scholarly journals Insights into bacterial lipoprotein trafficking from a structure of LolA bound to the LolC periplasmic domain

2018 ◽  
Vol 115 (31) ◽  
pp. E7389-E7397 ◽  
Author(s):  
Elise Kaplan ◽  
Nicholas P. Greene ◽  
Allister Crow ◽  
Vassilis Koronakis
Keyword(s):  
Outer Membrane ◽  
Abc Transporter ◽  
Gram Negative Bacteria ◽  
Outer Membrane Receptor ◽  
Outer Leaflet ◽  
Periplasmic Domain ◽  
Cellular Integrity ◽  
To Receive ◽  
Membrane Lipoproteins ◽  
Important System

In Gram-negative bacteria, outer-membrane lipoproteins are essential for maintaining cellular integrity, transporting nutrients, establishing infections, and promoting the formation of biofilms. The LolCDE ABC transporter, LolA chaperone, and LolB outer-membrane receptor form an essential system for transporting newly matured lipoproteins from the outer leaflet of the cytoplasmic membrane to the innermost leaflet of the outer membrane. Here, we present a crystal structure of LolA in complex with the periplasmic domain of LolC. The structure reveals how a solvent-exposed β-hairpin loop (termed the “Hook”) and trio of surface residues (the “Pad”) of LolC are essential for recruiting LolA from the periplasm and priming it to receive lipoproteins. Experiments with purified LolCDE complex demonstrate that association with LolA is independent of nucleotide binding and hydrolysis, and homology models based on the MacB ABC transporter predict that LolA recruitment takes place at a periplasmic site located at least 50 Å from the inner membrane. Implications for the mechanism of lipoprotein extraction and transfer are discussed. The LolA–LolC structure provides atomic details on a key protein interaction within the Lol pathway and constitutes a vital step toward the complete molecular understanding of this important system.

scholarly journals Robust Suppression of Lipopolysaccharide Deficiency in Acinetobacter baumannii by Growth in Minimal Medium

2019 ◽  
Vol 201 (22) ◽  
Author(s):  
Emma Nagy ◽  
Richard Losick ◽  
Daniel Kahne
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Minimal Medium ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Growth Defects ◽  
Outer Leaflet ◽  
Morphological Defects ◽  
Outer Membrane Biogenesis

ABSTRACT Lipopolysaccharide (LPS) is normally considered to be essential for viability in Gram-negative bacteria but can be removed in Acinetobacter baumannii. Mutant cells lacking this component of the outer membrane show growth and morphological defects. Here, we report that growth rates equivalent to the wild type can be achieved simply by propagation in minimal medium. The loss of LPS requires that cells rely on phospholipids for both leaflets of the outer membrane. We show that growth rate in the absence of LPS is not limited by nutrient availability but by the rate of outer membrane biogenesis. We hypothesize that because cells grow more slowly, outer membrane synthesis ceases to be rate limiting in minimal medium. IMPORTANCE Gram-negative bacteria are defined by their asymmetric outer membrane that consists of phospholipids on the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. LPS is essential in all but a few Gram-negative species; the reason for this differential essentiality is not well understood. One species that can survive without LPS, Acinetobacter baumannii, shows characteristic growth and morphology phenotypes. We show that these phenotypes can be suppressed under conditions of slow growth and describe how LPS loss is connected to the growth defects. In addition to better defining the challenges A. baumannii cells face in the absence of LPS, we provide a new hypothesis that may explain the species-dependent conditional essentiality.

A new mechanism for membrane iron transport in Pseudomonas aeruginosa

2002 ◽  
Vol 30 (4) ◽  
pp. 702-705 ◽  
Author(s):  
I.J. Schalk ◽  
M. A. Abdallah ◽  
F. Pattus
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Iron Uptake ◽  
Iron Transport ◽  
Membrane Receptor ◽  
Gram Negative Bacteria ◽  
Outer Membrane Receptor ◽  
Uptake Mechanism ◽  
Atp Binding Cassette Transporter ◽  
Biophysical Studies

Various biochemical and biophysical studies have demonstrated the existence of a novel iron-uptake mechanism in Pseudomonas aeruginosa, different from that generally described for ferrichrome and ferric-enterobactin in Escherichia coli. This new iron-uptake mechanism involves all the proteins generally reported to be involved in the uptake of ferric-siderophore complexes in Gram-negative bacteria (i.e. the outer membrane receptor, periplasmic binding protein and ATP-binding-cassette transporter), but differs in the behaviour of the siderophore. One of the key features of this process is the binding of iron-free pyoverdin to the outer membrane receptor FpvA in conditions of iron deficiency.

scholarly journals Functional Analysis of the Protein Machinery Required for Transport of Lipopolysaccharide to the Outer Membrane of Escherichia coli

2008 ◽  
Vol 190 (13) ◽  
pp. 4460-4469 ◽  
Author(s):  
Paola Sperandeo ◽  
Fion K. Lau ◽  
Andrea Carpentieri ◽  
Cristina De Castro ◽  
Antonio Molinaro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Gram Negative Bacteria ◽  
Membrane Structures ◽  
Cellular Compartment ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Assembly Pathway

ABSTRACT Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.

scholarly journals The essential inner membrane protein YejM is a metalloenzyme

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Uma Gabale ◽  
Perla Arianna Peña Palomino ◽  
HyunAh Kim ◽  
Wenya Chen ◽  
Susanne Ressl
Keyword(s):  
Antibiotic Resistance ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Critical Role ◽  
Membrane Properties ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Periplasmic Domain ◽  
Inner Membrane Protein

Abstract Recent recurrent outbreaks of Gram-negative bacteria show the critical need to target essential bacterial mechanisms to fight the increase of antibiotic resistance. Pathogenic Gram-negative bacteria have developed several strategies to protect themselves against the host immune response and antibiotics. One such strategy is to remodel the outer membrane where several genes are involved. yejM was discovered as an essential gene in E. coli and S. typhimurium that plays a critical role in their virulence by changing the outer membrane permeability. How the inner membrane protein YejM with its periplasmic domain changes membrane properties remains unknown. Despite overwhelming structural similarity between the periplasmic domains of two YejM homologues with hydrolases like arylsulfatases, no enzymatic activity has been previously reported for YejM. Our studies reveal an intact active site with bound metal ions in the structure of YejM periplasmic domain. Furthermore, we show that YejM has a phosphatase activity that is dependent on the presence of magnesium ions and is linked to its function of regulating outer membrane properties. Understanding the molecular mechanism by which YejM is involved in outer membrane remodeling will help to identify a new drug target in the fight against the increased antibiotic resistance.

scholarly journals A Bilayer-Couple Model of Bacterial Outer Membrane Vesicle Biogenesis

mBio ◽  
2012 ◽  
Vol 3 (2) ◽  
Author(s):  
Jeffrey W. Schertzer ◽  
Marvin Whiteley
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicle ◽  
Membrane Curvature ◽  
Couple Model ◽  
Gram Negative Bacteria ◽  
Outer Membrane Vesicle ◽  
Gram Negative ◽  
Content Type ◽  
Outer Leaflet ◽  
Bilayer Couple

ABSTRACTGram-negative bacteria naturally produce outer membrane vesicles (OMVs) that arise through bulging and pinching off of the outer membrane. OMVs have several biological functions for bacteria, most notably as trafficking vehicles for toxins, antimicrobials, and signaling molecules. While their biological roles are now appreciated, the mechanism of OMV formation has not been fully elucidated. We recently demonstrated that the signaling molecule 2-heptyl-3-hydroxy-4-quinolone (PQS) is required for OMV biogenesis inP. aeruginosa. We hypothesized that PQS stimulates OMV formation through direct interaction with the outer leaflet of the outer membrane. To test this hypothesis, we employed a red blood cell (RBC) model that has been used extensively to study small-molecule–membrane interactions. Our results revealed that addition of PQS to RBCs induced membrane curvature, resulting in the formation of membrane spicules (spikes), consistent with small molecules that are inserted stably into the outer leaflet of the membrane. Radiotracer experiments demonstrated that sufficient PQS was inserted into the membrane to account for this curvature and that curvature induction was specific to PQS structure. These data suggest that a low rate of interleaflet flip-flop forces PQS to accumulate in and expand the outer leaflet relative to the inner leaflet, thus inducing membrane curvature. In support of PQS-mediated outer leaflet expansion, the PQS effect was antagonized by chlorpromazine, a molecule known to be preferentially inserted into the inner leaflet. Based on these data, we propose a bilayer-couple model to describeP. aeruginosaOMV biogenesis and suggest that this is a general mechanism for bacterial OMV formation.IMPORTANCEDespite the ubiquity and importance of outer membrane vesicle (OMV) production in Gram-negative bacteria, the molecular details of OMV biogenesis are not fully understood. Early experiments showed that 2-heptyl-3-hydroxy-4-quinolone (PQS) induces OMV formation through physical interaction with the membrane but did not elucidate the mechanism. The present study demonstrates that PQS specifically and reversibly promotes blebbing of model membranes dependent upon the same properties that are required for OMV formation inP. aeruginosa. These results are consistent with a mechanism where expansion of the outer leaflet relative to the inner leaflet induces localized membrane curvature. This “bilayer-couple” model can account for OMV formation under all conditions and is easily generalized to other Gram-negative bacteria. The model therefore raises the possibility of a universal paradigm for vesicle production in prokaryotes with features strikingly different from what is known in eukaryotes.

scholarly journals Mutation and Suppressor Analysis of the Essential Lipopolysaccharide Transport Protein LptA Reveals Strategies To Overcome Severe Outer Membrane Permeability Defects in Escherichia coli

2017 ◽  
Vol 200 (2) ◽  
Author(s):  
Federica A. Falchi ◽  
Elisa A. Maccagni ◽  
Simone Puccio ◽  
Clelia Peano ◽  
Cristina De Castro ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Antibiotic Sensitivity ◽  
Major Constituent ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Increased Sensitivity

ABSTRACTIn Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane (OM), preventing the entry of toxic molecules, such as detergents and antibiotics. LPS is transported from the inner membrane (IM) to the OM by the Lpt multiprotein machinery. Defects in LPS transport compromise LPS assembly at the OM and result in increased antibiotic sensitivity. LptA is a key component of the Lpt machine that interacts with the IM protein LptC and chaperones LPS through the periplasm. We report here the construction oflptA41, a quadruple mutant in four conserved amino acids potentially involved in LPS or LptC binding. Although viable, the mutant displays increased sensitivity to several antibiotics (bacitracin, rifampin, and novobiocin) and the detergent SDS, suggesting thatlptA41affects LPS transport. Indeed,lptA41is defective in Lpt complex assembly, and its lipid A carries modifications diagnostic of LPS transport defects. We also selected and characterized two phenotypic bacitracin-resistant suppressors oflptA41. One mutant, in which only bacitracin sensitivity is suppressed, harbors a small in-frame deletion inmlaA, which codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other mutant, in which bacitracin, rifampin, and SDS sensitivity is suppressed, harbors an additional amino acid substitution in LptA41 and a nonsense mutation inopgH, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharide synthesis. Characterization of the suppressor mutants highlights different strategies adopted by the cell to overcome OM defects caused by impaired LPS transport.IMPORTANCELipopolysaccharide (LPS) is the major constituent of the outer membrane (OM) of most Gram-negative bacteria, forming a barrier against antibiotics. LPS is synthesized at the inner membrane (IM), transported across the periplasm, and assembled at the OM by the multiprotein Lpt complex. LptA is the periplasmic component of the Lpt complex, which bridges IM and OM and ferries LPS across the periplasm. How the cell coordinates the processes involved in OM biogenesis is not completely understood. We generated a mutant partially defective inlptAthat exhibited increased sensitivity to antibiotics and selected for suppressors of the mutant. The analysis of two independent suppressors revealed different strategies adopted by the cell to overcome defects in LPS biogenesis.

scholarly journals Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis inEscherichia coli

2016 ◽  
Vol 113 (11) ◽  
pp. 3108-3113 ◽  
Author(s):  
Akintunde Emiola ◽  
Steven S. Andrews ◽  
Carolin Heller ◽  
John George
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Unsaturated Fatty Acids ◽  
Gram Negative Bacteria ◽  
Strong Immune Response ◽  
Outer Leaflet ◽  
Outer Membrane Biogenesis ◽  
Ulosonic Acid ◽  
Feedback Source

The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression ofwaaAresulted in increased levels of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of theEscherichia coliouter membrane.

scholarly journals Structure-guided enzymology of the lipid A acyltransferase LpxM reveals a dual activity mechanism

2016 ◽  
Vol 113 (41) ◽  
pp. E6064-E6071 ◽  
Author(s):  
Dustin Dovala ◽  
Christopher M. Rath ◽  
Qijun Hu ◽  
William S. Sawyer ◽  
Steven Shia ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Structural Information ◽  
Mechanistic Model ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Label Free ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Domains Of Life

Gram-negative bacteria possess a characteristic outer membrane, of which the lipid A constituent elicits a strong host immune response through the Toll-like receptor 4 complex, and acts as a component of the permeability barrier to prevent uptake of bactericidal compounds. Lipid A species comprise the bulk of the outer leaflet of the outer membrane and are produced through a multistep biosynthetic pathway conserved in most Gram-negative bacteria. The final steps in this pathway involve the secondary acylation of lipid A precursors. These are catalyzed by members of a superfamily of enzymes known as lysophospholipid acyltransferases (LPLATs), which are present in all domains of life and play important roles in diverse biological processes. To date, characterization of this clinically important class of enzymes has been limited by a lack of structural information and the availability of only low-throughput biochemical assays. In this work, we present the structure of the bacterial LPLAT protein LpxM, and we describe a high-throughput, label-free mass spectrometric assay to characterize acyltransferase enzymatic activity. Using our structure and assay, we identify an LPLAT thioesterase activity, and we provide experimental evidence to support an ordered-binding and “reset” mechanistic model for LpxM function. This work enables the interrogation of other bacterial acyltransferases’ structure–mechanism relationships, and the assay described herein provides a foundation for quantitatively characterizing the enzymology of any number of clinically relevant LPLAT proteins.

scholarly journals TheAcinetobacter baumanniiMla system and glycerophospholipid transport to the outer membrane

2018 ◽  
Author(s):  
Cassandra Kamischke ◽  
Junping Fan ◽  
Julien Bergeron ◽  
Hemantha D. Kulasekara ◽  
Zachary D. Dalebroux ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Atp Hydrolysis ◽  
Antibiotic Sensitivity ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Selective Permeability ◽  
Transposon Mutants

ABSTRACTThe outer membrane (OM) of Gram-negative bacteria serves as a selective permeability barrier that allows entry of essential nutrients while excluding toxic compounds, including antibiotics. The OM is asymmetric and contains an outer leaflet of lipopolysaccharides (LPS) or lipooligosaccharides (LOS) and an inner leaflet of glycerophospholipids (GPL). We screenedAcinetobacter baumanniitransposon mutants and identified a number of mutants with OM defects, including an ABC transporter system homologous to the Mla system inE. coli. We further show that this opportunistic, antibiotic-resistant pathogen uses this multicomponent protein complex and ATP hydrolysis at the inner membrane to promote GPL export to the OM. The broad conservation of the Mla system in Gram-negative bacteria suggests the system may play a conserved role in OM biogenesis. The importance of the Mla system toAcinetobacter baumanniiOM integrity and antibiotic sensitivity suggests that its components may serve as new antimicrobial therapeutic targets.

scholarly journals Reconstitution of surface lipoprotein translocation reveals Slam as an outer membrane translocon in Gram-negative bacteria

2021 ◽  
Author(s):  
Minh Sang Huynh ◽  
Yogesh Hooda ◽  
Yuzi Raina Li ◽  
Maciej Jagielnicki ◽  
Christine Chieh-Lin Lai ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Bacterial Surface ◽  
Outer Leaflet ◽  
Translocation Efficiency ◽  
Translocation Assay ◽  
Periplasmic Chaperone ◽  
Dependent Pathway

Surface lipoproteins (SLPs) are peripherally attached to the outer leaflet of the outer membrane in many Gram-negative bacteria, playing significant roles in nutrient acquisition and immune evasion in the host. While the factors that are involved in the synthesis and delivery of SLPs in the inner membrane are well characterized, the molecular machineries required for the movement of SLPs to the surface are still not fully elucidated. In this study, we investigated the translocation of a surface lipoprotein TbpB through a Slam1-dependent pathway. Using purified components, we developed an in vitro translocation assay where unfolded TbpB is transported through Slam1 containing proteoliposomes, confirming Slam1 as an outer membrane translocon. While looking to identify factors to increase translocation efficiency, we discovered the periplasmic chaperone Skp interacted with TbpB in the periplasm of Escherichia coli. The presence of Skp was found to increase the translocation efficiency of TbpB in the reconstituted translocation assays. A knockout of Skp in Neisseria meningitidis revealed that Skp is essential for functional translocation of TbpB to the bacterial surface. Taken together, we propose a pathway for surface destined lipoproteins, where Skp acts as a holdase for Slam-mediated TbpB translocation across the outer membrane.

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