Background:The mechanism of coordination between LPS synthesis and translocation is unknown. Results: Two new proteins, LapA and LapB, co-purify with LPS transport proteins. lapB mutants display defects in lipid A and core assembly. Conclusion: lapB mutants accumulate precursor LPS core species and exhibit elevated levels of LpxC. Significance: Coordinated assembly of LPS is a critical step for targeting to the outer membrane.

To elucidate the minimal lipopolysaccharide (LPS) structure needed for the viability of Escherichia coli, suppressor-free strains lacking either the 3-deoxy-D-manno-oct-2-ulosonic acid transferase waaA gene or derivatives of the heptosyltransferase I waaC deletion with lack of one or all late acyltransferases (lpxL/M/P) and/or various outer membrane biogenesis factors were constructed. ⌬(waaC lpxL lpxM lpxP) and waaA mutants exhibited highly attenuated growth, whereas simultaneous deletion of waaC and surA was lethal. Analyses of LPS of suppressorfree waaA mutants grown at 21°C, besides showing accumulation of free lipid IV A precursor, also revealed the presence of its pentaacylated and hexaacylated derivatives, indicating in vivo late acylation can occur without Kdo. In contrast, LPS of ⌬(waaC lpxL lpxM lpxP) strains showed primarily Kdo 2 -lipid IV A , indicating that these minimal LPS structures are sufficient to support growth of E. coli under slow-growth conditions at 21/23°C. These lipid IV A derivatives could be modified biosynthetically by phosphoethanolamine, but not by 4-amino-4-deoxy-L-arabinose, indicating export defects of such minimal LPS. ⌬waaA and ⌬(waaC lpxL lpxM lpxP) exhibited cell-division defects with a decrease in the levels of FtsZ and OMP-folding factor PpiD. These mutations led to strong constitutive additive induction of envelope responsive CpxR/A and E signal transduction pathways. ⌬(lpxL lpxM lpxP) mutant, with intact waaC, synthesized tetraacylated lipid A and constitutively incorporated a third Kdo in growth medium inducing synthesis of P-EtN and L-Ara4N. Overexpression of msbA restored growth of ⌬(lpxL lpxM lpxP) under fast-growing conditions, but only partially that of the ⌬(waaC lpxL lpxM lpxP) mutant. This suppression could be alleviated by overexpression of certain mutant msbA alleles or the single-copy chromosomal MsbA-498V variant in the vicinity of Walker-box II.

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.

The RpoE factor is essential for the viability of Escherichia coli. RpoE regulates extracytoplasmic functions including lipopolysaccharide (LPS) translocation and some of its non-stoichiometric modifications. Transcription of the rpoE gene is positively autoregulated by E E and by unknown mechanisms that control the expression of its distally located promoter(s). Mapping of 5 ends of rpoE mRNA identified five new transcriptional initiation sites (P1 to P5) located distal to E E -regulated promoter. These promoters are activated in response to unique signals. Of these P2, P3, and P4 defined major promoters, recognized by RpoN, RpoD, and RpoS factors, respectively. Isolation of trans-acting factors, in vitro transcriptional and gel retardation assays revealed that the RpoN-recognized P2 promoter is positively regulated by a QseE/F two-component system and NtrC activator, whereas the RpoD-regulated P3 promoter is positively regulated by a Rcs system in response to defects in LPS core biosynthesis, overproduction of certain lipoproteins, and the global regulator CRP. Strains synthesizing Kdo 2 -LA LPS caused up to 7-fold increase in the rpoEP3 activity, which was abrogated in ⌬(waaC rcsB). Overexpression of a novel 73-nucleotide sRNA rirA (RfaH interacting RNA) generated by the processing of 5 UTR of the waaQ mRNA induces the rpoEP3 promoter activity concomitant with a decrease in LPS content and defects in the O-antigen incorporation. In the presence of RNA polymerase, RirA binds LPS regulator RfaH known to prevent premature transcriptional termination of waaQ and rfb operons. RirA in excess could titrate out RfaH causing LPS defects and the activation of rpoE transcription.The cell envelope of Gram-negative bacteria, including Escherichia coli, contains two distinct membranes, an inner (IM) 3 and an outer (OM) membrane separated by the periplasm, a hydrophilic compartment that includes a layer of peptidoglycan. The OM is an asymmetric lipid bilayer with phospholipids forming the inner leaflet and LPS forming the outer leaflet. LPSs are highly heterogeneous in composition. However, they share a common architecture composed of a membrane-anchored phosphorylated and acylated ␤(136)-linked GlcN disaccharide, termed lipid A, to which a carbohydrate moiety of varying size is attached (1, 2).During the analyses of signals that induce the RpoE-dependent extracytoplasmic stress response, we showed that strains synthesizing heptoseless LPS due to the absence of either GmhD (RfaD/HtrM) or WaaC exhibited a constitutive induction of RpoE (3-6). Such strains also display constitutive synthesis of exopolysaccharide that is regulated by the Rcs twocomponent system (5,7,8). Subsequently, we showed that strains synthesizing the minimal LPS structure composed of either Kdo 2 -lipid IV A or only free lipid IV A exhibited hyperelevated levels of the RpoE activity (6). The activity of RpoE is highly induced when the LPS assembly is severely compromised or when there is an imbalance between LPS and phospholipids (9). Lack of many LPS co...