scholarly journals Fine-Tuning of σEActivation Suppresses Multiple Assembly-Defective Mutations inEscherichia coli

2019 ◽  
Vol 201 (11) ◽  
Author(s):  
Elizabeth M. Hart ◽  
Aileen O’Connell ◽  
Kimberly Tang ◽  
Joseph S. Wzorek ◽  
Marcin Grabowicz ◽  
...  
Keyword(s):  
Stress Response ◽  
Cell Viability ◽  
Outer Membrane ◽  
Stress Responses ◽  
Expression Profiles ◽  
Outer Membrane Proteins ◽  
Fine Tuning ◽  
Permeability Barrier ◽  
Gram Negative ◽  
Envelope Stress

ABSTRACTThe Gram-negative outer membrane (OM) is a selectively permeable asymmetric bilayer that allows vital nutrients to diffuse into the cell but prevents toxins and hydrophobic molecules from entering. Functionally and structurally diverse β-barrel outer membrane proteins (OMPs) build and maintain the permeability barrier, making the assembly of OMPs crucial for cell viability. In this work, we characterize an assembly-defective mutant of the maltoporin LamB, LamBG439D. We show that the folding defect of LamBG439Dresults in an accumulation of unfolded substrate that is toxic to the cell when the periplasmic protease DegP is removed. Selection for suppressors of this toxicity identified the novel mutantdegSA323Eallele. The mutant DegSA323Eprotein contains an amino acid substitution at the PDZ/protease domain interface that results in a partially activated conformation of this protein. This activation increases basal levels of downstream σEstress response signaling. Furthermore, the enhanced σEactivity of DegSA323Esuppresses a number of other assembly-defective conditions without exhibiting the toxicity associated with high levels of σEactivity. We propose that the increased basal levels of σEsignaling primes the cell to respond to envelope stress before OMP assembly defects threaten cell viability. This finding addresses the importance of envelope stress responses in monitoring the OMP assembly process and underpins the critical balance between envelope defects and stress response activation.IMPORTANCEGram-negative bacteria, such asEscherichia coli, inhabit a natural environment that is prone to flux. In order to cope with shifting growth conditions and the changing availability of nutrients, cells must be capable of quickly responding to stress. Stress response pathways allow cells to rapidly shift gene expression profiles to ensure survival in this unpredictable environment. Here we describe a mutant that partially activates the σEstress response pathway. The elevated basal level of this stress response allows the cell to quickly respond to overwhelming stress to ensure cell survival.

scholarly journals Inhibitor of intramembrane protease RseP blocks the σE response causing lethal accumulation of unfolded outer membrane proteins

2018 ◽  
Vol 115 (28) ◽  
pp. E6614-E6621 ◽  
Author(s):  
Anna Konovalova ◽  
Marcin Grabowicz ◽  
Carl J. Balibar ◽  
Juliana C. Malinverni ◽  
Ronald E. Painter ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Nutrient Uptake ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Selective Inhibitor ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Underlying Basis

The outer membrane (OM) of Gram-negative bacteria forms a robust permeability barrier that blocks entry of toxins and antibiotics. Most OM proteins (OMPs) assume a β-barrel fold, and some form aqueous channels for nutrient uptake and efflux of intracellular toxins. The Bam machine catalyzes rapid folding and assembly of OMPs. Fidelity of OMP biogenesis is monitored by the σE stress response. When OMP folding defects arise, the proteases DegS and RseP act sequentially to liberate σE into the cytosol, enabling it to activate transcription of the stress regulon. Here, we identify batimastat as a selective inhibitor of RseP that causes a lethal decrease in σE activity in Escherichia coli, and we further identify RseP mutants that are insensitive to inhibition and confer resistance. Remarkably, batimastat treatment allows the capture of elusive intermediates in the OMP biogenesis pathway and offers opportunities to better understand the underlying basis for σE essentiality.

scholarly journals Hfq Modulates the σE-Mediated Envelope Stress Response and the σ32-Mediated Cytoplasmic Stress Response in Escherichia coli

2006 ◽  
Vol 189 (5) ◽  
pp. 1963-1973 ◽  
Author(s):  
Eric Guisbert ◽  
Virgil A. Rhodius ◽  
Nidhi Ahuja ◽  
Emily Witkin ◽  
Carol A. Gross
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Stress Responses ◽  
Outer Membrane Proteins ◽  
Sugar Uptake ◽  
Acid Uptake ◽  
General Role ◽  
Envelope Stress

ABSTRACT Hfq, a chaperone for small noncoding RNAs, regulates many processes in Escherichia coli, including the σS-mediated general stress response. Here we used microarray analysis to identify the changes in gene expression resulting from lack of Hfq. We identify several potential new targets for Hfq regulation, including genes encoding outer membrane proteins, enzymes, factors, and transporters. Many of these genes are involved in amino acid uptake and biosynthesis, sugar uptake and metabolism, and cell energetics. In addition, we find altered regulation of the σE- and σ32-mediated stress responses, which we analyze further. We show that cells lacking Hfq induce the σE-mediated envelope stress response and are defective in σE-mediated repression of outer membrane proteins. We also show that the σ32-mediated cytoplasmic stress response is repressed in cells lacking Hfq due to increased expression of DnaK. Furthermore, we show that cells lacking Hfq are defective in the “long-term adaptation” of σ32 to chronic chaperone overexpression. Together, our results indicate that Hfq may play a general role in stress response regulation in E. coli.

scholarly journals Recognition of β-Strand Motifs by RseB Is Required for σEActivity in Escherichia coli

2011 ◽  
Vol 193 (22) ◽  
pp. 6179-6186 ◽  
Author(s):  
Adam Kulp ◽  
Meta J. Kuehn
Keyword(s):  
Stress Response ◽  
Stress Responses ◽  
Outer Membrane Proteins ◽  
Harsh Environments ◽  
Gram Negative Bacteria ◽  
Dependent Manner ◽  
Gram Negative ◽  
Stress Response Pathway ◽  
Envelope Stress ◽  
Different Types

Gram-negative bacteria react to misfolded proteins in the envelope through a myriad of different stress response pathways. This cohort of pathways allows the bacteria to specifically respond to different types of damage, and many of these have been discovered to have key roles in the virulence of bacterial pathogens. Misfolded outer membrane proteins (OMPs) are typically recognized by the σEpathway, a highly conserved envelope stress response pathway. We examined the features of misfolded OMPs with respect to their ability to generate envelope stress responses. We determined that the secondary structure, particularly the potential to form β strands, is critical to inducing the σEresponse in an RseB-dependent manner. The sequence of the potential β-strand motif modulates the strength of the σEresponse generated by the constructs. By understanding the details of how such stress response pathways are activated, we can gain a greater understanding of how bacteria survive in harsh environments.

scholarly journals A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli

2015 ◽  
Vol 291 (4) ◽  
pp. 1921-1932 ◽  
Author(s):  
Matthias Urfer ◽  
Jasmina Bogdanovic ◽  
Fabio Lo Monte ◽  
Kerstin Moehle ◽  
Katja Zerbe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Permeability Barrier ◽  
Gram Negative ◽  
Fluorescence Studies ◽  
E Coli ◽  
Interaction Sites ◽  
External Stresses ◽  
Antibiotic Discovery

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.

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 The CpxQ sRNA Negatively Regulates Skp To Prevent Mistargeting of β-Barrel Outer Membrane Proteins into the Cytoplasmic Membrane

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Marcin Grabowicz ◽  
Daria Koren ◽  
Thomas J. Silhavy
Keyword(s):  
Stress Response ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Protein Misfolding ◽  
Outer Membrane Proteins ◽  
Inner Membrane ◽  
Bam Complex ◽  
Envelope Stress ◽  
Periplasmic Chaperone

ABSTRACT The promoter most strongly induced upon activation of the Cpx two-component envelope stress response is the cpxP promoter. The 3′ untranscribed region (UTR) of the cpxP transcript is shown to produce a small RNA (sRNA), CpxQ. We investigated the role of CpxQ in combating envelope stress. Remarkably, the two effectors specified by the transcript are deployed to combat distinct stresses in different cellular compartments. CpxP acts in both a regulatory negative-feedback loop and as an effector that combats periplasmic protein misfolding. We find that CpxQ combats toxicity at the inner membrane (IM) by downregulating the synthesis of the periplasmic chaperone Skp. Our data indicate that this regulation prevents Skp from inserting β-barrel outer membrane proteins (OMPs) into the IM, a lethal event that likely collapses the proton motive force. Our findings suggest that Skp can fold and directly insert OMPs into a lipid bilayer in vivo without the aid of the Bam complex. IMPORTANCE Skp is a well-characterized periplasmic chaperone that binds unfolded OMPs. Surprisingly, we find that Skp can catalyze the folding and mistargeting of OMPs into the inner membrane without the aid of the other cellular proteins that normally assemble OMPs. Several OMPs function as diffusion pores. Accordingly, their mistargeting is lethal because it depolarizes the inner membrane. We show that the most highly expressed transcript of the Cpx stress response produces an sRNA from the 3′ UTR, CpxQ, which combats this potential toxicity by downregulating Skp production. Defects in OMP assembly trigger the σ E response to upregulate factors, including Skp, that promote OMP folding. The Cpx response downregulates σ E . Our findings reveal that this heretofore puzzling hierarchy exists to protect the inner membrane.

scholarly journals Lipoprotein DolP supports proper folding of BamA in the bacterial outer membrane promoting fitness upon envelope stress

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
David Ranava ◽  
Yiying Yang ◽  
Luis Orenday-Tapia ◽  
François Rousset ◽  
Catherine Turlan ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Transcriptional Response ◽  
Permeability Barrier ◽  
Cell Recruitment ◽  
Bam Complex ◽  
Outer Membrane Lipoprotein ◽  
Envelope Stress ◽  
Late Step ◽  
Membrane Lipoprotein

In Proteobacteria, integral outer membrane proteins (OMPs) are crucial for the maintenance of the envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OMPs activates the sigmaE (σE) transcriptional response. σE upregulates OMP biogenesis factors, including the β-barrel assembly machinery (BAM) that catalyses OMP folding. Here we report that DolP (formerly YraP), a σE-upregulated and poorly understood outer membrane lipoprotein, is crucial for fitness in cells that undergo envelope stress. We demonstrate that DolP interacts with the BAM complex by associating with outer membrane-assembled BamA. We provide evidence that DolP is important for proper folding of BamA that overaccumulates in the outer membrane, thus supporting OMP biogenesis and envelope integrity. Notably, mid-cell recruitment of DolP had been linked to regulation of septal peptidoglycan remodelling by an unknown mechanism. We now reveal that, during envelope stress, DolP loses its association with the mid-cell, thereby suggesting a mechanistic link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.

scholarly journals A Stress Response Monitoring Lipoprotein Trafficking to the Outer Membrane

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Kerrie L. May ◽  
Kelly M. Lehman ◽  
Angela M. Mitchell ◽  
Marcin Grabowicz
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Outer Membrane ◽  
Stress Responses ◽  
Stress System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Essential Components ◽  
Trafficking Defects

ABSTRACTGram-negative bacteria produce lipid-anchored lipoproteins that are trafficked to their outer membrane (OM). These lipoproteins are essential components in each of the molecular machines that build the OM, including the Bam machine that assembles β-barrel proteins and the Lpt pathway that transports lipopolysaccharide. Stress responses are known to monitor Bam and Lpt function, yet no stress system has been found that oversees the fundamental process of lipoprotein trafficking. We used genetic and chemical biology approaches to induce several different lipoprotein trafficking stresses inEscherichia coli. Our results identified the Cpx two-component system as a stress response for monitoring trafficking. Cpx is activated by trafficking defects and is required to protect the cell against the consequence of the resulting stress. The OM-targeted lipoprotein NlpE acts as a sensor that allows Cpx to gauge trafficking efficiency. We reveal that NlpE signals to Cpx while it is transiting the inner membrane (IM)en routeto the OM and that only a small highly conserved N-terminal domain is required for signaling. We propose that defective trafficking causes NlpE to accumulate in the IM, activating Cpx to mount a transcriptional response that protects cells. Furthermore, we reconcile this new role of NlpE in signaling trafficking defects with its previously proposed role in sensing copper (Cu) stress by demonstrating that Cu impairs acylation of lipoproteins and, consequently, their trafficking to the OM.IMPORTANCEThe outer membrane built by Gram-negative bacteria such asEscherichia coliforms a barrier that prevents antibiotics from entering the cell, limiting clinical options at a time of prevalent antibiotic resistance. Stress responses ensure that barrier integrity is continuously maintained. We have identified the Cpx signal transduction system as a stress response that monitors the trafficking of lipid-anchored lipoproteins to the outer membrane. These lipoproteins are needed by every machine that builds the outer membrane. Cpx monitors just one lipoprotein, NlpE, to detect the efficiency of lipoprotein trafficking in the cell. NlpE and Cpx were previously shown to play a role in resistance to copper. We show that copper blocks lipoprotein trafficking, reconciling old and new observations. Copper is an important element in innate immunity against pathogens, and our findings suggest that NlpE and Cpx helpE. colisurvive the assault of copper on a key outer membrane assembly pathway.

scholarly journals Fine-Tuning of the Cpx Envelope Stress Response Is Required for Cell Wall Homeostasis inEscherichia coli

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Antoine Delhaye ◽  
Jean-François Collet ◽  
Géraldine Laloux
Keyword(s):  
Quality Control ◽  
Cell Wall ◽  
Control System ◽  
Stress Response ◽  
Cell Shape ◽  
Protein Quality ◽  
Fine Tuning ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Envelope Stress

ABSTRACTThe envelope of Gram-negative bacteria is an essential compartment that constitutes a protective and permeability barrier between the cell and its environment. The envelope also hosts the cell wall, a mesh-like structure made of peptidoglycan (PG) that determines cell shape and provides osmotic protection. Since the PG must grow and divide in a cell-cycle-synchronized manner, its synthesis and remodeling are tightly regulated. Here, we discovered that PG homeostasis is intimately linked to the levels of activation of the Cpx system, an envelope stress response system traditionally viewed as being involved in protein quality control in the envelope. We first show that Cpx is activated when PG integrity is challenged and that this activation provides protection to cells exposed to antibiotics inhibiting PG synthesis. By rerouting the outer membrane lipoprotein NlpE, a known Cpx activator, to a different envelope subcompartment, we managed to manipulate Cpx activation levels. We found that Cpx overactivation leads to aberrant cellular morphologies, to an increased sensitivity to β-lactams, and to dramatic division and growth defects, consistent with a loss of PG homeostasis. Remarkably, these phenotypes were largely abrogated by the deletion ofldtD, a Cpx-induced gene involved in noncanonical PG cross-linkage, suggesting that this transpeptidase is an important link between PG homeostasis and the Cpx system.Altogether our data show that fine-tuning of an envelope quality control system constitutes an important layer of regulation of the highly organized cell wall structure.IMPORTANCEThe envelope of Gram-negative bacteria is essential for viability. First, it includes the cell wall, a continuous polymer of peptidoglycan (PG) that determines cell morphology and protects against osmotic stress. Moreover, the envelope constitutes a protective barrier between the cell interior and the environment. Therefore, mechanisms called envelope stress response systems (ESRS) exist to monitor and defend envelope integrity against harmful conditions. Cpx is a major ESRS that detects and manages the accumulation of misfolded proteins in the envelope ofEscherichia coli. We found that this protein quality control system also plays a fundamental role in the regulation of PG assembly. Strikingly, the level of Cpx response is critical, as an excessive activation leads to phenotypes associated with a loss of cell wall integrity. Thus, by contributing to PG homeostasis, the Cpx system lies at the crossroads between key processes of bacterial life, including cell shape, growth, division, and antibiotic resistance.

scholarly journals Classifying β-Barrel Assembly Substrates by Manipulating Essential Bam Complex Members

2016 ◽  
Vol 198 (14) ◽  
pp. 1984-1992 ◽  
Author(s):  
Tara F. Mahoney ◽  
Dante P. Ricci ◽  
Thomas J. Silhavy
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Mechanistic Model ◽  
Outer Membrane Proteins ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Bam Complex ◽  
Periplasmic Chaperones

ABSTRACTThe biogenesis of the outer membrane (OM) ofEscherichia coliis a conserved and vital process. The assembly of integral β-barrel outer membrane proteins (OMPs), which represent a major component of the OM, depends on periplasmic chaperones and the heteropentameric β-barrel assembly machine (Bam complex) in the OM. However, not all OMPs are affected by null mutations in the same chaperones or nonessential Bam complex members, suggesting there are categories of substrates with divergent requirements for efficient assembly. We have previously demonstrated two classes of substrates, one comprising large, low-abundance, and difficult-to-assemble substrates that are heavily dependent on SurA and also Skp and FkpA, and the other comprising relatively simple and abundant substrates that are not as dependent on SurA but are strongly dependent on BamB for assembly. Here, we describe novel mutations inbamDthat lower levels of BamD 10-fold and >25-fold without altering the sequence of the mature protein. We utilized these mutations, as well as a previously characterized mutation that lowers wild-type BamA levels, to reveal a third class of substrates. These mutations preferentially cause a marked decrease in the levels of multimeric proteins. This susceptibility of multimers to lowered quantities of Bam machines in the cell may indicate that multiple Bam complexes are needed to efficiently assemble multimeric proteins into the OM.IMPORTANCEThe outer membrane (OM) of Gram-negative bacteria, such asEscherichia coli, serves as a selective permeability barrier that prevents the uptake of toxic molecules and antibiotics. Integral β-barrel proteins (OMPs) are assembled by the β-barrel assembly machine (Bam), components of which are conserved in mitochondria, chloroplasts, and all Gram-negative bacteria, including many clinically relevant pathogenic species. Bam is essential for OM biogenesis and accommodates a diverse array of client proteins; however, a mechanistic model that accounts for the selectivity and broad substrate range of Bam is lacking. Here, we show that the assembly of multimeric OMPs is more strongly affected than that of monomeric OMPs when essential Bam complex components are limiting, suggesting that multiple Bam complexes are needed to assemble multimeric proteins.

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