scholarly journals Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Sara Alvira ◽  
Daniel W Watkins ◽  
Lucy Troman ◽  
William J Allen ◽  
James S Lorriman ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Conformational Changes ◽  
Major Constituent ◽  
Gram Negative Bacteria ◽  
Surface Adhesion ◽  
Core Complex ◽  
Inner Membrane ◽  
Bacterial Outer Membrane ◽  
Periplasmic Chaperones ◽  
Outer Membrane Biogenesis

The outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent – hydrophobic β-barrel Outer-Membrane Proteins (OMPs) – are first secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperones, for example SurA, which prevent aggregation. OMPs are then offloaded to the β-Barrel Assembly Machinery (BAM) in the outer-membrane for insertion and folding. We show the Holo-TransLocon (HTL) – an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane ‘insertase’ YidC – contacts BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Furthermore, the proton-motive force (PMF) across the inner-membrane acts at distinct stages of protein secretion: (1) SecA-driven translocation through SecYEG and (2) communication of conformational changes via SecDF across the periplasm to BAM. The latter presumably drives efficient passage of OMPs. These interactions provide insights of inter-membrane organisation and communication, the importance of which is becoming increasingly apparent.

scholarly journals Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis

2019 ◽  
Author(s):  
Sara Alvira ◽  
Daniel W. Watkins ◽  
Lucy Troman ◽  
William J. Allen ◽  
James Lorriman ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Conformational Changes ◽  
Major Constituent ◽  
Gram Negative Bacteria ◽  
Surface Adhesion ◽  
Core Complex ◽  
Inner Membrane ◽  
Bacterial Outer Membrane ◽  
Periplasmic Chaperones ◽  
Outer Membrane Biogenesis

SUMMARYThe outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent – hydrophobic β-barrel Outer-Membrane Proteins (OMPs) – are secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperones e.g. SurA, which prevent aggregation. OMPs are then offloaded to the β-Barrel Assembly Machinery (BAM) in the outer-membrane for insertion and folding. We show the Holo-TransLocon (HTL: an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane ‘insertase’ YidC) contacts SurA and BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Our results show the trans-membrane proton-motive-force (PMF) acts at distinct stages of protein secretion: for SecA-driven translocation across the inner-membrane through SecYEG; and to communicate conformational changes via SecDF to the BAM machinery. The latter presumably ensures efficient passage of OMPs. These interactions provide insights of inter-membrane organisation, the importance of which is becoming increasingly apparent.

scholarly journals Emerging Roles for NlpE as a Sensor for Lipoprotein Maturation and Transport to the Outer Membrane inEscherichia coli

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Brent W. Simpson ◽  
M. Stephen Trent
Keyword(s):  
Outer Membrane ◽  
Copper Stress ◽  
Gram Negative Bacteria ◽  
Two Component System ◽  
Inner Membrane ◽  
Complex Process ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Outer Membrane Biogenesis ◽  
Stress Pathway

ABSTRACTOuter membrane biogenesis is a complex process for Gram-negative bacteria as the components are synthesized in the cytoplasm or at the inner membrane and then transported to the outer membrane. Stress pathways monitor and respond to problems encountered in assembling the outer membrane. The two-component system CpxAR was recently reported to be a stress pathway for transport of lipoproteins to the outer membrane, but it was unclear how this stress is sensed. May et al. [K. L. May, K. M. Lehman, A. M. Mitchell, and M. Grabowicz, mBio 10(3):e00618-19, 2019,https://doi.org/10.1128/mBio.00618-19] determined that an outer membrane lipoprotein, NlpE, is the sensor for lipoprotein biogenesis stress. The group demonstrated that CpxAR is activated by the N-terminal domain of NlpE when the lipoprotein accumulates at the inner membrane. Further, this work resolved a previously debated role for NlpE in sensing copper stress; copper was shown to inhibit acylation of lipoproteins, preventing them from being transported to the outer membrane.

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 NMR Structures and Interactions of Temporin-1Tl and Temporin-1Tb with Lipopolysaccharide Micelles

2011 ◽  
Vol 286 (27) ◽  
pp. 24394-24406 ◽  
Author(s):  
Anirban Bhunia ◽  
Rathi Saravanan ◽  
Harini Mohanram ◽  
Maria L. Mangoni ◽  
Surajit Bhattacharjya
Keyword(s):  
Antimicrobial Peptides ◽  
Outer Membrane ◽  
Conformational Changes ◽  
Close Contact ◽  
Resonance Energy ◽  
Helical Structure ◽  
Major Constituent ◽  
Gram Negative Bacteria ◽  
Nmr Studies ◽  
Gram Negative

Temporins are a group of closely related short antimicrobial peptides from frog skin. Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, plays important roles in the activity of temporins. Earlier studies have found that LPS induces oligomerization of temporin-1Tb (TB) thus preventing its translocation across the outer membrane and, as a result, reduces its activity on Gram-negative bacteria. On the other hand, temporin-1Tl (TL) exhibits higher activity, presumably because of lack of such oligomerization. A synergistic mechanism was proposed, involving TL and TB in overcoming the LPS-mediated barrier. Here, to gain insights into interactions of TL and TB within LPS, we investigated the structures and interactions of TL, TB, and TL+TB in LPS micelles, using NMR and fluorescence spectroscopy. In the context of LPS, TL assumes a novel antiparallel dimeric helical structure sustained by intimate packing between aromatic-aromatic and aromatic-aliphatic residues. By contrast, independent TB has populations of helical and aggregated conformations in LPS. The LPS-induced aggregated states of TB are largely destabilized in the presence of TL. Saturation transfer difference NMR studies have delineated residues of TL and TB in close contact with LPS and enhanced interactions of these two peptides with LPS, when combined together. Fluorescence resonance energy transfer and 31P NMR have pointed out the proximity of TL and TB in LPS and conformational changes of LPS, respectively. Importantly, these results provide the first structural insights into the mode of action and synergism of antimicrobial peptides at the level of the LPS-outer membrane.

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

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
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.

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.

scholarly journals Dynamic interaction of fluoroquinolones with magnesium ions monitored using bacterial outer membrane nanopores

2020 ◽  
Vol 11 (38) ◽  
pp. 10344-10353
Author(s):  
Jiajun Wang ◽  
Jigneshkumar Dahyabhai Prajapati ◽  
Ulrich Kleinekathöfer ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Divalent Cations ◽  
Dynamic Interaction ◽  
Gram Negative Bacteria ◽  
Magnesium Ions ◽  
Gram Negative ◽  
Bacterial Outer Membrane

Divalent cations alter the translocation of antibiotic molecules through the Gram-negative bacteria outer membrane nanopores.

Advances in understanding bacterial outer-membrane biogenesis

2006 ◽  
Vol 4 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Natividad Ruiz ◽  
Daniel Kahne ◽  
Thomas J. Silhavy
Keyword(s):  
Outer Membrane ◽  
Membrane Biogenesis ◽  
Bacterial Outer Membrane ◽  
Outer Membrane Biogenesis

scholarly journals Conformational Changes That Coordinate the Activity of BamA and BamD Allowing β-Barrel Assembly

2017 ◽  
Vol 199 (20) ◽  
Author(s):  
Anne L. McCabe ◽  
Dante Ricci ◽  
Modupe Adetunji ◽  
Thomas J. Silhavy
Keyword(s):  
Outer Membrane ◽  
Conformational Changes ◽  
Signal Sequence ◽  
Outer Membrane Proteins ◽  
Gram Negative Bacteria ◽  
Complex Stability ◽  
Essential Proteins ◽  
Bam Complex ◽  
Sequence Recognition ◽  
Charged Residues

ABSTRACT Most integral outer membrane proteins (OMPs) of Gram-negative bacteria, such as Escherichia coli, assume a β-barrel structure. The β-barrel assembly machine (Bam), a five-member complex composed of β-barrel OMP BamA and four associated lipoproteins, BamB, BamC, BamD, and BamE, folds and inserts OMPs into the outer membrane. The two essential proteins BamA and BamD interact to stabilize two subcomplexes, BamAB and BamCDE, and genetic and structural evidence suggests that interactions between BamA and BamD occur via an electrostatic interaction between a conserved aspartate residue in a periplasmic domain of BamA and a conserved arginine in BamD. In this work, we characterize charge-change mutations at these key BamA and BamD residues and nearby charged residues in BamA with respect to OMP assembly and Bam complex stability. We show that Bam complex stability does not correlate with function, that BamA and BamD must adopt at least two active conformational states during OMP assembly, and that these charged residues are not required for function. Rather, these charged residues are important for coordinating the activities of BamA and BamD to allow efficient OMP assembly. We present a model of OMP assembly wherein recognition and binding of unfolded OMP substrate by BamA and BamD induce a signaling interaction between the two proteins, causing conformational changes necessary for the assembly reaction to proceed. By analogy to signal sequence recognition by SecYEG, we believe these BamA-BamD interactions ensure that both substrate and complex are competent for OMP assembly before the assembly reaction commences. IMPORTANCE Conformational changes in the proteins of the β-barrel assembly machine (Bam complex) are associated with the folding and assembly of outer membrane proteins (OMPs) in Gram-negative bacteria. We show that electrostatic interactions between the two essential proteins BamA and BamD coordinate conformational changes upon binding of unfolded substrate that allow the assembly reaction to proceed. Mutations affecting this interaction are lethal not because they destabilize the Bam complex but rather because they disrupt this coordination. Our model of BamA-BamD interactions regulating conformation in response to proper substrate interaction is reminiscent of conformational changes the secretory (Sec) machinery undergoes after signal sequence recognition that ensure protein quality control.

Epitope mapping of monoclonal antibodies specific for the directly cross-linkedmeso-diaminopimelic acid peptidoglycan found in the anaerobic beer spoilage bacteriumPectinatus cerevisiiphilus

1999 ◽  
Vol 45 (9) ◽  
pp. 779-785 ◽  
Author(s):  
Barry Ziola ◽  
Sheryl L Gares ◽  
Brandene Lorrain ◽  
Lori Gee ◽  
W M Ingledew ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Outer Membrane ◽  
Epitope Mapping ◽  
Sodium Dodecyl ◽  
Diaminopimelic Acid ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Spoilage Bacteria ◽  
Beer Spoilage ◽  
Bacterial Outer Membrane

Nineteen monoclonal antibodies (Mabs) were isolated based on reactivity with disrupted Pectinatus cerevisiiphilus cells. All of the Mabs reacted with cells from which the outer membrane had been stripped by incubation with sodium dodecyl sulphate, suggesting the peptidoglycan (PG) layer was involved in binding. Mab reactivity with purified PG confirmed this. Epitope mapping revealed the Mabs in total recognize four binding sites on the PG. Mabs specific for each of the four sites also bound strongly to disrupted Pectinatus frisingensis, Selenomonas lacticifix, Zymophilus paucivorans, and Zymophilus raffinosivorans cells, but weakly to disrupted Megasphaera cerevisiae cells. No antibody reactivity was seen with disrupted cells of 11 other species of Gram-negative bacteria. These results confirm that a common PG structure is used by several species of anaerobic Gram-negative beer spoilage bacteria. These results also indicate that PG-specific Mabs can be used to rapidly detect a range of anaerobic Gram-negative beer spoilage bacteria, provided the bacterial outer membrane is first removed to allow antibody binding.Key words: beer spoilage, epitope mapping, monoclonal antibodies, Pectinatus, peptidoglycan.

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