Evidence for phospholipid export from the gram-negative inner membrane: time to rethink the Mla pathway?

AbstractThe Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a novel pivoting β-sheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the Mla pathway functions in an anterograde rather than a retrograde direction by showing the inner membrane MlaFEDB machinery exports phospholipids and transfers them to MlaC in the periplasm. We confirm that the lipid export process occurs through the MlaD component of the MlaFEDB complex. This lipid export process is shown to be independent of ATP. Our data provides, for the first time, evidence of an apparatus for lipid export to the outer membrane.

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Structural insights into a novel family of integral membrane siderophore reductases

10.1101/2021.01.28.428567 ◽

2021 ◽

Author(s):

Inokentijs Josts ◽

Katharina Veith ◽

Vincent Normant ◽

Isabelle J. Schalk ◽

Henning Tidow

Keyword(s):

Crystal Structure ◽

Membrane Protein ◽

Outer Membrane ◽

Bacterial Species ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Functional Studies ◽

Inner Membrane Protein ◽

Structural Insights

AbstractGram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from P. aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a novel fold with two tightly-bound heme molecules. In combination with functional studies these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.

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MsbA Is Not Required for Phospholipid Transport in Neisseria meningitidis

Journal of Biological Chemistry ◽

10.1074/jbc.m509026200 ◽

2005 ◽

Vol 280 (43) ◽

pp. 35961-35966 ◽

Cited By ~ 42

Author(s):

Boris Tefsen ◽

Martine P. Bos ◽

Frank Beckers ◽

Jan Tommassen ◽

Hans de Cock

Keyword(s):

Neisseria Meningitidis ◽

Outer Membrane ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Outer Leaflet ◽

Growth Phenotype ◽

Inner Membrane Protein ◽

Phospholipid Transport

The outer membrane of Gram-negative bacteria contains phospholipids and lipopolysaccharide (LPS) in the inner and outer leaflet, respectively. Little is known about the transport of the phospholipids from their site of synthesis to the outer membrane. The inner membrane protein MsbA of Escherichia coli, which is involved in the transport of LPS across the inner membrane, has been reported to be involved in phospholipid transport as well. Here, we have reported the construction and the characterization of a Neisseria meningitidis msbA mutant. The mutant was viable, and it showed a retarded growth phenotype and contained very low amounts of LPS. However, it produced an outer membrane, demonstrating that phospholipid transport was not affected by the mutation. Notably, higher amounts of phospholipids were produced in the msbA mutant than in its isogenic parental strain, provided that capsular biosynthesis was also disrupted. Although these results confirmed that MsbA functions in LPS transport, they also demonstrated that it is not required for phospholipid transport, at least not in N. meningitidis.

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MlaFEDB displays flippase activity to promote phospholipid transport towards the outer membrane of Gram-negative bacteria

10.1101/2020.06.06.138008 ◽

2020 ◽

Cited By ~ 4

Author(s):

Gareth W. Hughes ◽

Pooja Sridhar ◽

Stephanie A. Nestorow ◽

Peter J. Wotherspoon ◽

Benjamin F. Cooper ◽

...

Keyword(s):

Outer Membrane ◽

Membrane Trafficking ◽

Atp Hydrolysis ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Membrane Protein Complex ◽

Gram Negative ◽

Inner Membrane Protein ◽

Phospholipid Transport

AbstractMlaFEDB is a Gram-negative inner membrane protein complex involved in the inter membrane trafficking of phospholipids. Originally proposed to transport phospholipids in a retrograde direction, recent evidence suggests MlaFEDB may actually export phospholipids from the inner membrane to the periplasmic carrier protein, MlaC, potentially suggesting a role in either anterograde trafficking of phospholipids to the outer membrane or bidirectional phospholipid movement. MlaFEDB is part of the ABC transporter superfamily of proteins and has been shown to hydrolyse ATP through the cytoplasmic facing MlaF component. However, the movement of PLs from FEDB to MlaC has been shown to occur in an ATP independent fashion hence the role of ATP hydrolysis within this complex remains unclear. In this study we sought to elucidate the role of ATP and provide evidence to suggest MlaFEDB has flippase activity, utilising ATP hydrolysis to translocate phospholipids from the outer to the inner leaflet of the IM. We also show that in the absence of ATP MlaFEDB mediates the loading of MlaC with phospholipids directly from the inner leaflet only. Our data provides a novel role for MlaFEDB and presents a link between Mla driven phospholipid transport and ATP hydrolysis.

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Shadowing the Actions of a Predator: Backlit Fluorescent Microscopy Reveals Synchronous Nonbinary Septation of Predatory Bdellovibrio inside Prey and Exit through Discrete Bdelloplast Pores

Journal of Bacteriology ◽

10.1128/jb.00914-10 ◽

2010 ◽

Vol 192 (24) ◽

pp. 6329-6335 ◽

Cited By ~ 40

Author(s):

A. K. Fenton ◽

M. Kanna ◽

R. D. Woods ◽

S.-I. Aizawa ◽

R. E. Sockett

Keyword(s):

Cell Division ◽

Outer Membrane ◽

Fluorescent Microscopy ◽

Gram Negative Bacteria ◽

Bacterial Cell Division ◽

Prey Cell ◽

Gram Negative ◽

A Genome ◽

Predatory Bacteria ◽

First Time

ABSTRACT The Bdellovibrio are miniature “living antibiotic” predatory bacteria which invade, reseal, and digest other larger Gram-negative bacteria, including pathogens. Nutrients for the replication of Bdellovibrio bacteria come entirely from the digestion of the single invaded bacterium, now called a bdelloplast, which is bound by the original prey outer membrane. Bdellovibrio bacteria are efficient digesters of prey cells, yielding on average 4 to 6 progeny from digestion of a single prey cell of a genome size similar to that of the Bdellovibrio cell itself. The developmental intrabacterial cycle of Bdellovibrio is largely unknown and has never been visualized “live.” Using the latest motorized xy stage with a very defined z-axis control and engineered periplasmically fluorescent prey allows, for the first time, accurate return and visualization without prey bleaching of developing Bdellovibrio cells using solely the inner resources of a prey cell over several hours. We show that Bdellovibrio bacteria do not follow the familiar pattern of bacterial cell division by binary fission. Instead, they septate synchronously to produce both odd and even numbers of progeny, even when two separate Bdellovibrio cells have invaded and develop within a single prey bacterium, producing two different amounts of progeny. Evolution of this novel septation pattern, allowing odd progeny yields, allows optimal use of the finite prey cell resources to produce maximal replicated, predatory bacteria. When replication is complete, Bdellovibrio cells exit the exhausted prey and are seen leaving via discrete pores rather than by breakdown of the entire outer membrane of the prey.

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Membrane fusion during phage lysis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1420588112 ◽

2015 ◽

Vol 112 (17) ◽

pp. 5497-5502 ◽

Cited By ~ 30

Author(s):

Manoj Rajaure ◽

Joel Berry ◽

Rohit Kongari ◽

Jesse Cahill ◽

Ry Young

Keyword(s):

Membrane Fusion ◽

Outer Membrane ◽

Cytoplasmic Membrane ◽

Bacterial Host ◽

Inner Membrane ◽

Gram Negative ◽

Encoded Proteins ◽

Coliphage Lambda ◽

Necessary And Sufficient

In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of μm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG.

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YejM Modulates Activity of the YciM/FtsH Protease Complex To Prevent Lethal Accumulation of Lipopolysaccharide

mBio ◽

10.1128/mbio.00598-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 10

Author(s):

Randi L. Guest ◽

Daniel Samé Guerra ◽

Maria Wissler ◽

Jacqueline Grimm ◽

Thomas J. Silhavy

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Content Type ◽

Essential Protein ◽

Ftsh Protease ◽

Acyl Chains ◽

Lipid Balance

ABSTRACT Lipopolysaccharide (LPS) is an essential glycolipid present in the outer membrane (OM) of many Gram-negative bacteria. Balanced biosynthesis of LPS is critical for cell viability; too little LPS weakens the OM, while too much LPS is lethal. In Escherichia coli, this balance is maintained by the YciM/FtsH protease complex, which adjusts LPS levels by degrading the LPS biosynthesis enzyme LpxC. Here, we provide evidence that activity of the YciM/FtsH protease complex is inhibited by the essential protein YejM. Using strains in which LpxC activity is reduced, we show that yciM is epistatic to yejM, demonstrating that YejM acts upstream of YciM to prevent toxic overproduction of LPS. Previous studies have shown that this toxicity can be suppressed by deleting lpp, which codes for a highly abundant OM lipoprotein. It was assumed that deletion of lpp restores lipid balance by increasing the number of acyl chains available for glycerophospholipid biosynthesis. We show that this is not the case. Rather, our data suggest that preventing attachment of lpp to the peptidoglycan sacculus allows excess LPS to be shed in vesicles. We propose that this loss of OM material allows continued transport of LPS to the OM, thus preventing lethal accumulation of LPS within the inner membrane. Overall, our data justify the commitment of three essential inner membrane proteins to avoid toxic over- or underproduction of LPS. IMPORTANCE Gram-negative bacteria are encapsulated by an outer membrane (OM) that is impermeable to large and hydrophobic molecules. As such, these bacteria are intrinsically resistant to several clinically relevant antibiotics. To better understand how the OM is established or maintained, we sought to clarify the function of the essential protein YejM in Escherichia coli. Here, we show that YejM inhibits activity of the YciM/FtsH protease complex, which regulates synthesis of the essential OM glycolipid lipopolysaccharide (LPS). Our data suggest that disrupting proper communication between LPS synthesis and transport to the OM leads to accumulation of LPS within the inner membrane (IM). The lethality associated with this event can be suppressed by increasing OM vesiculation. Our research has identified a completely novel signaling pathway that we propose coordinates LPS synthesis and transport.

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New Type of Outer Membrane Vesicle Produced by the Gram-Negative Bacterium Shewanella vesiculosa M7T: Implications for DNA Content

Applied and Environmental Microbiology ◽

10.1128/aem.03657-12 ◽

2013 ◽

Vol 79 (6) ◽

pp. 1874-1881 ◽

Cited By ~ 102

Author(s):

Carla Pérez-Cruz ◽

Ornella Carrión ◽

Lidia Delgado ◽

Gemma Martinez ◽

Carmen López-Iglesias ◽

...

Keyword(s):

Outer Membrane ◽

Dna Content ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Freeze Substitution ◽

Outer Membrane Vesicles ◽

Negative Bacterium ◽

Inner Membrane ◽

Gram Negative ◽

Content Type

ABSTRACTOuter membrane vesicles (OMVs) from Gram-negative bacteria are known to be involved in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. An ultrastructural study of the Antarctic psychrotolerant bacteriumShewanella vesiculosaM7Thas revealed that this Gram-negative bacterium naturally releases conventional one-bilayer OMVs through a process in which the outer membrane is exfoliated and only the periplasm is entrapped, together with a more complex type of OMV, previously undescribed, which on formation drag along inner membrane and cytoplasmic content and can therefore also entrap DNA. These vesicles, with a double-bilayer structure and containing electron-dense material, were visualized by transmission electron microscopy (TEM) after high-pressure freezing and freeze-substitution (HPF-FS), and their DNA content was fluorometrically quantified as 1.8 ± 0.24 ng DNA/μg OMV protein. The new double-bilayer OMVs were estimated by cryo-TEM to represent 0.1% of total vesicles. The presence of DNA inside the vesicles was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. In addition, a proteomic study of purified membrane vesicles confirmed the presence of plasma membrane and cytoplasmic proteins in OMVs from this strain. Our data demonstrate the existence of a previously unobserved type of double-bilayer OMV in the Gram-negative bacteriumShewanella vesiculosaM7Tthat can incorporate DNA, for which we propose the name outer-inner membrane vesicle (O-IMV).

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Crystal structure of calcium bound outer membrane phospholipase A (OmpLA) from Salmonella typhi and in silico anti-microbial screening

10.1101/2020.01.08.898262 ◽

2020 ◽

Author(s):

Perumal Perumal ◽

Rahul Raina ◽

Sundara Baalaji Narayanan ◽

Arulandu Arockiasamy

Keyword(s):

Crystal Structure ◽

Outer Membrane ◽

In Silico ◽

Membrane Integrity ◽

Acid Tolerance ◽

Salmonella Typhi ◽

Phospholipase A ◽

Gram Negative ◽

Activated State ◽

Outer Membrane Phospholipase A

AbstractAntimicrobial resistance is widespread in Salmonella infections that affect millions worldwide. Salmonella typhi and other Gram-negative bacterial pathogens encode an outer membrane phospholipase A (OmpLA), crucial for their membrane integrity. Further, OmpLA is implicated in pathogen internalization, haemolysis, acid tolerance, virulence and sustained infection in human hosts. OmpLA is an attractive drug target for developing novel anti-microbials that attenuate virulence, as the abrogation of OmpLA encoding pldA gene causes loss of virulence. Here, we present the crystal structure of Salmonella typhi OmpLA in dimeric calcium bound activated state at 2.95 Å. Structure analysis suggests that OmpLA is a potential druggable target. Further, we have identified and shortlisted small molecules that bind at the dimer interface using structure based in silico screening, docking and molecular dynamics. While it requires further experimental validation, anti-microbial discovery targeting OmpLA from gram-negative pathogens offers an advantage as OmpLA is required for virulence.

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Formation of a β-barrel membrane protein is catalyzed by the interior surface of the assembly machine protein BamA

eLife ◽

10.7554/elife.49787 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 8

Author(s):

James Lee ◽

David Tomasek ◽

Thiago MA Santos ◽

Mary D May ◽

Ina Meuskens ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Molecular Mechanisms ◽

Gram Negative Bacteria ◽

Site Specific ◽

Gram Negative ◽

Interior Surface ◽

Essential Component ◽

Bam Complex ◽

Β Sheet

The β-barrel assembly machine (Bam) complex in Gram-negative bacteria and its counterparts in mitochondria and chloroplasts fold and insert outer membrane β-barrel proteins. BamA, an essential component of the complex, is itself a β-barrel and is proposed to play a central role in assembling other barrel substrates. Here, we map the path of substrate insertion by the Bam complex using site-specific crosslinking to understand the molecular mechanisms that control β-barrel folding and release. We find that the C-terminal strand of the substrate is stably held by BamA and that the N-terminal strands of the substrate are assembled inside the BamA β-barrel. Importantly, we identify contacts between the assembling β-sheet and the BamA interior surface that determine the rate of substrate folding. Our results support a model in which the interior wall of BamA acts as a chaperone to catalyze β-barrel assembly.

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The essential inner membrane protein YejM is a metalloenzyme

Scientific Reports ◽

10.1038/s41598-020-73660-6 ◽

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.

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