scholarly journals The Mla pathway in Acinetobacter baumannii has no demonstrable role in anterograde lipid transport

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Matthew J Powers ◽  
Brent W Simpson ◽  
M Stephen Trent
Keyword(s):  
Acinetobacter Baumannii ◽  
Cell Envelope ◽  
Stringent Response ◽  
Lipid Homeostasis ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Anterograde Transport ◽  
Lipid Asymmetry ◽  
Outer Leaflet ◽  
Selective Permeability

The asymmetric outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier to the environment. Perturbations to OM lipid asymmetry sensitize the cell to antibiotics. As such, mechanisms involved in lipid asymmetry are fundamental to our understanding of OM lipid homeostasis. One such mechanism, the Maintenance of lipid asymmetry (Mla) pathway has been proposed to extract mislocalized glycerophospholipids from the outer leaflet of the OM and return them to the inner membrane (IM). Work on this pathway in Acinetobacter baumannii support conflicting models for the directionality of the Mla system being retrograde (OM to IM) or anterograde (IM to OM). Here, we show conclusively that A. baumannii mla mutants exhibit no defects in anterograde transport. Furthermore, we identify an allele of the GTPase obgE that is synthetically sick in the absence of Mla; providing another link between cell envelope homeostasis and stringent response.

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 The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane

eLife ◽  
2019 ◽  
Vol 8 ◽  
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

The 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 screened Acinetobacter baumannii transposon mutants and identified a number of mutants with OM defects, including an ABC transporter system homologous to the Mla system in E. 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 to Acinetobacter baumannii OM integrity and antibiotic sensitivity suggests that its components may serve as new antimicrobial therapeutic targets.

scholarly journals Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Shuhua Thong ◽  
Bilge Ercan ◽  
Federico Torta ◽  
Zhen Yang Fong ◽  
Hui Yi Alvina Wong ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Abc Transporter ◽  
Hydrolytic Activity ◽  
Membrane Lipid ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Lipid Asymmetry ◽  
Selective Permeability ◽  
Complex Functions ◽  
Bacterial Outer Membrane

In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry.

scholarly journals TheEscherichia coliPhospholipase PldA Regulates Outer Membrane Homeostasis via Lipid Signaling

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Kerrie L. May ◽  
Thomas J. Silhavy
Keyword(s):  
Fatty Acids ◽  
Outer Membrane ◽  
Coenzyme A ◽  
Second Messengers ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Long Distance ◽  
Lipid Asymmetry ◽  
Gram Negative ◽  
Outer Leaflet

ABSTRACTThe outer membrane (OM) bilayer of Gram-negative bacteria is biologically unique in its asymmetrical organization of lipids, with an inner leaflet composed of glycerophospholipids (PLs) and a surface-exposed outer leaflet composed of lipopolysaccharide (LPS). This lipid organization is integral to the OM’s barrier properties. Perturbations of the outer leaflet by antimicrobial peptides or defects in LPS biosynthesis or transport to the OM cause a compensatory flipping of PLs to the outer leaflet. As a result, lipid asymmetry is disrupted and OM integrity is compromised. Recently, we identified anEscherichia colimutant that exhibits aberrant accumulation of surface PLs accompanied by a cellular increase in LPS production. Remarkably, the observed hyperproduction of LPS is PldA dependent. Here we provide evidence that the fatty acids generated by PldA at the OM are transported into the cytoplasm and simultaneously activated by thioesterification to coenzyme A (CoA) by FadD. The acyl-CoAs produced ultimately inhibit LpxC degradation by FtsH. The increased levels of LpxC, the enzyme that catalyzes the first committed step in LPS biosynthesis, increases the amount of LPS produced. Our data suggest that PldA acts as a sensor for lipid asymmetry in the OM. PldA protects the OM barrier by both degrading mislocalized PLs and generating lipid second messengers that enable long-distance signaling that prompts the cell to restore homeostasis at a distant organelle.IMPORTANCEThe outer membrane of Gram-negative bacteria is an effective permeability barrier that protects the cell from toxic agents, including antibiotics. Barrier defects are often manifested by phospholipids present in the outer leaflet of this membrane that take up space normally occupied by lipopolysaccharide. We have discovered a signaling mechanism that operates across the entire cell envelope used by the cell to detect these outer membrane defects. A phospholipase, PldA, that functions to degrade these mislocalized phospholipids has a second, equally important function as a sensor. The fatty acids produced by hydrolysis of the phospholipids act as second messengers to signal the cell that more lipopolysaccharide is needed. These fatty acids diffuse across the periplasm and are transported into the cytoplasm by a process that attaches coenzyme A. The acyl-CoA molecule produces signals to inhibit the degradation of the critical enzyme LpxC by the ATP-dependent protease FtsH, increasing lipopolysaccharide production.

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 How the assembly and protection of the bacterial cell envelope depend on cysteine residues

2020 ◽  
Vol 295 (34) ◽  
pp. 11984-11994 ◽  
Author(s):  
Jean-François Collet ◽  
Seung-Hyun Cho ◽  
Bogdan I. Iorga ◽  
Camille V. Goemans
Keyword(s):  
Amino Acid ◽  
Cell Envelope ◽  
Multilayered Structure ◽  
Cysteine Residues ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Amino Acid Residues ◽  
Gram Negative ◽  
Oxidative Inactivation ◽  
Peptidoglycan Cell Wall

The cell envelope of Gram-negative bacteria is a multilayered structure essential for bacterial viability; the peptidoglycan cell wall provides shape and osmotic protection to the cell, and the outer membrane serves as a permeability barrier against noxious compounds in the external environment. Assembling the envelope properly and maintaining its integrity are matters of life and death for bacteria. Our understanding of the mechanisms of envelope assembly and maintenance has increased tremendously over the past two decades. Here, we review the major achievements made during this time, giving central stage to the amino acid cysteine, one of the least abundant amino acid residues in proteins, whose unique chemical and physical properties often critically support biological processes. First, we review how cysteines contribute to envelope homeostasis by forming stabilizing disulfides in crucial bacterial assembly factors (LptD, BamA, and FtsN) and stress sensors (RcsF and NlpE). Second, we highlight the emerging role of enzymes that use cysteine residues to catalyze reactions that are necessary for proper envelope assembly, and we also explain how these enzymes are protected from oxidative inactivation. Finally, we suggest future areas of investigation, including a discussion of how cysteine residues could contribute to envelope homeostasis by functioning as redox switches. By highlighting the redox pathways that are active in the envelope of Escherichia coli, we provide a timely overview of the assembly of a cellular compartment that is the hallmark of Gram-negative bacteria.

scholarly journals Acinetobacter baumannii Can Survive with an Outer Membrane Lacking Lipooligosaccharide Due to Structural Support from Elongasome Peptidoglycan Synthesis

mBio ◽  
2021 ◽  
Author(s):  
Brent W. Simpson ◽  
Marta Nieckarz ◽  
Victor Pinedo ◽  
Amanda B. McLean ◽  
Felipe Cava ◽  
...  
Keyword(s):  
Mechanical Strength ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Structural Support ◽  
Peptidoglycan Synthesis

Gram-negative bacteria have a multilayered cell envelope with a layer of cross-linked polymers (peptidoglycan) sandwiched between two membranes. Peptidoglycan was long thought to exclusively provide rigidity to the cell providing mechanical strength.

scholarly journals DpaA detaches Braun’s lipoprotein from peptidoglycan

2021 ◽  
Author(s):  
Matthias Winkle ◽  
Víctor M. Hernández-Rocamora ◽  
Karthik Pullela ◽  
Emily C. A. Goodall ◽  
Alessandra M. Martorana ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Stress Conditions ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Sequence Motifs ◽  
Gram Negative ◽  
E Coli ◽  
Impermeable Barrier ◽  
Unique Cell

ABSTRACTGram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun’s lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB and LdtC. LdtD and LdtE are members of the same family of LD-transpeptidases but they catalyse a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF remains unclear, although it has been shown to become essential in cells with inhibited LPS export to the outer membrane. We now show that LdtF hydrolyses the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product down-regulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated.IMPORTANCEGram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun’s lipoprotein (Lpp) is the most abundant protein in E. coli and about one third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from PG and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

scholarly journals DpaA Detaches Braun’s Lipoprotein from Peptidoglycan

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Matthias Winkle ◽  
Víctor M. Hernández-Rocamora ◽  
Karthik Pullela ◽  
Emily C. A. Goodall ◽  
Alessandra M. Martorana ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Stress Conditions ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Sequence Motifs ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Impermeable Barrier

ABSTRACT Gram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun’s lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB, and LdtC. LdtD and LdtE are members of the same family of ld-transpeptidases but they catalyze a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF, remains unclear, although it has been shown to become essential in cells with inhibited lipopolysaccharide export to the outer membrane. We now show that LdtF hydrolyzes the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product downregulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria, of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated. IMPORTANCE Gram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun’s lipoprotein (Lpp) is the most abundant protein in E. coli, and about one-third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far, the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from peptidoglycan, and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

scholarly journals Degradation of Components of the Lpt Transenvelope Machinery Reveals LPS-Dependent Lpt Complex Stability in Escherichia coli

2021 ◽  
Vol 8 ◽  
Author(s):  
Alessandra M. Martorana ◽  
Elisabete C. C. M. Moura ◽  
Paola Sperandeo ◽  
Flavia Di Vincenzo ◽  
Xiaofei Liang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Envelope ◽  
Barrier Properties ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Complex Stability ◽  
Gram Negative ◽  
Essential Proteins ◽  
Assembly Result ◽  
The Stability

Lipopolysaccharide (LPS) is a peculiar component of the outer membrane (OM) of many Gram-negative bacteria that renders these bacteria highly impermeable to many toxic molecules, including antibiotics. LPS is assembled at the OM by a dedicated intermembrane transport system, the Lpt (LPS transport) machinery, composed of seven essential proteins located in the inner membrane (IM) (LptB2CFG), periplasm (LptA), and OM (LptDE). Defects in LPS transport compromise LPS insertion and assembly at the OM and result in an overall modification of the cell envelope and its permeability barrier properties. LptA is a key component of the Lpt machine. It connects the IM and OM sub-complexes by interacting with the IM protein LptC and the OM protein LptD, thus enabling the LPS transport across the periplasm. Defects in Lpt system assembly result in LptA degradation whose stability can be considered a marker of an improperly assembled Lpt system. Indeed, LptA recruitment by its IM and OM docking sites requires correct maturation of the LptB2CFG and LptDE sub-complexes, respectively. These quality control checkpoints are crucial to avoid LPS mistargeting. To further dissect the requirements for the complete Lpt transenvelope bridge assembly, we explored the importance of LPS presence by blocking its synthesis using an inhibitor compound. Here, we found that the interruption of LPS synthesis results in the degradation of both LptA and LptD, suggesting that, in the absence of the LPS substrate, the stability of the Lpt complex is compromised. Under these conditions, DegP, a major chaperone–protease in Escherichia coli, is responsible for LptD but not LptA degradation. Importantly, LptD and LptA stability is not affected by stressors disturbing the integrity of LPS or peptidoglycan layers, further supporting the notion that the LPS substrate is fundamental to keeping the Lpt transenvelope complex assembled and that LptA and LptD play a major role in the stability of the Lpt system.

Sign in / Sign up

Export Citation Format

Share Document