Outer Membrane Biogenesis Konovalova, Anna; Kahne, Daniel E; Silhavy, Thomas J
Annual review of microbiology,
09/2017, Letnik:
71, Številka:
1
Journal Article
Recenzirano
Odprti dostop
The hallmark of gram-negative bacteria and organelles such as mitochondria and chloroplasts is the presence of an outer membrane. In bacteria such as
Escherichia coli
, the outer membrane is a unique ...asymmetric lipid bilayer with lipopolysaccharide in the outer leaflet. Integral transmembrane proteins assume a β-barrel structure, and their assembly is catalyzed by the heteropentameric Bam complex containing the outer membrane protein BamA and four lipoproteins, BamB-E. How the Bam complex assembles a great diversity of outer membrane proteins into a membrane without an obvious energy source is a particularly challenging problem, because folding intermediates are predicted to be unstable in either an aqueous or a hydrophobic environment. Two models have been put forward: the budding model, based largely on structural data, and the BamA assisted model, based on genetic and biochemical studies. Here we offer a critical discussion of the pros and cons of each.
The outer membrane of Gram-negative bacteria is essential for their survival in harsh environments and provides intrinsic resistance to many antibiotics. This membrane is remarkable; it is a highly ...asymmetric lipid bilayer. The inner leaflet of the outer membrane contains phospholipids, whereas the fatty acyl chains attached to lipopolysaccharide (LPS) comprise the hydrophobic portion of the outer leaflet. This lipid asymmetry, and in particular the exclusion of phospholipids from the outer leaflet, is key to creating an almost impenetrable barrier to hydrophobic molecules that can otherwise pass through phospholipid bilayers. It has long been known that these lipids are not made in the outer membrane. It is now believed that conserved multisubunit protein machines extract these lipids after their synthesis is completed at the inner membrane and transport them to the outer membrane. A longstanding question is how the cell builds and maintains this asymmetric lipid bilayer in coordination with the assembly of the other components of the cell envelope. This Review describes the trans-envelope lipid transport systems that have been identified to participate in outer-membrane biogenesis: LPS transport via the Lpt machine, and phospholipid transport via the Mla pathway and several recently proposed transporters.
Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly by the glycan polymerase RodA and crosslinking enzyme ...PBP2. Using Escherichia coli as a model system, we identified a PBP2 variant that promotes Rod system function when essential accessory components of the machinery are inactivated. This PBP2 variant hyperactivates cell wall synthesis in vivo and stimulates the activity of RodA-PBP2 complexes in vitro. Cells with the activated synthase also exhibited enhanced polymerization of the actin-like MreB component of the Rod system. Our results define an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient cell wall assembly. In light of these results, previously isolated mutations that activate cytokinesis suggest that an analogous pathway may also control cell wall synthesis by the division machinery.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Highlights • We discuss the cellular role of lipopolysaccharide within Gram-negative bacteria. • We propose explanations for why lipopolysaccharide is essential in certain organisms. • We consider ...implications for developing lipopolysaccharide-targeting antibiotics.
The bacterial cell wall is conserved in prokaryotes, stabilizing cells against osmotic stress. Beta-lactams inhibit cell-wall synthesis and induce lysis through a bulge-mediated mechanism; however, ...little is known about the formation dynamics and stability of these bulges. To capture processes of different timescales, we developed an imaging platform combining automated image analysis with live-cell microscopy at high time resolution. Beta-lactam killing of Escherichia coli cells proceeded through four stages: elongation, bulge formation, bulge stagnation, and lysis. Both the cell wall and outer membrane (OM) affect the observed dynamics; damaging the cell wall with different beta-lactams and compromising OM integrity cause different modes and rates of lysis. Our results show that the bulge-formation dynamics are determined by how the cell wall is perturbed. The OM plays an independent role in stabilizing the bulge once it is formed. The stabilized bulge delays lysis and allows recovery upon drug removal.
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► Live-cell microscopy is used to monitor single-cell shape dynamics ► Beta-lactam-induced lysis proceeds through four distinct physical stages ► Dynamics of bulge formation and its stability vary for different beta-lactams ► Cells with stabilized bulges revert to rod shape upon drug removal
Gram-negative bacteria have a double-membrane cellular envelope that enables them to colonize harsh environments and prevents the entry of many clinically available antibiotics. A main component of ...most outer membranes is lipopolysaccharide (LPS), a glycolipid containing several fatty acyl chains and up to hundreds of sugars that is synthesized in the cytoplasm. In the past two decades, the proteins that are responsible for transporting LPS across the cellular envelope and assembling it at the cell surface in Escherichia coli have been identified, but it remains unclear how they function. In this Review, we discuss recent advances in this area and present a model that explains how energy from the cytoplasm is used to power LPS transport across the cellular envelope to the cell surface.
Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of many Gram-negative bacteria, providing a barrier against the entry of toxic molecules. In Escherichia coli, LPS is ...exported to the cell surface by seven essential proteins (LptA-G) that form a transenvelope complex. At the inner membrane, the ATP-binding cassette (ABC) transporter LptB
FG associates with LptC to power LPS extraction from the membrane and transfer to the periplasmic LptA protein, which is in complex with the OM translocon LptDE. LptC interacts both with LptB
FG and LptADE to mediate the formation of the transenvelope bridge and regulates the ATPase activity of LptB
FG. A genetic screen has previously identified suppressor mutants at a residue (R212) of LptF that are viable in the absence of LptC. Here, we present
evidence that the LptF R212G mutant assembles a six-protein transenvelope complex in which LptA mediates interactions with LptF and LptD in the absence of LptC. Furthermore, we present
evidence that the mutant LptB
FG complexes restore the regulation of ATP hydrolysis as it occurs in the LptB
FGC complex to achieve wild-type efficient coupling of ATP hydrolysis and LPS movement. We also show the suppressor mutations restore the wild-type levels of LPS transport both
and
, but remarkably, without restoring the affinity of the inner membrane complex for LptA. Based on the sensitivity of
suppressor mutants to selected stress conditions relative to wild-type cells, we show that there are additional regulatory functions of LptF and LptC that had not been identified.
The presence of an external LPS layer in the outer membrane makes Gram-negative bacteria intrinsically resistant to many antibiotics. Millions of LPS molecules are transported to the cell surface per generation by the Lpt molecular machine made, in E. coli, by seven essential proteins. LptC is the unconventional regulatory subunit of the LptB
FGC ABC transporter, involved in coordinating energy production and LPS transport. Surprisingly, despite being essential for bacterial growth, LptC can be deleted, provided that a specific residue in the periplasmic domain of LptF is mutated and LptA is overexpressed. Here, we apply biochemical techniques to investigate the suppression mechanism. The data produced in this work disclose an unknown regulatory function of LptF in the transporter that not only expands the knowledge about the Lpt complex but can also be targeted by novel LPS biogenesis inhibitors.
Gram-negative bacteria are impervious to many drugs and environmental stresses because they possess an outer membrane (OM) containing lipopolysaccharide (LPS). LPS is biosynthesized at the ...cytoplasmic (inner) membrane and is transported to the OM by an unknown mechanism involving the LPS transport proteins, LptA–G. These proteins have been proposed to form a bridge between the two membranes; however, it is not known how this bridge is assembled to prevent mistargeting of LPS. We use in vivo photo-cross-linking to reveal the specific protein–protein interaction sites that give rise to the Lpt bridge. We also show that the formation of this transenvelope bridge cannot proceed before the correct assembly of the LPS translocon in the OM. This ordered sequence of events may ensure that LPS is never transported to the OM if it cannot be translocated across it to the cell surface.
β-barrel membrane proteins in Gram-negative bacteria, mitochondria, and chloroplasts are assembled by highly conserved multi-protein complexes. The mechanism by which these molecular machines fold ...and insert their substrates is poorly understood. It has not been possible to dissect the folding and insertion pathway because the process has not been reproduced in a biochemical system. We purified the components that fold and insert Escherichia coli outer membrane proteins and reconstituted β-barrel protein assembly in proteoliposomes using the enzymatic activity of a protein substrate to report on its folding state. The assembly of this protein occurred without an energy source but required a soluble chaperone in addition to the multi-protein assembly complex.
Most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by polysaccharide polymerases called penicillin-binding proteins (PBPs). Because they are the targets of penicillin ...and related antibiotics, the structure and biochemical functions of the PBPs have been extensively studied. Despite this, we still know surprisingly little about how these enzymes build the PG layer in vivo. Here, we identify the Escherichia coli outer-membrane lipoproteins LpoA and LpoB as essential PBP cofactors. We show that LpoA and LpoB form specific trans-envelope complexes with their cognate PBP and are critical for PBP function in vivo. We further show that LpoB promotes PG synthesis by its partner PBP in vitro and that it likely does so by stimulating glycan chain polymerization. Overall, our results indicate that PBP accessory proteins play a central role in PG biogenesis, and like the PBPs they work with, these factors are attractive targets for antibiotic development.
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► Bacterial cell wall polymerases require protein cofactors for their in vivo function ► At least one of the E. coli cofactors, LpoA or LpoB, is required for cell wall assembly ► LpoA and LpoB form specific trans-envelope complexes with their cognate polymerase ► LpoB stimulates glycan chain polymerization by its partner polymerase
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