Mechanical stress compromises multicomponent efflux complexes in bacteria

Physical forces have a profound effect on growth, morphology, locomotion, and survival of organisms. At the level of individual cells, the role of mechanical forces is well recognized in eukaryotic physiology, but much less is known about prokaryotic organisms. Recent findings suggest an effect of physical forces on bacterial shape, cell division, motility, virulence, and biofilm initiation, but it remains unclear how mechanical forces applied to a bacterium are translated at the molecular level. In Gram-negative bacteria, multicomponent protein complexes can form rigid links across the cell envelope and are therefore subject to physical forces experienced by the cell. Here we manipulate tensile and shear mechanical stress in the bacterial cell envelope and use single-molecule tracking to show that octahedral shear (but not hydrostatic) stress within the cell envelope promotes disassembly of the tripartite efflux complex CusCBA, a system used byEscherichia colito resist copper and silver toxicity. By promoting disassembly of this protein complex, mechanical forces within the cell envelope make the bacteria more susceptible to metal toxicity. These findings demonstrate that mechanical forces can inhibit the function of cell envelope protein assemblies in bacteria and suggest the possibility that other multicomponent, transenvelope efflux complexes may be sensitive to mechanical forces including complexes involved in antibiotic resistance, cell division, and translocation of outer membrane components. By modulating the function of proteins within the cell envelope, mechanical stress has the potential to regulate multiple processes required for bacterial survival and growth.

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Mechanical stress compromises multicomponent efflux complexes in bacteria

10.1101/571398 ◽

2019 ◽

Author(s):

Lauren A. Genova ◽

Melanie F. Roberts ◽

Yu-Chern Wong ◽

Christine E. Harper ◽

Ace George Santiago ◽

...

Keyword(s):

Cell Division ◽

Mechanical Stress ◽

Single Molecule ◽

Protein Complexes ◽

Cell Envelope ◽

Mechanical Forces ◽

Growth And Survival ◽

Physical Forces ◽

Bacterial Growth And Survival ◽

Membrane Components

Physical forces have long been recognized for their effects on the growth, morphology, locomotion, and survival of eukaryotic organisms1. Recently, mechanical forces have been shown to regulate processes in bacteria, including cell division2, motility3, virulence4, biofilm initiation5,6, and cell shape7,8, although it remains unclear how mechanical forces in the cell envelope lead to changes in molecular processes. In Gram-negative bacteria, multicomponent protein complexes that form rigid links across the cell envelope directly experience physical forces and mechanical stresses applied to the cell. Here we manipulate tensile and shear mechanical stress in the bacterial cell envelope and use single-molecule tracking to show that shear (but not tensile) stress within the cell envelope promotes disassembly of the tripartite efflux complex CusCBA, a system used by E. coli to resist copper and silver toxicity, thereby making bacteria more susceptible to metal toxicity. These findings provide the first demonstration that mechanical forces, such as those generated during colony overcrowding or bacterial motility through submicron pores, can inhibit the contact and function of multicomponent complexes in bacteria. As multicomponent, trans-envelope efflux complexes in bacteria are involved in many processes including antibiotic resistance9, cell division10, and translocation of outer membrane components11, our findings suggest that the mechanical environment may regulate multiple processes required for bacterial growth and survival.

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The Type IVa Pilus Machinery Is Recruited to Sites of Future Cell Division

mBio ◽

10.1128/mbio.02103-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 17

Author(s):

Tyson Carter ◽

Ryan N. C. Buensuceso ◽

Stephanie Tammam ◽

Ryan P. Lamers ◽

Hanjeong Harvey ◽

...

Keyword(s):

Cell Division ◽

Bacterial Cell ◽

Protein Complexes ◽

Cell Envelope ◽

Assembly System ◽

Physical Barrier ◽

Large Protein ◽

Binding Motifs ◽

Daughter Cells ◽

Cell Envelopes

ABSTRACT Type IVa pili (T4aP) are ubiquitous microbial appendages used for adherence, twitching motility, DNA uptake, and electron transfer. Many of these functions depend on dynamic assembly and disassembly of the pilus by a megadalton-sized, cell envelope-spanning protein complex located at the poles of rod-shaped bacteria. How the T4aP assembly complex becomes integrated into the cell envelope in the absence of dedicated peptidoglycan (PG) hydrolases is unknown. After ruling out the potential involvement of housekeeping PG hydrolases in the installation of the T4aP machinery in Pseudomonas aeruginosa, we discovered that key components of inner (PilMNOP) and outer (PilQ) membrane subcomplexes are recruited to future sites of cell division. Midcell recruitment of a fluorescently tagged alignment subcomplex component, mCherry-PilO, depended on PilQ secretin monomers—specifically, their N-terminal PG-binding AMIN domains. PilP, which connects PilO to PilQ, was required for recruitment, while PilM, which is structurally similar to divisome component FtsA, was not. Recruitment preceded secretin oligomerization in the outer membrane, as loss of the PilQ pilotin PilF had no effect on localization. These results were confirmed in cells chemically blocked for cell division prior to outer membrane invagination. The hub protein FimV and a component of the polar organelle coordinator complex—PocA—were independently required for midcell recruitment of PilO and PilQ. Together, these data suggest an integrated, energy-efficient strategy for the targeting and preinstallation—rather than retrofitting—of the T4aP system into nascent poles, without the need for dedicated PG-remodeling enzymes. IMPORTANCE The peptidoglycan (PG) layer of bacterial cell envelopes has limited porosity, representing a physical barrier to the insertion of large protein complexes involved in secretion and motility. Many systems include dedicated PG hydrolase components that create space for their insertion, but the ubiquitous type IVa pilus (T4aP) system lacks such an enzyme. Instead, we found that components of the T4aP system are recruited to future sites of cell division, where they could be incorporated into the cell envelope during the formation of new poles, eliminating the need for PG hydrolases. Targeting depends on the presence of septal PG-binding motifs in specific components, as removal of those motifs causes delocalization. This preinstallation strategy for the T4aP assembly system would ensure that both daughter cells are poised to extrude pili from new poles as soon as they separate from one another. IMPORTANCE The peptidoglycan (PG) layer of bacterial cell envelopes has limited porosity, representing a physical barrier to the insertion of large protein complexes involved in secretion and motility. Many systems include dedicated PG hydrolase components that create space for their insertion, but the ubiquitous type IVa pilus (T4aP) system lacks such an enzyme. Instead, we found that components of the T4aP system are recruited to future sites of cell division, where they could be incorporated into the cell envelope during the formation of new poles, eliminating the need for PG hydrolases. Targeting depends on the presence of septal PG-binding motifs in specific components, as removal of those motifs causes delocalization. This preinstallation strategy for the T4aP assembly system would ensure that both daughter cells are poised to extrude pili from new poles as soon as they separate from one another.

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Movement Dynamics of Divisome and Penicillin-Binding Proteins (PBPs) in Cells of Streptococcus pneumoniae

10.1101/429217 ◽

2018 ◽

Author(s):

Amilcar J. Perez ◽

Yann Cesbron ◽

Sidney L. Shaw ◽

Jesus Bazan Villicana ◽

Ho-Ching T. Tsui ◽

...

Keyword(s):

Cell Division ◽

Streptococcus Pneumoniae ◽

Single Molecule ◽

Protein Complexes ◽

Bacterial Cell Division ◽

Dynamic Movement ◽

Movement Dynamics ◽

Directional Movement ◽

Tightly Coupled ◽

Filament Bundles

ABSTRACTBacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae (Spn). Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase binding partner relative to FtsZ treadmilling in Spn cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in Spn.SignificanceThis study answers two long-standing questions about FtsZ dynamics and its relationship to septal PG synthesis in Spn for the first time. In previous models, FtsZ concertedly moves from midcell septa to MapZ rings that have reached the equators of daughter cells. Instead, the results presented here show that FtsZ, FtsA, and EzrA filaments/bundles move continuously out from early septa as part of MapZ rings. In addition, this study establishes that the movement of bPBP2x:FtsW complexes in septal PG synthesis depends on and likely mirrors new PG synthesis and is not correlated with the treadmilling of FtsZ filaments/bundles. These findings are consistent with a mechanism where septal FtsZ rings organize directional movement of bPBP2x:FtsW complexes dependent on PG substrate availability.

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Peptidoglycan layer and disruption processes in Bacillus subtilis cells visualized using quick-freeze, deep-etch electron microscopy

Microscopy ◽

10.1093/jmicro/dfz033 ◽

2019 ◽

Vol 68 (6) ◽

pp. 441-449 ◽

Cited By ~ 5

Author(s):

Isil Tulum ◽

Yuhei O Tahara ◽

Makoto Miyata

Keyword(s):

Electron Microscopy ◽

Bacillus Subtilis ◽

Cell Division ◽

Cell Envelope ◽

Critical Role ◽

Turgor Pressure ◽

Cell Mass ◽

Fluorescence Labeling ◽

Bacterial Survival ◽

Division Site

Abstract Peptidoglycan, which is the main component of the bacterial cell wall, is a heterogeneous polymer of glycan strands cross-linked with short peptides and is synthesized in cooperation with the cell division cycle. Although it plays a critical role in bacterial survival, its architecture is not well understood. Herein, we visualized the architecture of the peptidoglycan surface in Bacillus subtilis at the nanometer resolution, using quick-freeze, deep-etch electron microscopy (EM). Filamentous structures were observed on the entire surface of the cell, where filaments about 11 nm wide formed concentric circles on cell poles, filaments about 13 nm wide formed a circumferential mesh-like structure on the cylindrical part and a ‘piecrust’ structure was observed at the boundary. When growing cells were treated with lysozyme, the entire cell mass migrated to one side and came out from the cell envelope. Fluorescence labeling showed that lysozyme preferentially bound to a cell pole and cell division site, where the peptidoglycan synthesis was not complete. Ruffling of surface structures was observed during EM. When cells were treated with penicillin, the cell mass came out from a cleft around the cell division site. Outward curvature of the protoplast at the cleft seen using EM suggested that turgor pressure was applied as the peptidoglycan was not damaged at other positions. When muropeptides were depleted, surface filaments were lost while the rod shape of the cell was maintained. These changes can be explained on the basis of the working points of the chemical structure of peptidoglycan.

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Uniaxial loading induces a scalable switch in cortical actomyosin flow polarization and reveals mechanosensitive regulation of cytokinesis

10.1101/560433 ◽

2019 ◽

Author(s):

Deepika Singh ◽

Devang Odedra ◽

Christian Pohl

Keyword(s):

Cell Division ◽

Stress Response ◽

Mechanical Stress ◽

Rotational Flow ◽

Mechanical Forces ◽

Longitudinal Flow ◽

Contractile Ring ◽

Animal Development ◽

C Elegans ◽

Cortical Fibers

AbstractDuring animal development, it is crucial that cells can sense and adapt to mechanical forces from their environment. Ultimately, these forces are transduced through the actomyosin cortex. How the cortex can simultaneously respond to and create forces during cytokinesis is not well understood. Here we show that under mechanical stress, cortical actomyosin flow switches its polarization during cytokinesis in the C. elegans embryo. In unstressed embryos, longitudinal cortical flows contribute to contractile ring formation, while rotational cortical flow is additionally induced in uniaxially loaded embryos. Rotational cortical flow is required for the redistribution of the actomyosin cortex in loaded embryos. Rupture of longitudinally aligned cortical fibers during cortex rotation releases tension, initiates orthogonal longitudinal flow and thereby contributes to furrowing in loaded embryos. A targeted screen for factors required for rotational flow revealed that actomyosin regulators involved in RhoA regulation, cortical polarity and chirality are all required for rotational flow and become essential for cytokinesis under mechanical stress. In sum, our findings extend the current framework of mechanical stress response during cell division and show scaling of orthogonal cortical flows to the amount of mechanical stress.

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Movement dynamics of divisome proteins and PBP2x:FtsW in cells of Streptococcus pneumoniae

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816018116 ◽

2019 ◽

Vol 116 (8) ◽

pp. 3211-3220 ◽

Cited By ~ 36

Author(s):

Amilcar J. Perez ◽

Yann Cesbron ◽

Sidney L. Shaw ◽

Jesus Bazan Villicana ◽

Ho-Ching T. Tsui ◽

...

Keyword(s):

Cell Division ◽

Streptococcus Pneumoniae ◽

Single Molecule ◽

Protein Complexes ◽

Bacterial Cell Division ◽

Binding Partner ◽

Dynamic Movement ◽

Movement Dynamics ◽

Tightly Coupled ◽

Filament Bundles

Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae. Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase-binding partner relative to FtsZ treadmilling in S. pneumoniae cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in S. pneumoniae.

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The dimerization equilibrium of a ClC Cl−/H+ antiporter in lipid bilayers

eLife ◽

10.7554/elife.17438 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 19

Author(s):

Rahul Chadda ◽

Venkatramanan Krishnamani ◽

Kacey Mersch ◽

Jason Wong ◽

Marley Brimberry ◽

...

Keyword(s):

Membrane Protein ◽

Lipid Bilayers ◽

Single Molecule ◽

Protein Complexes ◽

New Approach ◽

Membrane Protein Folding ◽

Protein Interfaces ◽

New Type ◽

Membrane Protein Complexes ◽

Physical Forces

Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states – monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.

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Peptidoglycan layer and disruption processes inBacillus subtiliscells visualized using quick-freeze, deep-etch electron microscopy

10.1101/600171 ◽

2019 ◽

Author(s):

Isil Tulum ◽

Yuhei O Tahara ◽

Makoto Miyata

Keyword(s):

Electron Microscopy ◽

Cell Division ◽

Cell Envelope ◽

Critical Role ◽

Turgor Pressure ◽

Cell Mass ◽

Cylindrical Part ◽

Fluorescence Labeling ◽

Bacterial Survival ◽

Division Site

ABSTRACTPeptidoglycan, which is the main component of the bacterial cell wall, is a heterogeneous polymer of glycan strands crosslinked with short peptides and is synthesized in cooperation with the cell division cycle. Although it plays a critical role in bacterial survival, its architecture is not well understood. Herein, we visualized the architecture of the peptidoglycan surface inBacillus subtilisat the nanometer resolution, using quick-freeze, deep-etch electron microscopy. Filamentous structures were observed on the entire surface of the cell, where filaments about 11-nm wide formed concentric circles on cell poles, filaments about 13-nm wide formed a circumferential mesh-like structure on the cylindrical part, and a “piecrust” structure was observed at the boundary. When growing cells were treated with lysozyme, the entire cell mass migrated to one side and came out from the cell envelope. Fluorescence labeling showed that lysozyme preferentially bound to a cell pole and cell division site, where the peptidoglycan synthesis was not complete. Ruffling of surface structures was observed during electron microscopy. When cells were treated with penicillin, the cell mass came out from a cleft around the cell division site. Outward curvature of the protoplast at the cleft seen using electron microscopy suggested that turgor pressure was applied as the peptidoglycan was not damaged at other positions. When muropeptides were depleted, surface filaments were lost while the rod shape of the cell was maintained. These changes can be explained on the basis of the working points of the chemical structure of peptidoglycan.

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