scholarly journals High-throughput suppressor screen demonstrates that RcsF monitors outer membrane integrity and not Bam complex function

2021 ◽  
Vol 118 (32) ◽  
pp. e2100369118
Author(s):  
Muralidhar Tata ◽  
Santosh Kumar ◽  
Sarah R. Lach ◽  
Shreya Saha ◽  
Elizabeth M. Hart ◽  
...  
Keyword(s):  
Outer Membrane ◽  
High Throughput ◽  
Cell Envelope ◽  
Complex Function ◽  
Membrane Integrity ◽  
Active Role ◽  
Central Component ◽  
Loss Of Function ◽  
Bam Complex ◽  
Suppressor Screen

The regulator of capsule synthesis (Rcs) is a complex signaling cascade that monitors gram-negative cell envelope integrity. The outer membrane (OM) lipoprotein RcsF is the sensory component, but how RcsF functions remains elusive. RcsF interacts with the β-barrel assembly machinery (Bam) complex, which assembles RcsF in complex with OM proteins (OMPs), resulting in RcsF’s partial cell surface exposure. Elucidating whether RcsF/Bam or RcsF/OMP interactions are important for its sensing function is challenging because the Bam complex is essential, and partial loss-of-function mutations broadly compromise the OM biogenesis. Our recent discovery that, in the absence of nonessential component BamE, RcsF inhibits function of the central component BamA provided a genetic tool to select mutations that specifically prevent RcsF/BamA interactions. We employed a high-throughput suppressor screen to isolate a collection of such rcsF and bamA mutants and characterized their impact on RcsF/OMP assembly and Rcs signaling. Using these mutants and BamA inhibitors MRL-494L and darobactin, we provide multiple lines of evidence against the model in which RcsF senses Bam complex function. We show that Rcs activation in bam mutants results from secondary OM and lipopolysaccharide defects and that RcsF/OMP assembly is required for this activation, supporting an active role of RcsF/OMP complexes in sensing OM stress.

scholarly journals Stress-Based High-Throughput Screening Assays to Identify Inhibitors of Cell Envelope Biogenesis

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 808
Author(s):  
Maurice Steenhuis ◽  
Corinne M. ten Hagen-Jongman ◽  
Peter van Ulsen ◽  
Joen Luirink
Keyword(s):  
Outer Membrane ◽  
High Throughput ◽  
Stress Responses ◽  
High Throughput Screening ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Compound Screening ◽  
Outer Membrane Biogenesis

The structural integrity of the Gram-negative cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. coli upon addition of antibacterial compounds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compound screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes.

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 Penetration of Enveloped Double-Stranded RNA Bacteriophages φ13 and φ6 into Pseudomonas syringae Cells

2005 ◽  
Vol 79 (8) ◽  
pp. 5017-5026 ◽  
Author(s):  
Rimantas Daugelavičius ◽  
Virginija Cvirkaitė ◽  
Aušra Gaidelytė ◽  
Elena Bakienė ◽  
Rasa Gabrėnaitė-Verkhovskaya ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Outer Membrane ◽  
Pseudomonas Syringae ◽  
Cell Envelope ◽  
Active Role ◽  
Lytic Enzyme ◽  
Virus Infections ◽  
Virus Binding ◽  
Double Stranded Rna

ABSTRACT Bacteriophages φ6 and φ13 are related enveloped double-stranded RNA viruses that infect gram-negative Pseudomonas syringae cells. φ6 uses a pilus as a receptor, and φ13 attaches to the host lipopolysaccharide. We compared the entry-related events of these two viruses, including receptor binding, envelope fusion, peptidoglycan penetration, and passage through the plasma membrane. The infection-related events are dependent on the multiplicity of infection in the case of φ13 but not with φ6. A temporal increase of host outer membrane permeability to lipophilic ions was observed from 1.5 to 4 min postinfection in both virus infections. This enhanced permeability period coincided with the fast dilution of octadecyl rhodamine B-labeled virus-associated lipid molecules. This result is in agreement with membrane fusion, and the presence of temporal virus-derived membrane patches on the outer membrane. Similar to φ6, φ13 contains a thermosensitive lytic enzyme involved in peptidoglycan penetration. The phage entry also caused a limited depolarization of the plasma membrane. Inhibition of host respiration considerably decreased the efficiency of irreversible virus binding and membrane fusion. An active role of cell energy metabolism in restoring the infection-induced defects in the cell envelope was also observed.

scholarly journals Assembly of outer-membrane proteins in bacteria and mitochondria

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2587-2596 ◽  
Author(s):  
Jan Tommassen
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Basic Mechanism ◽  
Eukaryotic Cell ◽  
Mitochondrial Outer Membrane ◽  
Central Component ◽  
Cell Organelles ◽  
C Terminus

The cell envelope of Gram-negative bacteria consists of two membranes separated by the periplasm. In contrast with most integral membrane proteins, which span the membrane in the form of hydrophobic α-helices, integral outer-membrane proteins (OMPs) form β-barrels. Similar β-barrel proteins are found in the outer membranes of mitochondria and chloroplasts, probably reflecting the endosymbiont origin of these eukaryotic cell organelles. How these β-barrel proteins are assembled into the outer membrane has remained enigmatic for a long time. In recent years, much progress has been reached in this field by the identification of the components of the OMP assembly machinery. The central component of this machinery, called Omp85 or BamA, is an essential and highly conserved bacterial protein that recognizes a signature sequence at the C terminus of its substrate OMPs. A homologue of this protein is also found in mitochondria, where it is required for the assembly of β-barrel proteins into the outer membrane as well. Although accessory components of the machineries are different between bacteria and mitochondria, a mitochondrial β-barrel OMP can be assembled into the bacterial outer membrane and, vice versa, bacterial OMPs expressed in yeast are assembled into the mitochondrial outer membrane. These observations indicate that the basic mechanism of OMP assembly is evolutionarily highly conserved.

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 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 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.

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