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 Stress Reduction, Bacterial Style

2017 ◽  
Vol 199 (20) ◽  
Author(s):  
Susan Gottesman
Keyword(s):  
Escherichia Coli ◽  
Stress Reduction ◽  
Stress Responses ◽  
Cell Envelope ◽  
Content Type ◽  
E Coli ◽  
Envelope Stress ◽  
Multiple Cell ◽  
As Stress ◽  
Sigma E

ABSTRACT Bacteria have robust responses to a variety of stresses. In particular, bacteria like Escherichia coli have multiple cell envelope stress responses, and generally we evaluate what these responses are doing by the repair systems they induce. However, probably at least as important in interpreting what is being sensed as stress are the genes that these stress systems downregulate, directly or indirectly. This is discussed here for the Cpx and sigma E systems of E. coli.

scholarly journals Transcriptional Responses of Escherichia coli to a Small-Molecule Inhibitor of LolCDE, an Essential Component of the Lipoprotein Transport Pathway

2016 ◽  
Vol 198 (23) ◽  
pp. 3162-3175 ◽  
Author(s):  
Christian Lorenz ◽  
Thomas J. Dougherty ◽  
Stephen Lory
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Small Molecule ◽  
Stress Responses ◽  
Transport Pathway ◽  
Transcript Levels ◽  
Content Type ◽  
E Coli ◽  
Molecule Inhibitor ◽  
Envelope Stress

ABSTRACTIn Gram-negative bacteria, a dedicated machinery consisting of LolABCDE components targets lipoproteins to the outer membrane. We used a previously identified small-molecule inhibitor of the LolCDE complex ofEscherichia colito assess the global transcriptional consequences of interference with lipoprotein transport. Exposure ofE. colito the LolCDE inhibitor at concentrations leading to minimal and significant growth inhibition, followed by transcriptome sequencing, identified a small group of genes whose transcript levels were decreased and a larger group whose mRNA levels increased 10- to 100-fold compared to those of untreated cells. The majority of the genes whose mRNA concentrations were reduced were part of the flagellar assembly pathway, which contains an essential lipoprotein component. Most of the genes whose transcript levels were elevated encode proteins involved in selected cell stress pathways. Many of these genes are involved with envelope stress responses induced by the mislocalization of outer membrane lipoproteins. Although several of the genes whose RNAs were induced have previously been shown to be associated with the general perturbation of the cell envelope by antibiotics, a small subset was affected only by LolCDE inhibition. Findings from this work suggest that the efficiency of the Lol system function may be coupled to a specific monitoring system, which could be exploited in the development of reporter constructs suitable for use for screening for additional inhibitors of lipoprotein trafficking.IMPORTANCEInhibition of the lipoprotein transport pathway leads toE. colideath and subsequent lysis. Early significant changes in the levels of RNA for a subset of genes identified to be associated with some periplasmic and envelope stress responses were observed. Together these findings suggest that disruption of this key pathway can have a severe impact on balanced outer membrane synthesis sufficient to affect viability.

scholarly journals Role ofrpoSin the Development of Cell Envelope Resilience and Pressure Resistance in Stationary-Phase Escherichia coli

2011 ◽  
Vol 77 (15) ◽  
pp. 5220-5229 ◽  
Author(s):  
Duangkamol Charoenwong ◽  
Simon Andrews ◽  
Bernard Mackey
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Fatty Acyl ◽  
Content Type ◽  
Pressure Sensitive ◽  
Membrane Resealing ◽  
Pressure Resistance ◽  
Regulated Functions

ABSTRACTThis work investigated the role ofrpoSin the development of increased cell envelope resilience and enhanced pressure resistance in stationary-phase cells ofEscherichia coli. Loss of both colony-forming ability and membrane integrity, measured as uptake of propidium iodide (PI), occurred at lower pressures inE. coliBW3709 (rpoS) than in the parental strain (BW2952). TherpoSmutant also released much higher concentrations of protein under pressure than the parent. We propose that RpoS-regulated functions are responsible for the increase in membrane resilience as cells enter stationary phase and that this plays a major role in the development of pressure resistance. Strains from the Keio collection with mutations in two RpoS-regulated genes,cfa(cyclopropane fatty acyl phospholipid synthase) andosmB(outer membrane lipoprotein), were significantly more pressure sensitive and took up more PI than the parent strain, withcfahaving the greatest effect. Mutations in thebolAmorphogene and other RpoS-regulated lipoprotein genes (osmC,osmE,osmY, andybaY) had no effect on pressure resistance. The cytoplasmic membranes of therpoSmutant failed to reseal after pressure treatment, and strains with mutations inosmBandnlpI(new lipoprotein) were also somewhat impaired in the ability to reseal their membranes. Thecfamutant, though pressure sensitive, was unaffected in membrane resealing, implying that the initial transient permeabilization event is critical for loss of viability rather than the failure to reseal. The enhanced pressure sensitivity ofpolA,recA, andxthAmutants suggested that DNA may be a target of oxidative stress in pressure-treated cells.

scholarly journals Activation of Toxin-Antitoxin System Toxins Suppresses Lethality Caused by the Loss of σEin Escherichia coli

2015 ◽  
Vol 197 (14) ◽  
pp. 2316-2324 ◽  
Author(s):  
Yasushi Daimon ◽  
Shin-ichiro Narita ◽  
Yoshinori Akiyama
Keyword(s):  
Escherichia Coli ◽  
Stress Responses ◽  
Ectopic Expression ◽  
Growth Media ◽  
Content Type ◽  
E Coli ◽  
Envelope Stress ◽  
Genes Encoding ◽  
Σ Factor ◽  
Mutant Forms

ABSTRACTσE, an alternative σ factor that governs a major signaling pathway in envelope stress responses in Gram-negative bacteria, is essential for growth ofEscherichia colinot only under stressful conditions, such as elevated temperature, but also under normal laboratory conditions. A mutational inactivation of thehicBgene has been reported to suppress the lethality caused by the loss of σE.hicBencodes the antitoxin of the HicA-HicB toxin-antitoxin (TA) system; overexpression of the HicA toxin, which exhibits mRNA interferase activity, causes cleavage of mRNAs and an arrest of cell growth, while simultaneous expression of HicB neutralizes the toxic effects of overproduced HicA. To date, however, how the loss of HicB rescues the cell lethality in the absence of σEand, more specifically, whether HicA is involved in this process remain unknown. Here we showed that simultaneous disruption ofhicAabolished suppression of the σEessentiality in the absence ofhicB, while ectopic expression of wild-type HicA, but not that of its mutant forms without mRNA interferase activity, restored the suppression. Furthermore, HicA and two other mRNA interferase toxins, HigB and YafQ, suppressed the σEessentiality even in the presence of chromosomally encoded cognate antitoxins when these toxins were overexpressed individually. Interestingly, when the growth media were supplemented with low levels of antibiotics that are known to activate toxins,E. colicells with no suppressor mutations grew independently of σE. Taken together, our results indicate that the activation of TA system toxins can suppress the σEessentiality and affect the extracytoplasmic stress responses.IMPORTANCEσEis an alternative σ factor involved in extracytoplasmic stress responses. Unlike other alternative σ factors, σEis indispensable for the survival ofE. colieven under unstressed conditions, although the exact reason for its essentiality remains unknown. Toxin-antitoxin (TA) systems are widely distributed in prokaryotes and are composed of two adjacent genes, encoding a toxin that exerts harmful effects on the toxin-producing bacterium itself and an antitoxin that neutralizes the cognate toxin. Curiously, it is known that inactivation of an antitoxin rescues the σEessentiality, suggesting a connection between TA systems and σEfunction. We demonstrate here that toxin activation is necessary for this rescue and suggest the possible involvement of TA systems in extracytoplasmic stress responses.

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 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 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 Experimental Evolution ofEscherichia coliK-12 at High pH and with RpoS Induction

2018 ◽  
Vol 84 (15) ◽  
Author(s):  
Issam Hamdallah ◽  
Nadia Torok ◽  
Katarina M. Bischof ◽  
Nadim Majdalani ◽  
Sriya Chadalavada ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Experimental Evolution ◽  
Sigma Factor ◽  
Point Mutations ◽  
Envelope Proteins ◽  
Content Type ◽  
High Ph ◽  
K 12 ◽  
Sigma Factor Rpos

ABSTRACTExperimental evolution ofEscherichia coliK-12 W3110 by serial dilutions for 2,200 generations at high pH extended the range of sustained growth from pH 9.0 to pH 9.3. pH 9.3-adapted isolates showed mutations in DNA-binding regulators and envelope proteins. One population showed an IS1knockout ofphoB(encoding the positive regulator of the phosphate regulon). AphoB::kanRknockout increased growth at high pH.phoBmutants are known to increase production of fermentation acids, which could enhance fitness at high pH. Mutations inpcnB[poly(A) polymerase] also increased growth at high pH. Three out of four populations showed deletions oftorI, an inhibitor of TorR, which activates expression oftorCAD(trimethylamineN-oxide respiration) at high pH. All populations showed point mutations affecting the stationary-phase sigma factor RpoS, either in the coding gene or in genes for regulators of RpoS expression. RpoS is required for survival at extremely high pH. In our microplate assay,rpoSdeletion slightly decreased growth at pH 9.1. RpoS protein accumulated faster at pH 9 than at pH 7. The RpoS accumulation at high pH required the presence of one or more antiadaptors that block degradation (IraM, IraD, and IraP). Other genes with mutations after high-pH evolution encode regulators, such as those encoded byyobG(mgrB) (PhoPQ regulator),rpoN(nitrogen starvation sigma factor),malI, andpurR, as well as envelope proteins, such as those encoded byompTandyahO. Overall,E. colievolution at high pH selects for mutations in key transcriptional regulators, includingphoBand the stationary-phase sigma factor RpoS.IMPORTANCEEscherichia coliin its native habitat encounters high-pH stress such as that of pancreatic secretions. Experimental evolution over 2,000 generations showed selection for mutations in regulatory factors, such as deletion of the phosphate regulator PhoB and mutations that alter the function of the global stress regulator RpoS. RpoS is induced at high pH via multiple mechanisms.

scholarly journals Complex Physiology and Compound Stress Responses during Fermentation of Alkali-Pretreated Corn Stover Hydrolysate by an Escherichia coli Ethanologen

2012 ◽  
Vol 78 (9) ◽  
pp. 3442-3457 ◽  
Author(s):  
Michael S. Schwalbach ◽  
David H. Keating ◽  
Mary Tremaine ◽  
Wesley D. Marner ◽  
Yaoping Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Amino Acids ◽  
Stationary Phase ◽  
Gene Expression Profiling ◽  
Stress Responses ◽  
Expression Profiling ◽  
Content Type ◽  
E Coli ◽  
Cell Maintenance

ABSTRACTThe physiology of ethanologenicEscherichia coligrown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into howE. coliresponds to such hydrolysates, we studied anE. coliK-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (∼6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate,E. coliceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.

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