scholarly journals The pathway and spatial scale for MscS inactivation

2011 ◽  
Vol 138 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Kishore Kamaraju ◽  
Vladislav Belyy ◽  
Ian Rowe ◽  
Andriy Anishkin ◽  
Sergei Sukharev
Keyword(s):  
Escherichia Coli ◽  
Mechanosensitive Channel ◽  
Activation Curve ◽  
The Core ◽  
Closed State ◽  
Complete Inactivation ◽  
Excised Patches ◽  
Sequential Processes ◽  
Eukaryotic Organisms ◽  
Small Conductance

The mechanosensitive channel of small conductance (MscS) is a bacterial tension-driven osmolyte release valve with homologues in many walled eukaryotic organisms. When stimulated by steps of tension in excised patches, Escherichia coli MscS exhibits transient opening followed by reversible adaptation and then complete inactivation. Here, we study properties of the inactivation transition, which renders MscS nonconductive and tension insensitive. Using special pressure protocols we demonstrate that adaptation and inactivation are sequential processes with opposite tension dependencies. In contrast to many eukaryotic channels, which inactivate from the open state, MscS inactivates primarily from the closed state because full openings by preconditioning pulses do not influence the degree of inactivation, and saturating tensions keeping channels open prevent inactivation. The easily opened A98S mutant lacks inactivation completely, whereas the L111S mutant with a right-shifted activation curve inactivates silently before reaching the threshold for opening. This suggests that opening and inactivation are two independent tension-driven pathways, both starting from the closed state. Analysis of tension dependencies for inactivation and recovery rates estimated the in-plane expansion (ΔA) associated with inactivation as 8.5 nm2, which is about half of the area change for opening. Given that the interhelical contact between the outer TM1–TM2 pairs and the core TM3s is the force-transmitting path from the periphery to the gate, the determined ΔA now can be used as a constraining parameter for the models of the inactivated state in which the lipid-facing TM1–TM2 pairs are displaced and uncoupled from the gate.

scholarly journals The cytoplasmic cage domain of the mechanosensitive channel MscS is a sensor of macromolecular crowding

2014 ◽  
Vol 143 (5) ◽  
pp. 543-557 ◽  
Author(s):  
Ian Rowe ◽  
Andriy Anishkin ◽  
Kishore Kamaraju ◽  
Kenjiro Yoshimura ◽  
Sergei Sukharev
Keyword(s):  
Transmembrane Domain ◽  
Feedback Mechanism ◽  
Axial Position ◽  
Mechanosensitive Channel ◽  
Membrane Tension ◽  
The Core ◽  
Specific Feedback ◽  
Dynamics Simulations ◽  
Association Result ◽  
Small Conductance

Cells actively regulate the macromolecular excluded volume of the cytoplasm to maintain the reciprocal fraction of free aqueous solution that is optimal for intracellular processes. However, the mechanisms whereby cells sense this critical parameter remain unclear. The mechanosensitive channel of small conductance (MscS channel), which is the major regulator of turgor in bacteria, mediates efflux of small osmolytes in response to increased membrane tension. At moderate sustained tensions produced by a decrease in external osmolarity, MscS undergoes slow adaptive inactivation; however, it inactivates abruptly in the presence of cytoplasmic crowding agents. To understand the mechanism underlying this rapid inactivation, we combined extrapolated and equilibrium molecular dynamics simulations with electrophysiological analyses of MscS mutants to explore possible transitions of MscS and generated models of the resting and inactivated states. Our models suggest that the coupling of the gate formed by TM3 helices to the peripheral TM1–TM2 pairs depends on the axial position of the core TM3 barrel relative to the TM1–TM2 shaft and the state of the associated hollow cytoplasmic domain (“cage”). They also indicate that the tension-driven inactivation transition separates the gate from the peripheral helices and promotes kinks in TM3s at G113 and that this conformation is stabilized by association of the TM3b segment with the β domain of the cage. We found that mutations destabilizing the TM3b–β interactions preclude inactivation and make the channel insensitive to crowding agents and voltage; mutations that strengthen this association result in a stable closed state and silent inactivation. Steered simulations showed that pressure exerted on the cage bottom in the inactivated state reduces the volume of the cage in the cytoplasm and at the same time increases the footprint of the transmembrane domain in the membrane, implying coupled sensitivity to both membrane tension and crowding pressure. The cage, therefore, provides feedback on the increasing crowding that disengages the gate and prevents excessive draining and condensation of the cytoplasm. We discuss the structural mechanics of cells surrounded by an elastic cell wall where this MscS-specific feedback mechanism may be necessary.

scholarly journals Cyclodextrins increase membrane tension and are universal activators of mechanosensitive channels

2021 ◽  
Vol 118 (36) ◽  
pp. e2104820118
Author(s):  
Charles D. Cox ◽  
Yixiao Zhang ◽  
Zijing Zhou ◽  
Thomas Walz ◽  
Boris Martinac
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Functional Characterization ◽  
Mechanosensitive Channel ◽  
Mechanical Forces ◽  
Mechanosensitive Channels ◽  
Membrane Tension ◽  
Lipid Environment ◽  
Small Conductance

The bacterial mechanosensitive channel of small conductance (MscS) has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted Escherichia coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel’s sensitivity to membrane tension. Importantly, cholesterol-loaded CDs do not activate MscS. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. While many MS channels respond to membrane forces, generalized by the “force-from-lipids” principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. To test whether CDs can also be used to activate other MS channels, we chose to investigate the mechanosensitive channel of large conductance (MscL) and demonstrate that CDs can also activate this structurally unrelated channel. Since CDs can open the least tension-sensitive MS channel, MscL, they should be able to open any MS channel that responds to membrane tension. Thus, CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.

Mass Spectrometry of Escherichia coli RNA PolymeraseInteractions of the Core Enzyme with σ70 and Rsd Protein

Structure ◽  
2004 ◽  
Vol 12 (2) ◽  
pp. 269-275 ◽  
Author(s):  
L ILAG ◽  
L WESTBLADE ◽  
C DESHAYES ◽  
A KOLB ◽  
S BUSBY ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
The Core ◽  
Core Enzyme

scholarly journals Identification of mutations that alter the gating of the Escherichia coli mechanosensitive channel protein, MscK

2007 ◽  
Vol 66 (2) ◽  
pp. 552-552
Author(s):  
Chan Li ◽  
Michelle D. Edwards ◽  
Hotcherl Jeong ◽  
John Roth ◽  
Ian R. Booth
Keyword(s):  
Escherichia Coli ◽  
Mechanosensitive Channel ◽  
Channel Protein

scholarly journals To catch a hijacker: abundance, evolution and genetic diversity of P4-like bacteriophage satellites

2021 ◽  
Vol 377 (1842) ◽  
Author(s):  
Jorge A. Moura de Sousa ◽  
Eduardo P. C. Rocha
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Core Genome ◽  
Mobile Genetic Elements ◽  
Abundance Distribution ◽  
Theme Issue ◽  
Genetic Elements ◽  
The Core ◽  
Antagonistic Interactions ◽  
Variable Genes

Bacteriophages (phages) are bacterial parasites that can themselves be parasitized by phage satellites. The molecular mechanisms used by satellites to hijack phages are sometimes understood in great detail, but the origins, abundance, distribution and composition of these elements are poorly known. Here, we show that P4-like elements are present in more than 30% of the genomes of Enterobacterales, and in almost half of those of Escherichia coli , sometimes in multiple distinct copies. We identified over 1000 P4-like elements with very conserved genetic organization of the core genome and a few hotspots with highly variable genes. These elements are never found in plasmids and have very little homology to known phages, suggesting an independent evolutionary origin. Instead, they are scattered across chromosomes, possibly because their integrases are often exchanged with other elements. The rooted phylogenies of hijacking functions are correlated and suggest longstanding coevolution. They also reveal broad host ranges in P4-like elements, as almost identical elements can be found in distinct bacterial genera. Our results show that P4-like phage satellites constitute a very distinct, widespread and ancient family of mobile genetic elements. They pave the way for studying the molecular evolution of antagonistic interactions between phages and their satellites. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

scholarly journals Defining the Role of the Tension Sensor in the Mechanosensitive Channel of Small Conductance

2011 ◽  
Vol 101 (2) ◽  
pp. 345-352 ◽  
Author(s):  
Hannah R. Malcolm ◽  
Yoon-Young Heo ◽  
Donald E. Elmore ◽  
Joshua A. Maurer
Keyword(s):  
Mechanosensitive Channel ◽  
Tension Sensor ◽  
Small Conductance

Temperature-sensitive mutants of MscL mechanosensitive channel

2019 ◽  
Vol 166 (3) ◽  
pp. 281-288 ◽  
Author(s):  
Naoto Owada ◽  
Megumi Yoshida ◽  
Kohei Morita ◽  
Kenjiro Yoshimura
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Structural Change ◽  
Cell Growth ◽  
Thermodynamic Stability ◽  
Transmembrane Helix ◽  
Mechanosensitive Channel ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants ◽  
Membrane Stretching

Abstract MscL is a mechanosensitive channel that undergoes a global conformational change upon application of membrane stretching. To elucidate how the structural stability and flexibility occur, we isolated temperature-sensitive (Ts) mutants of Escherichia coli MscL that allowed cell growth at 32°C but not at 42°C. Two Ts mutants, L86P and D127V, were identified. The L86P mutation occurred in the second transmembrane helix, TM2. Substitution of residues neighbouring L86 with proline also led to a Ts mutation, but the substitution of L86 with other amino acids did not result in a Ts phenotype, indicating that the Ts phenotype was due to a structural change of TM2 helix by the introduction of a proline residue. The D127V mutation was localized in the electrostatic belt of the bundle of cytoplasmic helices, indicating that stability of the pentameric bundle of the cytoplasmic helix affects MscL structure. Together, this study described a novel class of MscL mutations that were correlated with the thermodynamic stability of the MscL structure.

scholarly journals A Role for Mediator Core in Limiting Coactivator Recruitment in Saccharomyces cerevisiae

Genetics ◽  
2020 ◽  
Vol 215 (2) ◽  
pp. 407-420 ◽  
Author(s):  
Robert M. Yarrington ◽  
Yaxin Yu ◽  
Chao Yan ◽  
Lu Bai ◽  
David J. Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Regulation ◽  
Transcription Factors ◽  
Gene Activation ◽  
Mediator Complex ◽  
Transcriptional Coactivator ◽  
The Core ◽  
Link Type ◽  
Upstream Activating Sequence ◽  
Eukaryotic Organisms

Mediator is an essential, multisubunit complex that functions as a transcriptional coactivator in yeast and other eukaryotic organisms. Mediator has four conserved modules, Head, Middle, Tail, and Kinase, and has been implicated in nearly all aspects of gene regulation. The Tail module has been shown to recruit the Mediator complex to the enhancer or upstream activating sequence (UAS) regions of genes via interactions with transcription factors, and the Kinase module facilitates the transition of Mediator from the UAS/enhancer to the preinitiation complex via protein phosphorylation. Here, we analyze expression of the Saccharomyces cerevisiae HO gene using a sin4 Mediator Tail mutation that separates the Tail module from the rest of the complex; the sin4 mutation permits independent recruitment of the Tail module to promoters without the rest of Mediator. Significant increases in recruitment of the SWI/SNF and SAGA coactivators to the HO promoter UAS were observed in a sin4 mutant, along with increased gene activation. These results are consistent with recent studies that have suggested that the Kinase module functions negatively to inhibit activation by the Tail. However, we found that Kinase module mutations did not mimic the effect of a sin4 mutation on HO expression. This suggests that at HO the core Mediator complex (Middle and Head modules) must play a role in limiting Tail binding to the promoter UAS and gene activation. We propose that the core Mediator complex helps modulate Mediator binding to the UAS regions of genes to limit coactivator recruitment and ensure proper regulation of gene transcription.

scholarly journals Escherichia coli ST131: a multidrug-resistant clone primed for global domination

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 195 ◽  
Author(s):  
Johann D.D. Pitout ◽  
Rebekah DeVinney
Keyword(s):  
Escherichia Coli ◽  
Core Genome ◽  
Multidrug Resistant ◽  
Accessory Gene ◽  
The Core ◽  
Gene Profiles ◽  
Clade C ◽  
Compensatory Mutations ◽  
The 1960S ◽  
Sequential Acquisition

A single extra-intestinal pathogenic Escherichia coli (ExPEC) clone, named sequence type (ST) 131, is responsible for millions of global antimicrobial-resistant (AMR) infections annually. Population genetics indicate that ST131 consists of different clades (i.e. A, B, and C); however, clade C is the most dominant globally. A ST131 subclade, named C1-M27, is emerging in Japan and has been responsible for the recent increase in AMR ExPEC in that country. The sequential acquisition of several virulence and AMR genes associated with mobile genetic elements during the 1960s to 1980s primed clade C (and its subclades C1 and C2) for success in the 1990s to 2000s. IncF plasmids with F1:A2:B20 and F2:A1:B replicons have shaped the evolution of the C1 and C2 subclades. It is possible that ST131 is a host generalist with different accessory gene profiles. Compensatory mutations within the core genome of this clone have counterbalanced the fitness cost associated with IncF plasmids. ST131 clade C had dramatically changed the population structure of ExPEC, but it still remains unclear which features of this clade resulted in one of the most unprecedented AMR successes of the 2000s.

scholarly journals Inactivation of Escherichia coli and Staphyloccocus aureus in Litchi Juice by Dimethyl Dicarbonate (DMDC) Combined With Nisin

2014 ◽  
Vol 3 (3) ◽  
pp. 1 ◽  
Author(s):  
Yuanshan Yu ◽  
Yujuan Xu ◽  
Jijun Wu ◽  
Gengsheng Xiao ◽  
Jing Wen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Synergistic Effect ◽  
Ph Value ◽  
Inactivation Rate ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Complete Inactivation ◽  
Staphyloccocus Aureus ◽  
Addition Level

<p>Inactivation of Gram-negative <em>Escherichia coli</em> and Gram-positive <em>Staphyloccocus aureus</em> in litchi juice by DMDC combined with nisin was individually investigated. A 1.66 log cycles reduction of<em> E. coli </em>and 2.03 log cycles reduction of <em>S. aureus </em>in litchi juice (pH 4.5) added without nisin was achieved as exposed to 150 mg/l DMDC at 30 °C for 1 h, and the inactivation rate of <em>E. coli </em>and <em>S. aureus</em> during initial 1 h was far greater than during the remaining 5 h. As exposed to 150 mg/l DMDC at 30 °C for 1 h, the inactivation of <em>E. coli</em> and <em>S. aureus</em> in the litchi juice showed a trend toward increase with increasing of nisin addition level in the range from 0 to 200 IU/ml. Moreover, DMDC and nisin exhibited a synergistic effect on the inactivation of <em>E. coli</em> and <em>S. aureus</em> in litchi juice, and the inactivation of<em> E. coli</em> and <em>S. aureus</em> in the litchi juice also depends on the temperature of litchi juice, pH value of litchi juice and DMDC concentration when treated with DMDC and nisin. In addition, <em>E. coli</em> showed higher resistance to nisin as comparing with <em>S. aureus</em>. After <em>E. coli</em> and <em>S. aureus</em> in the litchi juice of pH 4.0 were individually treated with 150 mg/l DMDC combined with 200 IU/ml nisin at 30 °C for 1 h, a complete inactivation of <em>S. aureus</em> (6.59 log cycles) was achieved, but only 3.52 log cycles reduction of <em>E. coli</em> was observed.</p>

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