The lipopolysaccharide transport (Lpt) machinery : A nonconventional transporter for lipopolysaccharide assembly at the outer membrane of Gram-negative bacteria

The outer membrane (OM) of Gram-negative is a unique lipid bilayer containing LPS in its outer leaflet. Because of the presence of amphipathic LPS molecules, theOMbehaves as an effective permeability barrier that makes Gram-negative bacteria inherently resistant to many antibiotics. This review focuses on LPS biogenesis and discusses recent advances that have contributed to our understanding of how this complex molecule is transported across the cellular envelope and is assembled at the OM outer leaflet. Clearly, this knowledge represents an important platform for the development of novel therapeutic options to manage Gram-negative infections

Dissecting Escherichia coli outer membrane biogenesis using differential proteomics

The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality. \ua9 2014 Martorana et al

The Escherichia coli Lpt transenvelope protein complex for lipopolysaccharide export is assembled via conserved structurally homologous domains

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components

Peptidoglycan Remodeling Enables Escherichia coli To Survive Severe Outer Membrane Assembly Defect

Gram-negative bacteria have a tripartite cell envelope with the cytoplasmic membrane (CM), a stress-bearing peptidoglycan (PG) layer, and the asymmetric outer membrane (OM) containing lipopolysaccharide (LPS) in the outer leaflet. Cells must tightly coordinate the growth of their complex envelope to maintain cellular integrity and OM permeability barrier function. The biogenesis of PG and LPS relies on specialized macromolecular complexes that span the entire envelope. In this work, we show that Escherichia coli cells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG. This PG remodeling program relies mainly on the activities of the stress response LDT, LdtD, together with the major PG synthase PBP1B, its cognate activator LpoB, and the carboxypeptidase PBP6a. Our data support a model according to which these proteins cooperate to strengthen the PG in response to defective OM synthesis.IMPORTANCE In Gram-negative bacteria, the outer membrane protects the cell against many toxic molecules, and the peptidoglycan layer provides protection against osmotic challenges, allowing bacterial cells to survive in changing environments. Maintaining cell envelope integrity is therefore a question of life or death for a bacterial cell. Here we show that Escherichia coli cells activate the LD-transpeptidase LdtD to introduce 3-3 cross-links in the peptidoglycan layer when the integrity of the outer membrane is compromised, and this response is required to avoid cell lysis. This peptidoglycan remodeling program is a strategy to increase the overall robustness of the bacterial cell envelope in response to defects in the outer membrane

Lipopolysaccharide biogenesis and transport at the outer membrane of Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer containing a unique glycolipid, lipopolysaccharide (LPS) in its outer leaflet. LPS molecules confer to the OM peculiar permeability barrier properties enabling Gram-negative bacteria to exclude many toxic compounds, including clinically useful antibiotics, and to survive harsh environments. Transport of LPS poses several problems to the cells due to the amphipatic nature of this molecule. In this review we summarize the current knowledge on the LPS transport machinery, discuss the challenges associated with this process and present the solutions that bacterial cells have evolved to address the problem of LPS transport and assembly at the cell surface. Finally, we discuss how knowledge on LPS biogenesis can be translated for the development of novel antimicrobial therapies. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop

The Lpt ABC transporter for lipopolysaccharide export to the cell surface

The surface of the outer membrane of Gram-negative bacteria is covered by a tightly packed layer of lipopolysaccharide molecules which provide a barrier against many toxic compounds and antibiotics. Lipopolysaccharide, synthesized in the cytoplasm, is assembled in the periplasmic leaflet of the inner membrane where the intermembrane Lpt system mediates its transport to the cell surface. The first step of lipopolysaccharide transport is its extraction from the outer leaflet of inner membrane powered by the atypical LptB2FGC ABC transporter. Here we review latest advances leading to understanding at molecular level how lipopolysaccharide is transported irreversibly to the outer membrane

Transcriptional analysis of a locus involved in LPS biosynthesis and transport to the outer membrane of E. coli

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer that interacts with the environment and helps preserving the cell integrity. Lipopolysaccharide (LPS) is the major essential component of the OM. Although its structure and biosynthesis are well known, the transport mechanism of this molecule from the inner membrane to the OM are poorly understood. We previously showed that all genes except yrbG in a locus composed of yrbG-kdsD-kdsC-lptC-lptA-lptB are involved in LPS biosynthesis and transport and obtained genetic evidence that at least three promoters are scattered within this locus. We now analyzed in more detail the transcriptional organization of the locus. The picture emerging is that this region seems to be transcribed from five promoters: yrbGp, kdsCp1, kdsCp2, lptAp1 and lptAp2, with different sigma factors implicated in their regulation. This complex transcriptional organization suggests that the different genes of this locus may be finely and differentially regulated. Work is in progress to understand which environmental conditions may modulate the expression patterns of these genes

Complex transcriptional organization regulates an Escherichia coli locus implicated in lipopolysaccharide biogenesis

The Escherichia coli yrbG-lptB locus (yrbG kdsD kdsC lptC lptA lptB) encodes genes for outer membrane biogenesis, namely, kdsC and kdsD for biosynthesis of the lipopolysaccharide inner core sugar Kdo, and lptA, lptB, and lptC for lipopolysaccharide transport to the outer membrane. Three promoters (yrbGp, kdsCp and the \u3c3E-dependent lptAp) have been previously identified by genetic analysis. In this work, we show that transcription of this locus generates an array of overlapping mRNAs and we characterize the two intralocus promoter regions. In the kdsCp region, we identified three promoters (kdsCp1, kdsCp2, and kdsCp3) scattered within about 600nt in the 3\u2032-coding region of kdsD. The lptAp region is composed of two closely spaced promoters, lptAp1 and lptAp2. The former had been previously identified as a \u3c3E-dependent promoter. Interestingly, lptAp1 is not activated by several stressful conditions that normally induce the \u3c3E-dependent envelope stress response, whereas it seems to respond to conditions affecting lipopolysaccharide biogenesis, thus implying a specialized \u3c3E-dependent LPS stress signaling pathway