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 Regulation of Iron Storage by CsrA Supports Exponential Growth of Escherichia coli

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Christine Pourciau ◽  
Archana Pannuri ◽  
Anastasia Potts ◽  
Helen Yakhnin ◽  
Paul Babitzke ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Escherichia Coli ◽  
Exponential Growth ◽  
Iron Homeostasis ◽  
Iron Storage ◽  
Content Type ◽  
Mrna Targets ◽  
Cellular Iron ◽  
The Impact

ABSTRACT The global regulatory protein CsrA coordinates gene expression in response to physiological cues reflecting cellular stress and nutrition. CsrA binding to the 5′ segments of mRNA targets affects their translation, RNA stability, and/or transcript elongation. Recent studies identified probable mRNA targets of CsrA that are involved in iron uptake and storage in Escherichia coli, suggesting an unexplored role for CsrA in regulating iron homeostasis. Here, we assessed the impact of CsrA on iron-related gene expression, cellular iron, and growth under various iron levels. We investigated five new targets of CsrA regulation, including the genes for 4 ferritin or ferritin-like iron storage proteins (ISPs) and the stress-inducible Fe-S repair protein, SufA. CsrA bound with high affinity and specificity to ftnB, bfr, and dps mRNAs and inhibited their translation, while it modestly activated ftnA expression. Furthermore, CsrA was found to regulate cellular iron levels and support growth by repressing the expression of genes for ISPs, most importantly, ferritin B (FtnB) and bacterioferritin (Bfr). Iron starvation did not substantially affect cellular levels of CsrA or its small RNA (sRNA) antagonists, CsrB and CsrC. csrA disruption led to increased resistance to the lethal effects of H2O2 during exponential growth, consistent with a regulatory role in oxidative stress resistance. We propose that during exponential growth and under minimal stress, CsrA represses the deleterious expression of the ISPs that function under oxidative stress and stationary-phase conditions (FtnB, Bfr, and Dps), thus ensuring that cellular iron is available to processes that are required for growth. IMPORTANCE Iron is an essential micronutrient for nearly all living organisms but is toxic in excess. Consequently, the maintenance of iron homeostasis is a critical biological process, and the genes involved in this function are tightly regulated. Here, we explored a new role for the bacterial RNA binding protein CsrA in the regulation of iron homeostasis. CsrA was shown to be a key regulator of iron storage genes in Escherichia coli, with consequential effects on cellular iron levels and growth. Our findings establish a model in which robust CsrA activity during the exponential phase of growth leads to repression of genes whose products sequester iron or divert it to unnecessary stress response processes. In so doing, CsrA supports E. coli growth under iron-limiting laboratory conditions and may promote fitness in the competitive iron-limited environment of the host large intestine.

scholarly journals Compromised Outer Membrane Integrity in Vibrio cholerae Type II Secretion Mutants

2007 ◽  
Vol 189 (23) ◽  
pp. 8484-8495 ◽  
Author(s):  
Aleksandra E. Sikora ◽  
Suzanne R. Lybarger ◽  
Maria Sandkvist
Keyword(s):  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Culture Medium ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Polymyxin B ◽  
Growth Defect ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Infant Mouse

ABSTRACT The type II secretion (T2S) system of Vibrio cholerae is a multiprotein complex that spans the cell envelope and secretes proteins important for pathogenesis as well as survival in different environments. Here we report that, in addition to the loss of extracellular secretion, removal or inhibition of expression of the T2S genes, epsC-N, results in growth defects and a broad range of alterations in the outer membrane that interfere with its barrier function. Specifically, the sensitivity to membrane-perturbing agents such as bile salts and the antimicrobial peptide polymyxin B is increased, and periplasmic constituents leak out into the culture medium. As a consequence, the σE stress response is induced. Furthermore, due to the defects caused by inactivation of the T2S system, the Δeps deletion mutant of V. cholerae strain N16961 is incapable of surviving the passage through the infant mouse gastrointestinal tract. The growth defect and leaky outer membrane phenotypes are suppressed when the culture medium is supplemented with 5% glucose or sucrose, although the eps mutants remain sensitive to membrane-damaging agents. This suggests that the sugars do not restore the integrity of the outer membrane in the eps mutant strains per se but may provide osmoprotective functions.

scholarly journals The Irr and RirA Proteins Participate in a Complex Regulatory Circuit and Act in Concert To Modulate Bacterioferritin Expression in Ensifer meliloti 1021

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Daniela Costa ◽  
Vanesa Amarelle ◽  
Claudio Valverde ◽  
Mark R. O'Brian ◽  
Elena Fabiano
Keyword(s):  
Gene Expression ◽  
Small Rna ◽  
Iron Homeostasis ◽  
Sufficient Conditions ◽  
Iron Storage ◽  
Content Type ◽  
Reading Frame ◽  
Regulatory Circuit ◽  
Ensifer Meliloti ◽  
Rhizobial Species

ABSTRACT In this work we found that the bfr gene of the rhizobial species Ensifer meliloti, encoding a bacterioferritin iron storage protein, is involved in iron homeostasis and the oxidative stress response. This gene is located downstream of and overlapping the smc03787 open reading frame (ORF). No well-predicted RirA or Irr boxes were found in the region immediately upstream of the bfr gene although two presumptive RirA boxes and one presumptive Irr box were present in the putative promoter of smc03787. We demonstrate that bfr gene expression is enhanced under iron-sufficient conditions and that Irr and RirA modulate this expression. The pattern of bfr gene expression as well as the response to Irr and RirA is inversely correlated to that of smc03787. Moreover, our results suggest that the small RNA SmelC759 participates in RirA- and Irr-mediated regulation of bfr expression and that additional unknown factors are involved in iron-dependent regulation. IMPORTANCE E. meliloti belongs to the Alphaproteobacteria, a group of bacteria that includes several species able to associate with eukaryotic hosts, from mammals to plants, in a symbiotic or pathogenic manner. Regulation of iron homeostasis in this group of bacteria differs from that found in the well-studied Gammaproteobacteria. In this work we analyzed the effect of rirA and irr mutations on bfr gene expression. We demonstrate the effect of an irr mutation on iron homeostasis in this bacterial genus. Moreover, results obtained indicate a complex regulatory circuit where multiple regulators, including RirA, Irr, the small RNA SmelC759, and still unknown factors, act in concert to balance bfr gene expression.

scholarly journals aro Mutations in Salmonella enterica Cause Defects in Cell Wall and Outer Membrane Integrity

2008 ◽  
Vol 190 (9) ◽  
pp. 3155-3160 ◽  
Author(s):  
Alena Sebkova ◽  
Daniela Karasova ◽  
Magdalena Crhanova ◽  
Eva Budinska ◽  
Ivan Rychlik
Keyword(s):  
Gene Expression ◽  
Cell Wall ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Membrane Integrity ◽  
Innate Immune ◽  
Multicopy Plasmid ◽  
Oral Vaccines ◽  
Serovar Typhimurium ◽  
Serum Albumen

ABSTRACT In this study we characterized aro mutants of Salmonella enterica serovars Enteritidis and Typhimurium, which are frequently used as live oral vaccines. We found that the aroA, aroD, and aroC mutants were sensitive to blood serum, albumen, EDTA, and ovotransferrin, and this defect could be complemented by an appropriate aro gene cloned in a plasmid. Subsequent microarray analysis of gene expression in the aroD mutant in serovar Typhimurium indicated that the reason for this sensitivity might be the upregulation of murA. To confirm this, we artificially overexpressed murA from a multicopy plasmid, and this overexpression caused sensitivity of the strain to albumen and EDTA but not to serum and ovotransferrin. We concluded that attenuation of aro mutants is caused not only by their inability to synthesize aromatic metabolites but also by their defect in cell wall and outer membrane functions associated with decreased resistance to components of innate immune response.

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 PerR Controls Oxidative Stress Resistance and Iron Storage Proteins and Is Required for Virulence in Staphylococcus aureus

2001 ◽  
Vol 69 (6) ◽  
pp. 3744-3754 ◽  
Author(s):  
Malcolm J. Horsburgh ◽  
Mark O. Clements ◽  
Howard Crossley ◽  
Eileen Ingham ◽  
Simon J. Foster
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Stress Resistance ◽  
Iron Homeostasis ◽  
Storage Proteins ◽  
Iron Storage ◽  
Oxidative Stress Resistance ◽  
Genes Encoding ◽  
Starvation Survival ◽  
Iron Storage Proteins

ABSTRACT The Staphylococcus aureus genome encodes three ferric uptake regulator (Fur) homologues: Fur, PerR, and Zur. To determine the exact role of PerR, we inactivated the gene by allelic replacement using a kanamycin cassette, creating strain MJH001 (perR). PerR was found to control transcription of the genes encoding the oxidative stress resistance proteins catalase (KatA), alkyl hydroperoxide reductase (AhpCF), bacterioferritin comigratory protein (Bcp), and thioredoxin reductase (TrxB). Furthermore, PerR regulates transcription of the genes encoding the iron storage proteins ferritin (Ftn) and the ferritin-like Dps homologue, MrgA. Transcription of perR was autoregulated, and PerR repressed transcription of the iron homeostasis regulator Fur, which is a positive regulator of catalase expression. PerR functions as a manganese-dependent, transcriptional repressor of the identified regulon. Elevated iron concentrations produced induction of the PerR regulon. PerR may act as a peroxide sensor, since addition of external hydrogen peroxide to 8325-4 (wild type) resulted in increased transcription of most of the PerR regulon, except forfur and perR itself. The PerR-regulatedkatA gene encodes the sole catalase of S. aureus, which is an important starvation survival determinant but is surprisingly not required for pathogenicity in a murine skin abscess model of infection. In contrast, PerR is not necessary for starvation survival but is required for full virulence (P < 0.005) in this model of infection. PerR ofS. aureus may act as a redox sentinel protein during infection, analogous to the in vitro activities of OxyR and PerR ofEscherichia coli and Bacillus subtilis, respectively. However, it differs in its response to the metal balance within the cell and has the added capability of regulating iron uptake and storage.

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.

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