of waaQ and rfb LPS biosynthetic operons. The sRNA RirA binds to RfaH and abrogates its activity to maintain a balanced biosynthesis of LPS.

Figure 2

Schematic depiction of unmodified hexaacylated glycoform I and glycoform derivatives with various non-stoichiometric substitutions with a third Kdo-Rha disaccharide. Various genes whose products mediate different steps in LPS biosynthesis and incorporation of different modifications are indicated. Glycoform I constitutes the major LPS species under non-stress conditions (A). Upon the induction of RpoE sigma factor and conditions inducing BasS/R and PhoB/R TCSs, the lipid A part is modified by P-EtN and l-Ara4N at 1 and 4′ position by EptA and ArnT, respectively (B). The induction of RpoE causes increased synthesis of EptB overcoming MgrR silencing and incorporation of P-EtN on the second Kdo (panels B1 and B2). The induction of RpoE favors pathway of the third Kdo incorporation by increased synthesis of WaaZ and repression of WaaR by the RybB sRNA, leading to synthesis of glycoforms with a third Kdo with attachment of Rha on the third Kdo with a concomitant truncation of the terminal GlcIII-HepIV disaccharide shown in violet background. The PhoB/R induction leads to the incorporation of GlcUA at the expense of HepII phosphorylation (blue background). Lipid A can also be modified by PagP generating heptaacylated lipid A and by the removal of acyl chains by LpxR in the OM after translocation (light brown background). Various sRNA-mediated controls are shown in blue color.

Figure 3

Pathways of sensing defects in LPS biosynthesis/assembly: sensing via the Rcs phosphorelay system, which leads to the induction of transcription from the sigma 70-regulated rpoEP3 promoter requiring RcsB as activator and the activation of Qse TCS induces the rpoEP2 promoter with QseF acting as an activator of RpoN. Severe defects in LPS induce the Rcs phosphorelay cascade leading to phosphorylation of RcsB. Phosphorylated RcsB acts as an activator for the transcription directed from the rpoEP3 promoter leading to the induction of RpoE-mediated stress response. The overlapping rpoEP2 promoter also responds to LPS defects albeit to a lower extent. Severe defects in LPS also induce the CpxR/A TCS. Eσ^E is required to maintain envelope integrity by regulating expression of genes whose products are involved in protein folding in the periplasm, assembly of OMPs (bam genes) and certain genes whose products are involved in LPS biosynthesis/modifications. Release of LPS from the Lpt system can lead to association of LPS with RseB, which relieves inhibition of RseA proteolysis and can lead to accumulation of free RpoE without RseA.