scholarly journals Role of Ring6 in the function of the E. coli MCE protein LetB

2021 ◽  
Author(s):  
Casey Vieni ◽  
Nicolas Coudray ◽  
Georgia L Isom ◽  
Gira Bhabha ◽  
Damian Charles Ekiert
Keyword(s):  
Cell Envelope ◽  
Primary Sequence ◽  
Functional Roles ◽  
E Coli ◽  
Outer Membranes ◽  
Gating Mechanism ◽  
Pore Lining ◽  
Insight Into ◽  
Modular Nature

LetB is a tunnel-forming protein found in the cell envelope of some double-membraned bacteria, and is thought to be important for the transport of lipids between the inner and outer membranes. In Escherichia coli the LetB tunnel is formed from a stack of seven rings (Ring1 - Ring7), in which each ring is composed of a homo-hexameric assembly of MCE domains. The primary sequence of each MCE domain of the LetB protein is substantially divergent from the others, making each MCE ring unique in nature. The role of each MCE domain and how it contributes to the function of LetB is not well understood. Here we probed the importance of each MCE ring for the function of LetB, using a combination of bacterial growth assays and cryo-EM. Surprisingly, we find that ΔRing3 and ΔRing6 mutants, in which Ring3 and Ring6 have been deleted, confer increased resistance to membrane perturbing agents. Specific mutations in the pore-lining loops of Ring6 similarly confer increased resistance. A cryo-EM structure of the ΔRing6 mutant shows that despite the absence of Ring6, which leads to a shorter assembly, the overall architecture is maintained, highlighting the modular nature of MCE proteins. Previous work has shown that Ring6 is dynamic and in its closed state, may restrict the passage of substrate through the tunnel. Our work suggests that removal of Ring6 may relieve this restriction. The deletion of Ring6 combined with mutations in the pore-lining loops leads to a model for the tunnel gating mechanism of LetB. Together, these results provide insight into the functional roles of individual MCE domains and pore-lining loops in the LetB protein.

scholarly journals Binding From both sides: TolR and full-length OmpA bind and maintain the local structure of the E. coli cell wall

2018 ◽  
Author(s):  
Alister T. Boags ◽  
Firdaus Samsudin ◽  
Syma Khalid
Keyword(s):  
Cell Wall ◽  
Electrostatic Interactions ◽  
Cell Envelope ◽  
Binding Domain ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
E Coli ◽  
Non Covalent Interactions ◽  
Covalent Interactions

SUMMARYWe present a molecular modeling and simulation study of the of the E. coli cell envelope, with a particular focus on the role of TolR, a native protein of the E. coli inner membrane in interactions with the cell wall. TolR has been proposed to bind to peptidoglycan, but the only structure of this protein thus far is in a conformation in which the putative peptidoglycan binding domain is not accessible. We show that a model of the extended conformation of the protein in which this domain is exposed, binds peptidoglycan largely through electrostatic interactions. We show that non-covalent interactions of TolR and OmpA with the cell wall, from the inner membrane and outer membrane sides respectively, maintain the position of the cell wall even in the absence of Braun’s lipoprotein. When OmpA is truncated to remove the peptidoglycan binding domain, TolR is able to pull the cell wall down towards the inner membrane. The charged residues that mediate the cell-wall interactions of TolR in our simulations, are conserved across a number of species of Gram-negative bacteria.

scholarly journals Using a Chemical Genetic Screen to Enhance Our Understanding of the Antimicrobial Properties of Gallium against Escherichia coli

Genes ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 34 ◽  
Author(s):  
Natalie Gugala ◽  
Kate Chatfield-Reed ◽  
Raymond J. Turner ◽  
Gordon Chua
Keyword(s):  
Escherichia Coli ◽  
Cell Envelope ◽  
Antimicrobial Properties ◽  
Iron Sulfur Cluster ◽  
Nucleotide Biosynthesis ◽  
E Coli ◽  
Chemical Genetic ◽  
Iron Sulfur ◽  
Insight Into ◽  
Mutant Collection

The diagnostic and therapeutic agent gallium offers multiple clinical and commercial uses including the treatment of cancer and the localization of tumors, among others. Further, this metal has been proven to be an effective antimicrobial agent against a number of microbes. Despite the latter, the fundamental mechanisms of gallium action have yet to be fully identified and understood. To further the development of this antimicrobial, it is imperative that we understand the mechanisms by which gallium interacts with cells. As a result, we screened the Escherichia coli Keio mutant collection as a means of identifying the genes that are implicated in prolonged gallium toxicity or resistance and mapped their biological processes to their respective cellular system. We discovered that the deletion of genes functioning in response to oxidative stress, DNA or iron–sulfur cluster repair, and nucleotide biosynthesis were sensitive to gallium, while Ga resistance comprised of genes involved in iron/siderophore import, amino acid biosynthesis and cell envelope maintenance. Altogether, our explanations of these findings offer further insight into the mechanisms of gallium toxicity and resistance in E. coli.

scholarly journals The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

2002 ◽  
Vol 184 (10) ◽  
pp. 2850-2853 ◽  
Author(s):  
Annie Conter ◽  
Rachel Sturny ◽  
Claude Gutierrez ◽  
Kaymeuang Cam
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
E Coli ◽  
Induced Stress ◽  
Mutant Strains ◽  
Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

scholarly journals lncRNA Gm16410 Mediates PM2.5-Induced Macrophage Activation via PI3K/AKT Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Jingbin Xu ◽  
Henggui Xu ◽  
Kexin Ma ◽  
Yue Wang ◽  
Ben Niu ◽  
...  
Keyword(s):  
Lung Inflammation ◽  
Macrophage Activation ◽  
Biological Effects ◽  
Pulmonary Inflammation ◽  
Biological Processes ◽  
Functional Roles ◽  
Non Coding Rna ◽  
Macrophage Polarity ◽  
Insight Into

PM2.5 refers to atmospheric particulate matters with a diameter of less than 2.5 μm. The deposit of PM2.5 in lung cells can cause oxidative stress, leading to changes in macrophage polarity, which can subsequently cause pulmonary inflammation. Long-chain non-coding RNA (lncRNA) is a class of transcripts that regulate biological processes through multiple mechanisms. However, the role of lncRNA in PM2.5-induced lung inflammation has not been established. In this study, the biological effects and associated mechanism of lncRNA in PM2.5-induced change in macrophage polarity were investigated. The lncRNA-mediated PM2.5-induced macrophage inflammation and lung inflammation-associated injury were also determined. Mice were exposed to chronic levels of PM2.5, and changes in the expression of lncRNA in the lung were measured by lncRNA microarray. lncRNAs that showed significant changes in expression in response to PM2.5 were identified. lncRNA showing the biggest change was subjected to further analysis to determine its functional roles and mechanisms with respect to macrophage activation. The result showed that a significant reduction in expression of one lncRNA, identified as lncGm16410, was observed in the lung of mice and RAW264.7 cells following exposure to PM2.5. lncGm16410 suppressed PM2.5-induced macrophage activation via the SRC protein-mediated PI3K/AKT signaling pathway. PM2.5 promoted lung inflammation by downregulating the expression of lncGm16410, enhancing the activation of macrophages. Thus, lncGm16410 might provide new insight into the prevention of PM2.5 injury.

scholarly journals Architecturally complex O-glycopeptidases are customized for mucin recognition and hydrolysis

2021 ◽  
Vol 118 (10) ◽  
pp. e2019220118
Author(s):  
Benjamin Pluvinage ◽  
Elizabeth Ficko-Blean ◽  
Ilit Noach ◽  
Christopher Stuart ◽  
Nicole Thompson ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Peptide Bond ◽  
Molecular Organization ◽  
Dimensional Structure ◽  
Carbohydrate Binding ◽  
Functional Roles ◽  
Protein Substrates ◽  
Individual Domain ◽  
Insight Into

A challenge faced by peptidases is the recognition of highly diverse substrates. A feature of some peptidase families is the capacity to specifically use post-translationally added glycans present on their protein substrates as a recognition determinant. This is ultimately critical to enabling peptide bond hydrolysis. This class of enzyme is also frequently large and architecturally sophisticated. However, the molecular details underpinning glycan recognition by these O-glycopeptidases, the importance of these interactions, and the functional roles of their ancillary domains remain unclear. Here, using the Clostridium perfringens ZmpA, ZmpB, and ZmpC M60 peptidases as model proteins, we provide structural and functional insight into how these intricate proteins recognize glycans as part of catalytic and noncatalytic substrate recognition. Structural, kinetic, and mutagenic analyses support the key role of glycan recognition within the M60 domain catalytic site, though they point to ZmpA as an apparently inactive enzyme. Wider examination of the Zmp domain content reveals noncatalytic carbohydrate binding as a feature of these proteins. The complete three-dimensional structure of ZmpB provides rare insight into the overall molecular organization of a highly multimodular enzyme and reveals how the interplay of individual domain function may influence biological activity. O-glycopeptidases frequently occur in host-adapted microbes that inhabit or attack mucus layers. Therefore, we anticipate that these results will be fundamental to informing more detailed models of how the glycoproteins that are abundant in mucus are destroyed as part of pathogenic processes or liberated as energy sources during normal commensal lifestyles.

scholarly journals Effects of Sequential Campylobacter jejuni 81-176 Lipooligosaccharide Core Truncations on Biofilm Formation, Stress Survival, and Pathogenesis

2010 ◽  
Vol 192 (8) ◽  
pp. 2182-2192 ◽  
Author(s):  
Mizue Naito ◽  
Emilisa Frirdich ◽  
Joshua A. Fields ◽  
Mark Pryjma ◽  
Jianjun Li ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Biofilm Formation ◽  
Cell Envelope ◽  
Polymyxin B ◽  
Outer Core ◽  
Survival Strategies ◽  
Calcofluor White ◽  
Stress Survival ◽  
Insight Into

ABSTRACTCampylobacter jejuniis a highly prevalent human pathogen for which pathogenic and stress survival strategies remain relatively poorly understood. We previously found that aC. jejunistrain 81-176 mutant defective for key virulence and stress survival attributes was also hyper-biofilm and hyperreactive to the UV fluorescent dye calcofluor white (CFW). We hypothesized that screening for CFW hyperreactive mutants would identify additional genes required forC. jejunipathogenesis properties. Surprisingly, two such mutants harbored lesions in lipooligosaccharide (LOS) genes (waaFandlgtF), indicating a complete loss of the LOS outer core region. We utilized this as an opportunity to explore the role of each LOS core-specific moiety in the pathogenesis and stress survival of this strain and thus also constructed ΔgalTand ΔcstIImutants with more minor LOS truncations. Interestingly, we found that mutants lacking the LOS outer core (ΔwaaFand ΔlgtFbut not ΔgalTor ΔcstIImutants) exhibited enhanced biofilm formation. The presence of the complete outer core was also necessary for resistance to complement-mediated killing. In contrast, any LOS truncation, even that of the terminal sialic acid (ΔcstII), resulted in diminished resistance to polymyxin B. The cathelicidin LL-37 was found to be active againstC. jejuni, with the LOS mutants exhibiting modest but tiled alterations in LL-37 sensitivity. The ΔwaaFmutant but not the other LOS mutant strains also exhibited a defect in intraepithelial cell survival, an aspect ofC. jejunipathogenesis that has only recently begun to be clarified. Finally, using a mouse competition model, we now provide the first direct evidence for the importance of theC. jejuniLOS in host colonization. Collectively, this study has uncovered novel roles for theC. jejuniLOS, highlights the dynamic nature of theC. jejunicell envelope, and provides insight into the contribution of specific LOS core moieties to stress survival and pathogenesis.

scholarly journals Genome-Wide Analysis of Lipoprotein Expression in Escherichia coli MG1655

2004 ◽  
Vol 186 (10) ◽  
pp. 3254-3258 ◽  
Author(s):  
Stephen J. Brokx ◽  
Michael Ellison ◽  
Troy Locke ◽  
Drell Bottorff ◽  
Laura Frost ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Envelope ◽  
Global Gene Expression ◽  
Growth Conditions ◽  
Rt Pcr ◽  
E Coli ◽  
Genome Wide ◽  
Aerobic And Anaerobic ◽  
Insight Into ◽  
Good Agreement

ABSTRACT To gain insight into the cell envelope of Escherichia coli grown under aerobic and anaerobic conditions, lipoproteins were examined by using functional genomics. The mRNA expression levels of each of these genes under three growth conditions—aerobic, anaerobic, and anaerobic with nitrate—were examined by using both Affymetrix GeneChip E. coli antisense genome arrays and real-time PCR (RT-PCR). Many genes showed significant changes in expression level. The RT-PCR results were in very good agreement with the microarray data. The results of this study represent the first insights into the possible roles of unknown lipoprotein genes and broaden our understanding of the composition of the cell envelope under different environmental conditions. Additionally, these data serve as a test set for the refinement of high-throughput bioinformatic and global gene expression methods.

scholarly journals Advances of Regulatory B Cells in Autoimmune Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Qiugang Zhu ◽  
Ke Rui ◽  
Shengjun Wang ◽  
Jie Tian
Keyword(s):  
B Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Contact ◽  
Regulatory B Cells ◽  
Functional Roles ◽  
Direct Cell ◽  
Humoral Responses ◽  
Insight Into

With the ability to induce T cell activation and elicit humoral responses, B cells are generally considered as effectors of the immune system. However, the emergence of regulatory B cells (Bregs) has given new insight into the role of B cells in immune responses. Bregs exhibit immunosuppressive functions via diverse mechanisms, including the secretion of anti-inflammatory cytokines and direct cell contact. The balance between Bregs and effector B cells is important for the immune tolerance. In this review, we focus on recent advances in the characteristics of Bregs and their functional roles in autoimmunity.

scholarly journals YodL and YisK Possess Shape-Modifying Activities That Are Suppressed by Mutations in Bacillus subtilismreBandmbl

2016 ◽  
Vol 198 (15) ◽  
pp. 2074-2088 ◽  
Author(s):  
Yi Duan ◽  
Anthony M. Sperber ◽  
Jennifer K. Herman
Keyword(s):  
Cell Shape ◽  
Cell Envelope ◽  
Critical Role ◽  
Binding Pocket ◽  
Cell Width ◽  
Content Type ◽  
Structural Rigidity ◽  
Sporulation Efficiency ◽  
Insight Into

ABSTRACTMany bacteria utilize actin-like proteins to direct peptidoglycan (PG) synthesis. MreB and MreB-like proteins are thought to act as scaffolds, guiding the localization and activity of key PG-synthesizing proteins during cell elongation. Despite their critical role in viability and cell shape maintenance, very little is known about how the activity of MreB family proteins is regulated. Using aBacillus subtilismisexpression screen, we identified two genes,yodLandyisK, that when misexpressed lead to loss of cell width control and cell lysis. Expression analysis suggested thatyodLandyisKare previously uncharacterized Spo0A-regulated genes, and consistent with these observations, a ΔyodLΔyisKmutant exhibited reduced sporulation efficiency. Suppressors resistant to YodL's killing activity occurred primarily inmreBmutants and resulted in amino acid substitutions at the interface between MreB and the highly conserved morphogenic protein RodZ, whereas suppressors resistant to YisK occurred primarily inmblmutants and mapped to Mbl's predicted ATP-binding pocket. YodL's shape-altering activity appears to require MreB, as a ΔmreBmutant was resistant to the effects of YodL but not YisK. Similarly, YisK appears to require Mbl, as a Δmblmutant was resistant to the cell-widening effects of YisK but not of YodL. Collectively, our results suggest that YodL and YisK likely modulate MreB and Mbl activity, possibly during the early stages of sporulation.IMPORTANCEThe peptidoglycan (PG) component of the cell envelope confers structural rigidity to bacteria and protects them from osmotic pressure. MreB and MreB-like proteins are thought to act as scaffolds for PG synthesis and are essential in bacteria exhibiting nonpolar growth. Despite the critical role of MreB-like proteins, we lack mechanistic insight into how their activities are regulated. Here, we describe the discovery of twoB. subtilisproteins, YodL and YisK, which modulate MreB and Mbl activities. Our data suggest that YodL specifically targets MreB, whereas YisK targets Mbl. The apparent specificities with which YodL and YisK are able to differentially target MreB and Mbl make them potentially powerful tools for probing the mechanics of cytoskeletal function in bacteria.

Properties of haemolysin E (HlyE) from a pathogenic Escherichia coli avian isolate and studies of HlyE export

Microbiology ◽  
2004 ◽  
Vol 150 (5) ◽  
pp. 1495-1505 ◽  
Author(s):  
Neil R. Wyborn ◽  
Angela Clark ◽  
Ruth E. Roberts ◽  
Stuart J. Jamieson ◽  
Svetomir Tzokov ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Blood Cells ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Intrinsic Property ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Carboxyl Terminal ◽  
K 12

Haemolysin E (HlyE) is a novel pore-forming toxin first identified in Escherichia coli K-12. Analysis of the 3-D structure of HlyE led to the proposal that a unique hydrophobic β-hairpin structure (the β-tongue, residues 177–203) interacts with the lipid bilayer in target membranes. In seeming contradiction to this, the hlyE sequence from a pathogenic E. coli strain (JM4660) that lacks all other haemolysins has been reported to encode an Arg residue at position 188 that was difficult to reconcile with the proposed role of the β-tongue. Here it is shown that the JM4660 hlyE sequence encodes Gly, not Arg, at position 188 and that substitution of Gly188 by Arg in E. coli K-12 HlyE abolishes activity, emphasizing the importance of the head domain in HlyE function. Nevertheless, 76 other amino acid substitutions were confirmed compared to the HlyE protein of E. coli K-12. The JM4660 HlyE protein was dimeric, suggesting a mechanism for improving toxin solubility, and it lysed red blood cells from many species by forming 36–41 Å diameter pores. However, the haemolytic phenotype of JM4660 was found to be unstable due to defects in HlyE export, indicating that export of active HlyE is not an intrinsic property of the protein but requires additional components. TnphoA mutagenesis of hlyE shows that secretion from the cytoplasm to the periplasm does not require the carboxyl-terminal region of HlyE. Finally, disruption of genes associated with cell envelope function, including tatC, impairs HlyE export, indicating that outer membrane integrity is important for effective HlyE secretion.

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