scholarly journals The periplasmic space cannot be artificially enlarged due to homeostatic regulation maintaining spatial constraints essential for membrane spanning processes and cell viability

2021 ◽  
Author(s):  
Eric Mandela ◽  
Christopher J. Stubenrauch ◽  
David Ryoo ◽  
Hyea Hwang ◽  
Eli J. Cohen ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Lipid Transport ◽  
Cell Stiffness ◽  
Spatial Constraint ◽  
Spatial Constraints ◽  
Load Bearing ◽  
Membrane Spanning ◽  
Peptidoglycan Layer

ABSTRACTThe cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope dictate protein and lipid transport and drug resistance phenotypes, and depend on spatial constraints across the envelope and load-bearing forces across the cell surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics and a synthetic lethal screen we show that lengthening Lpp to the upper limit does not change periplasmic width and spatial constraint, but rather impacts the load-bearing capacity across the outer membrane. E. coli expressing elongated Lpp activate potent homeostatic mechanisms to enforce a wild-type spatial constraint: they increase steady-state levels of factors determining cell stiffness, decrease membrane integrity, increase membrane vesiculation and depend on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Our findings demonstrate complex regulatory mechanisms for tight control over periplasmic width to enable spatial constraint essential for membrane spanning processes. They further show that the periplasm cannot be widened by engineering approaches, with implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling and protein translocation.

scholarly journals The Rcs Phosphorelay Is a Cell Envelope Stress Response Activated by Peptidoglycan Stress and Contributes to Intrinsic Antibiotic Resistance

2008 ◽  
Vol 190 (6) ◽  
pp. 2065-2074 ◽  
Author(s):  
Mary E. Laubacher ◽  
Sarah E. Ades
Keyword(s):  
Outer Membrane ◽  
Single Molecule ◽  
Stress Responses ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Envelope Stress ◽  
Peptidoglycan Layer

ABSTRACTGram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identifyEscherichia colistress responses activated following inhibition of specific PBPs by the β-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance ofE. colito β-lactam antibiotics.

scholarly journals Cell Envelope Perturbation Induces Oxidative Stress and Changes in Iron Homeostasis in Vibrio cholerae

2009 ◽  
Vol 191 (17) ◽  
pp. 5398-5408 ◽  
Author(s):  
Aleksandra E. Sikora ◽  
Sinem Beyhan ◽  
Michael Bagdasarian ◽  
Fitnat H. Yildiz ◽  
Maria Sandkvist
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Iron Homeostasis ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Genetic Alterations ◽  
Iron Storage ◽  
Study Gene Expression

ABSTRACT The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189:8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.

scholarly journals Appropriate Regulation of the σE-Dependent Envelope Stress Response Is Necessary To Maintain Cell Envelope Integrity and Stationary-Phase Survival in Escherichia coli

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Hervé Nicoloff ◽  
Saumya Gopalkrishnan ◽  
Sarah E. Ades
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Outer Membrane ◽  
Stress Responses ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Point Mutations ◽  
Content Type ◽  
Envelope Stress ◽  
Outer Membrane Porins

ABSTRACT The alternative sigma factor σE is a key component of the Escherichia coli response to cell envelope stress and is required for viability even in the absence of stress. The activity of σE increases during entry into stationary phase, suggesting an important role for σE when nutrients are limiting. Elevated σE activity has been proposed to activate a pathway leading to the lysis of nonculturable cells that accumulate during early stationary phase. To better understand σE-directed cell lysis and the role of σE in stationary phase, we investigated the effects of elevated σE activity in cultures grown for 10 days. We demonstrate that high σE activity is lethal for all cells in stationary phase, not only those that are nonculturable. Spontaneous mutants with reduced σE activity, due primarily to point mutations in the region of σE that binds the −35 promoter motif, arise and take over cultures within 5 to 6 days after entry into stationary phase. High σE activity leads to large reductions in the levels of outer membrane porins and increased membrane permeability, indicating membrane defects. These defects can be counteracted and stationary-phase lethality delayed significantly by stabilizing membranes with Mg2+ and buffering the growth medium or by deleting the σE-dependent small RNAs (sRNAs) MicA, RybB, and MicL, which inhibit the expression of porins and Lpp. Expression of these sRNAs also reverses the loss of viability following depletion of σE activity. Our results demonstrate that appropriate regulation of σE activity, ensuring that it is neither too high nor too low, is critical for envelope integrity and cell viability. IMPORTANCE The Gram-negative cell envelope and cytoplasm differ significantly, and separate responses have evolved to combat stress in each compartment. An array of cell envelope stress responses exist, each of which is focused on different parts of the envelope. The σE response is conserved in many enterobacteria and is tuned to monitor pathways for the maturation and delivery of outer membrane porins, lipoproteins, and lipopolysaccharide to the outer membrane. The activity of σE is tightly regulated to match the production of σE regulon members to the needs of the cell. In E. coli, loss of σE results in lethality. Here we demonstrate that excessive σE activity is also lethal and results in decreased membrane integrity, the very phenotype the system is designed to prevent.

scholarly journals Mutational Analysis of the Escherichia coli K-12 TolA N-Terminal Region and Characterization of Its TolQ-Interacting Domain by Genetic Suppression

1998 ◽  
Vol 180 (24) ◽  
pp. 6433-6439 ◽  
Author(s):  
Pierre Germon ◽  
Thierry Clavel ◽  
Anne Vianney ◽  
Raymond Portalier ◽  
Jean Claude Lazzaroni
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Cell Envelope ◽  
Transmembrane Helix ◽  
Membrane Integrity ◽  
Genetic Suppression ◽  
K 12

ABSTRACT The Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. They form two complexes in the cell envelope. Transmembrane domains of TolQ, TolR, and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. The N-terminal transmembrane domain of TolA anchors the protein to the cytoplasmic membrane and interacts with TolQ and TolR. Extensive mutagenesis of the N-terminal part of TolA was carried out to characterize the residues involved in such processes. Mutations affecting the function of TolA resulted in a lack or an alteration in TolA-TolQ or TolR-TolA interactions but did not affect the formation of TolQ-TolR complexes. Our results confirmed the importance of residues serine 18 and histidine 22, which are part of an SHLS motif highly conserved in the TolA and the related TonB proteins from different organisms. Genetic suppression experiments were performed to restore the functional activity of some tolA mutants. The suppressor mutations all affected the first transmembrane helix of TolQ. These results confirmed the essential role of the transmembrane domain of TolA in triggering interactions with TolQ and TolR.

scholarly journals In Vitro Characterization of Peptidoglycan-Associated Lipoprotein (PAL)–Peptidoglycan and PAL–TolB Interactions

1999 ◽  
Vol 181 (20) ◽  
pp. 6306-6311 ◽  
Author(s):  
Emmanuelle Bouveret ◽  
Hélène Bénédetti ◽  
Alain Rigal ◽  
Erwann Loret ◽  
Claude Lazdunski
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Periplasmic Protein ◽  
Multiprotein Complex ◽  
In Vitro Characterization ◽  
In Vitro Interaction

ABSTRACT The Tol-peptidoglycan-associated lipoprotein (PAL) system ofEscherichia coli is a multiprotein complex of the envelope involved in maintaining outer membrane integrity. PAL and the periplasmic protein TolB, two components of this complex, are interacting with each other, and they have also been reported to interact with OmpA and the major lipoprotein, two proteins interacting with the peptidoglycan. All these interactions suggest a role of the Tol-PAL system in anchoring the outer membrane to the peptidoglycan. Therefore, we were interested in better understanding the interaction between PAL and the peptidoglycan. We designed an in vitro interaction assay based on the property of purified peptidoglycan to be pelleted by ultracentrifugation. Using this assay, we showed that a purified PAL protein interacted in vitro with pure peptidoglycan. A peptide competition experiment further demonstrated that the region from residues 89 to 130 of PAL was sufficient to bind the peptidoglycan. Moreover, the fact that this same region of PAL was also binding to TolB suggested that these two interactions were exclusive. Indeed, the TolB-PAL complex appeared not to be associated with the peptidoglycan. This led us to the conclusion that PAL may exist in two forms in the cell envelope, one bound to TolB and the other bound to the peptidoglycan.

scholarly journals Peptidoglycan recycling contributes to outer membrane integrity and carbapenem tolerance in Acinetobacter baumannii

2021 ◽  
Author(s):  
Nowrosh Islam ◽  
Misha I. Kazi ◽  
Katie N. Kang ◽  
Jacob Biboy ◽  
Joe Gray ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Normal Growth ◽  
Multidrug Resistant ◽  
Antibiotic Tolerance ◽  
Gram Negative ◽  
Tolerance Factors ◽  
The Impact

The Gram-negative cell envelope is an essential structure that not only protects the cell against lysis from the internal turgor, but also forms a barrier to limit entry of antibiotics. Some of our most potent bactericidal antibiotics, the β-lactams, exploit the essentiality of the cell envelope by inhibiting its biosynthesis, typically inducing lysis and rapid death. However, many Gram-negative bacteria exhibit antibiotic tolerance, the ability to sustain viability in the presence of β-lactams for extended time periods. Despite several studies showing that antibiotic tolerance contributes directly to treatment failure, and is a steppingstone in acquisition of true resistance, the molecular factors that promote intrinsic tolerance are not well-understood. Acinetobacter baumannii is a critical-threat nosocomial pathogen notorious for its ability to rapidly develop multidrug resistance. While typically reserved to combat multidrug resistant infections, carbapenem β-lactam antibiotics (i.e., meropenem) are first-line prescriptions to treat A. baumannii infections. Meropenem tolerance in Gram-negative pathogens is characterized by morphologically distinct populations of spheroplasts, but the impact of spheroplast formation is not fully understood. Here, we show that susceptible A. baumannii clinical isolates demonstrate high intrinsic tolerance to meropenem, form spheroplasts with the antibiotic and revert to normal growth after antibiotic removal. Using transcriptomics and genetics screens, we characterized novel tolerance factors and found that outer membrane integrity maintenance, drug efflux and peptidoglycan homeostasis collectively contribute to meropenem tolerance in A. baumannii. Furthermore, outer membrane integrity and peptidoglycan recycling are tightly linked in their contribution to meropenem tolerance in A. baumannii.

scholarly journals Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

2020 ◽  
Author(s):  
Nicolas Coudray ◽  
Georgia L. Isom ◽  
Mark R. MacRae ◽  
Mariyah N. Saiduddin ◽  
Gira Bhabha ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Sequence Similarity ◽  
Substrate Binding ◽  
Membrane Integrity ◽  
Binding Pocket ◽  
Pathogen Virulence ◽  
Phospholipid Transport ◽  
Distinct Features

In double-membraned bacteria, phospholipids must be transported across the cell envelope to maintain the outer membrane barrier, which plays a key role in antibiotic resistance and pathogen virulence. The Mla system has been implicated in phospholipid trafficking and outer membrane integrity, and includes an ABC transporter complex, MlaFEDB. The transmembrane subunit, MlaE, has minimal sequence similarity to other ABC transporters, and the structure of the entire inner membrane MlaFEDB complex remains unknown. Here we report the cryo-EM structure of the MlaFEDB complex at 3.05 Å resolution. Our structure reveals that while MlaE has many distinct features, it is distantly related to the LPS and MacAB transporters, as well as the eukaryotic ABCA/ABCG families. MlaE adopts an outward-open conformation, resulting in a continuous pathway for phospholipid transport from the MlaE substrate-binding site to the pore formed by the ring of MlaD. Unexpectedly, two phospholipids are bound in the substrate-binding pocket of MlaFEDB, raising the possibility that multiple lipid substrates may be translocated each transport cycle. Site-specific crosslinking confirms that lipids bind in this pocket in vivo. Our structure provides mechanistic insight into substrate recognition and transport by the MlaFEDB complex.

scholarly journals Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Nicolas Coudray ◽  
Georgia L Isom ◽  
Mark R MacRae ◽  
Mariyah N Saiduddin ◽  
Gira Bhabha ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Sequence Similarity ◽  
Substrate Recognition ◽  
Membrane Integrity ◽  
Transport Pathway ◽  
Mechanistic Insight ◽  
Membrane Barrier ◽  
Phospholipid Transport ◽  
Insight Into

In double-membraned bacteria, phospholipid transport across the cell envelope is critical to maintain the outer membrane barrier, which plays a key role in virulence and antibiotic resistance. An MCE transport system called Mla has been implicated in phospholipid trafficking and outer membrane integrity, and includes an ABC transporter, MlaFEDB. The transmembrane subunit, MlaE, has minimal sequence similarity to other transporters, and the structure of the entire inner-membrane MlaFEDB complex remains unknown. Here, we report the cryo-EM structure of MlaFEDB at 3.05 Å resolution, revealing distant relationships to the LPS and MacAB transporters, as well as the eukaryotic ABCA/ABCG families. A continuous transport pathway extends from the MlaE substrate-binding site, through the channel of MlaD, and into the periplasm. Unexpectedly, two phospholipids are bound to MlaFEDB, suggesting that multiple lipid substrates may be transported each cycle. Our structure provides mechanistic insight into substrate recognition and transport by MlaFEDB.

The Tol-Pal proteins of the Escherichia coli cell envelope: an energized system required for outer membrane integrity?

2001 ◽  
Vol 152 (6) ◽  
pp. 523-529 ◽  
Author(s):  
Roland Lloubès ◽  
Eric Cascales ◽  
Anne Walburger ◽  
Emmanuelle Bouveret ◽  
Claude Lazdunski ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Coli Cell

scholarly journals Intermembrane transport: Glycerophospholipid homeostasis of the Gram-negative cell envelope

2019 ◽  
Vol 116 (35) ◽  
pp. 17147-17155 ◽  
Author(s):  
Matthew J. Powers ◽  
M. Stephen Trent
Keyword(s):  
Outer Membrane ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Lipid Transport ◽  
Lipid Asymmetry ◽  
Gram Negative ◽  
Outer Membranes ◽  
Negative Cell ◽  
Initial Discovery ◽  
Structural Methods

This perspective addresses recent advances in lipid transport across the Gram-negative inner and outer membranes. While we include a summary of previously existing literature regarding this topic, we focus on the maintenance of lipid asymmetry (Mla) pathway. Discovered in 2009 by the Silhavy group [J. C. Malinverni, T. J. Silhavy, Proc. Natl. Acad. Sci. U.S.A. 106, 8009–8014 (2009)], Mla has become increasingly appreciated for its role in bacterial cell envelope physiology. Through the work of many, we have gained an increasingly mechanistic understanding of the function of Mla via genetic, biochemical, and structural methods. Despite this, there is a degree of controversy surrounding the directionality in which Mla transports lipids. While the initial discovery and subsequent studies have posited that it mediated retrograde lipid transport (removing glycerophospholipids from the outer membrane and returning them to the inner membrane), others have asserted the opposite. This Perspective aims to lay out the evidence in an unbiased, yet critical, manner for Mla-mediated transport in addition to postulation of mechanisms for anterograde lipid transport from the inner to outer membranes.

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