A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria.

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPSs) on the outer leaflet and phospholipids (PLs) on the inner leaflet. The loss of this asymmetry due to mutations in the LPS biosynthesis or transport pathways causes the externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we used metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase expression in Escherichia coli allowed for the incorporation of exogenous propargylcholine into phosphatidyl(propargyl)choline and exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC), as confirmed by LC/MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool for detecting a compromised bacterial OM, revealing aberrant PL externalization, and identifying or characterizing novel cell-active inhibitors of LPS biosynthesis or transport.

Download Full-text

On the Architecture of the Gram-Negative Bacterial Murein Sacculus

Journal of Bacteriology ◽

10.1128/jb.182.20.5925-5930.2000 ◽

2000 ◽

Vol 182 (20) ◽

pp. 5925-5930 ◽

Cited By ~ 36

Author(s):

David Pink ◽

Jeremy Moeller ◽

Bonnie Quinn ◽

Manfred Jericho ◽

Terry Beveridge

Keyword(s):

Gram Negative Bacteria ◽

Defect Structures ◽

Waste Products ◽

Gram Negative ◽

Large Molecules ◽

Glycan Chain ◽

Pass Through ◽

Murein Sacculus ◽

Chain Lengths ◽

Peptidoglycan Layer

ABSTRACT The peptidoglycan network of the murein sacculus must be porous so that nutrients, waste products, and secreted proteins can pass through. Using Escherichia coli and Pseudomonas aeruginosa as a baseline for gram-negative sacculi, the hole size distribution in the peptidoglycan network has been modeled by computer simulation to deduce the network's properties. By requiring that the distribution of glycan chain lengths predicted by the model be in accord with the distribution observed, we conclude that the holes are slits running essentially perpendicular to the local axis of the glycan chains (i.e., the slits run along the long axis of the cell). This result is in accord with previous permeability measurements of Beveridge and Jack and Demchik and Koch. We outline possible advantages that might accrue to the bacterium via this architecture and suggest ways in which such defect structures might be detected. Certainly, large molecules do penetrate the peptidoglycan layer of gram-negative bacteria, and the small slits that we suggest might be made larger by the bacterium.

Download Full-text

Outer Membranes and Efflux: The Path to Multidrug Resistance in Gram- Negative Bacteria

Current Pharmaceutical Biotechnology ◽

10.2174/1389201023378454 ◽

2002 ◽

Vol 3 (2) ◽

pp. 77-98 ◽

Cited By ~ 97

Author(s):

Keith Poole

Keyword(s):

Multidrug Resistance ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Outer Membranes

Download Full-text

Outer membranes of Gram-negative bacteria are permeable to steroid probes

Molecular Microbiology ◽

10.1111/j.1365-2958.1992.tb00853.x ◽

1992 ◽

Vol 6 (10) ◽

pp. 1323-1333 ◽

Cited By ~ 112

Author(s):

Patrick Plésiat ◽

Hiroshi Nikaido

Keyword(s):

Gram Negative Bacteria ◽

Gram Negative ◽

Outer Membranes

Download Full-text

Synergistic Effect of Propidium Iodide and Small Molecule Antibiotics with the Antimicrobial Peptide Dendrimer G3KL against Gram-Negative Bacteria

Molecules ◽

10.3390/molecules25235643 ◽

2020 ◽

Vol 25 (23) ◽

pp. 5643

Author(s):

Bee-Ha Gan ◽

Xingguang Cai ◽

Sacha Javor ◽

Thilo Köhler ◽

Jean-Louis Reymond

Keyword(s):

Synergistic Effect ◽

Propidium Iodide ◽

Synergistic Effects ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Gram Negative Bacteria ◽

Bacterial Killing ◽

Gram Negative ◽

Outer Membranes ◽

New Antibiotics

There is an urgent need to develop new antibiotics against multidrug-resistant bacteria. Many antimicrobial peptides (AMPs) are active against such bacteria and often act by destabilizing membranes, a mechanism that can also be used to permeabilize bacteria to other antibiotics, resulting in synergistic effects. We recently showed that G3KL, an AMP with a multibranched dendritic topology of the peptide chain, permeabilizes the inner and outer membranes of Gram-negative bacteria including multidrug-resistant strains, leading to efficient bacterial killing. Here, we show that permeabilization of the outer and inner membranes of Pseudomonas aeruginosa by G3KL, initially detected using the DNA-binding fluorogenic dye propidium iodide (PI), also leads to a synergistic effect between G3KL and PI in this bacterium. We also identify a synergistic effect between G3KL and six different antibiotics against the Gram-negative Klebsiella pneumoniae, against which G3KL is inactive.

Download Full-text

Interactions of an antimicrobial peptide, magainin 2, with lipopolysaccharide-containing liposomes as a model for outer membranes of Gram-negative bacteria

FEBS Letters ◽

10.1016/s0014-5793(99)00443-3 ◽

1999 ◽

Vol 449 (2-3) ◽

pp. 221-224 ◽

Cited By ~ 102

Author(s):

Katsumi Matsuzaki ◽

Ken-ichi Sugishita ◽

Koichiro Miyajima

Keyword(s):

Antimicrobial Peptide ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Outer Membranes

Download Full-text

Inhibition of the β-barrel assembly machine by a peptide that binds BamD

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1415955112 ◽

2015 ◽

Vol 112 (7) ◽

pp. 2011-2016 ◽

Cited By ~ 54

Author(s):

Christine L. Hagan ◽

Joseph S. Wzorek ◽

Daniel Kahne

Keyword(s):

Gram Negative Bacteria ◽

Gram Negative ◽

Outer Membranes ◽

Essential Proteins ◽

Growth Defects ◽

Substrate Protein ◽

Assembly Mechanism ◽

New Antibiotics

The protein complex that assembles integral membrane β-barrel proteins in the outer membranes of Gram-negative bacteria is an attractive target in the development of new antibiotics. This complex, the β-barrel assembly machine (Bam), contains two essential proteins, BamA and BamD. We have identified a peptide that inhibits the assembly of β-barrel proteins in vitro by characterizing the interaction of BamD with an unfolded substrate protein. This peptide is a fragment of the substrate protein and contains a conserved amino acid sequence. We have demonstrated that mutations of this sequence in the full-length substrate protein impair the protein’s assembly, implying that BamD’s interaction with this sequence is an important part of the assembly mechanism. Finally, we have found that in vivo expression of a peptide containing this sequence causes growth defects and sensitizes Escherichia coli to antibiotics to which they are normally resistant. Therefore, inhibiting the binding of substrates to BamD is a viable strategy for developing new antibiotics directed against Gram-negative bacteria.

Download Full-text

Requirements for the import of neisserial Omp85 into the outer membrane of human mitochondria

Bioscience Reports ◽

10.1042/bsr20130007 ◽

2013 ◽

Vol 33 (2) ◽

Cited By ~ 3

Author(s):

Christine Ott ◽

Mandy Utech ◽

Monika Goetz ◽

Thomas Rudel ◽

Vera Kozjak-Pavlovic

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Outer Membrane ◽

Gram Negative Bacteria ◽

Mitochondrial Targeting ◽

Gram Negative ◽

Outer Membranes ◽

Sorting Signal ◽

Targeting Signal ◽

And Function

β-Barrel proteins are present only in the outer membranes of Gram-negative bacteria, chloroplasts and mitochondria. Fungal mitochondria were shown to readily import and assemble bacterial β-barrel proteins, but human mitochondria exhibit certain selectivity. Whereas enterobacterial β-barrel proteins are not imported, neisserial ones are. Of those, solely neisserial Omp85 is integrated into the outer membrane of mitochondria. In this study, we wanted to identify the signal that targets neisserial β-barrel proteins to mitochondria. We exchanged parts of neisserial Omp85 and PorB with their Escherichia coli homologues BamA and OmpC. For PorB, we could show that its C-terminal quarter can direct OmpC to mitochondria. In the case of Omp85, we could identify several amino acids of the C-terminal β-sorting signal as crucial for mitochondrial targeting. Additionally, we found that at least two POTRA (polypeptide-transport associated) domains and not only the β-sorting signal of Omp85 are needed for its membrane integration and function in human mitochondria. We conclude that the signal that directs neisserial β-barrel proteins to mitochondria is not conserved between these proteins. Furthermore, a linear mitochondrial targeting signal probably does not exist. It is possible that the secondary structure of β-barrel proteins plays a role in directing these proteins to mitochondria.

Download Full-text

Structure of LetB reveals a tunnel for lipid transport across the bacterial envelope

10.1101/748145 ◽

2019 ◽

Author(s):

Georgia L. Isom ◽

Nicolas Coudray ◽

Mark R. MacRae ◽

Collin T. McManus ◽

Damian C. Ekiert ◽

...

Keyword(s):

Outer Membrane ◽

Lipid Transport ◽

Transport Systems ◽

Permeability Barrier ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

Outer Membranes ◽

Hydrophobic Tunnel ◽

Outer Membrane Permeability

Gram-negative bacteria are surrounded by an outer membrane composed of phospholipids and lipopolysaccharide (LPS), which acts as a barrier to the environment and contributes to antibiotic resistance. While mechanisms of LPS transport have been well characterised, systems that translocate phospholipids across the periplasm, such as MCE (Mammalian Cell Entry) transport systems, are less well understood. Here we show that E. coli MCE protein LetB (formerly YebT), forms a ∼0.6 megadalton complex in the periplasm. Our cryo-EM structure reveals that LetB consists of a stack of seven modular rings, creating a long hydrophobic tunnel through the centre of the complex. LetB is sufficiently large to span the gap between the inner and outer membranes, and mutations that shorten the tunnel abolish function. Lipids bind inside the tunnel, suggesting that it functions as a pathway for lipid transport. Cryo-EM structures in the open and closed states reveal a dynamic tunnel lining, with implications for gating or substrate translocation. Together, our results support a model in which LetB establishes a physical link between the bacterial inner and outer membranes, and creates a hydrophobic pathway for the translocation of lipids across the periplasm, to maintain the integrity of the outer membrane permeability barrier.

Download Full-text