The Persistence-Inducing Toxin HokB Forms Dynamic Pores That Cause ATP Leakage.

Bacterial populations harbor a small fraction of cells that display transient multidrug tolerance. These so-called persister cells are extremely difficult to eradicate and contribute to the recalcitrance of chronic infections. Several signaling pathways leading to persistence have been identified. However, it is poorly understood how the effectors of these pathways function at the molecular level. In a previous study, we reported that the conserved GTPase Obg induces persistence in Escherichia coli via transcriptional upregulation of the toxin HokB. In the present study, we demonstrate that HokB inserts in the cytoplasmic membrane where it forms pores. The pore-forming capacity of the HokB peptide is demonstrated by in vitro conductance measurements on synthetic and natural lipid bilayers, revealing an asymmetrical conductance profile. Pore formation is directly linked to persistence and results in leakage of intracellular ATP. HokB-induced persistence is strongly impeded in the presence of a channel blocker, thereby providing a direct link between pore functioning and persistence. Furthermore, the activity of HokB pores is sensitive to the membrane potential. This sensitivity presumably results from the formation of either intermediate or mature pore types depending on the membrane potential. Taken together, these results provide a detailed view on the mechanistic basis of persister formation through the effector HokB.IMPORTANCE There is increasing awareness of the clinical importance of persistence. Indeed, persistence is linked to the recalcitrance of chronic infections, and evidence is accumulating that persister cells constitute a pool of viable cells from which resistant mutants can emerge. Unfortunately, persistence is a poorly understood process at the mechanistic level. In this study, we unraveled the pore-forming activity of HokB in E. coli and discovered that these pores lead to leakage of intracellular ATP, which is correlated with the induction of persistence. Moreover, we established a link between persistence and pore activity, as the number of HokB-induced persister cells was strongly reduced using a channel blocker. The latter opens opportunities to reduce the number of persister cells in a clinical setting.

Measuring the Viscosity of the Escherichia coli Plasma Membrane Using Molecular Rotors.

The viscosity is a highly important parameter within the cell membrane, affecting the diffusion of small molecules and, hence, controlling the rates of intracellular reactions. There is significant interest in the direct, quantitative assessment of membrane viscosity. Here we report the use of fluorescence lifetime imaging microscopy of the molecular rotor BODIPY C10 in the membranes of live Escherichia coli bacteria to permit direct quantification of the viscosity. Using this approach, we investigated the viscosity in live E. coli cells, spheroplasts, and liposomes made from E. coli membrane extracts. For live cells and spheroplasts, the viscosity was measured at both room temperature (23°C) and the E. coli growth temperature (37°C), while the membrane extract liposomes were studied over a range of measurement temperatures (5-40°C). At 37°C, we recorded a membrane viscosity in live E. coli cells of 950 cP, which is considerably higher than that previously observed in other live cell membranes (e.g., eukaryotic cells, membranes of Bacillus vegetative cells). Interestingly, this indicates that E. coli cells exhibit a high degree of lipid ordering within their liquid-phase plasma membranes.

A study of SeqA subcellular localization in Escherichia coli using photo-activated localization microscopy.

Escherichia coli (E. coli) cells replicate their genome once per cell cycle to pass on genetic information to the daughter cells. The SeqA protein binds the origin of replication, oriC, after DNA replication initiation and sequesters it from new initiations in order to prevent overinitiation. Conventional fluorescence microscopy studies of SeqA localization in bacterial cells have shown that the protein is localized to discrete foci. In this study we have used photo-activated localization microscopy (PALM) to determine the localization of SeqA molecules, tagged with fluorescent proteins, with a localization precision of 20-30 nm with the aim to visualize the SeqA subcellular structures in more detail than previously possible. SeqA-PAmCherry was imaged in wild type E. coli, expressed from plasmid or genetically engineered into the bacterial genome, replacing the native seqA gene. Unsynchronized cells as well as cells with a synchronized cell cycle were imaged at various time points, in order to investigate the evolution of SeqA localization during the cell cycle. We found that SeqA indeed localized into discrete foci but these were not the only subcellular localizations of the protein. A significant amount of SeqA-PAmCherry molecules was localized outside the foci and in a fraction of cells we saw patterns indicating localization at the membrane. Using quantitative PALM, we counted protein copy numbers per cell, protein copy numbers per focus, the numbers of foci per cell and the sizes of the SeqA clusters. The data showed broad cell-to-cell variation and we did not observe a correlation between SeqA-PAmCherry protein numbers and the cell cycle under the experimental conditions of this study. The numbers of SeqA-PAmCherry molecules per focus as well as the foci sizes also showed broad distributions indicating that the foci are likely not characterized by a fixed number of molecules. We also imaged an E. coli strain devoid of the dam methylase (Δdam) and observed that SeqA-PAmCherry no longer formed foci, and was dispersed throughout the cell and localized to the plasma membrane more readily. We discuss our results in the context of the limitations of the technique.

Impact of osmotic stress on protein diffusion in Lactococcus lactis.

We measured translational diffusion of proteins in the cytoplasm and plasma membrane of the Gram-positive bacterium Lactococcus lactis and probed the effect of osmotic upshift. For cells in standard growth medium the diffusion coefficients for cytosolic proteins (27 and 582 kDa) and 12-transmembrane helix membrane proteins are similar to those in Escherichia coli. The translational diffusion of GFP in L. lactis drops by two orders of magnitude when the medium osmolality is increased by ∼ 1.9 Osm, and the decrease in mobility is partly reversed in the presence of osmoprotectants. We find a large spread in diffusion coefficients over the full population of cells but a smaller spread if only sister cells are compared. While in general the diffusion coefficients we measure under normal osmotic conditions in L. lactis are similar to those reported in E. coli, the decrease in translational diffusion upon osmotic challenge in L. lactis is smaller than in E. coli. An even more striking difference is that in L. lactis the GFP diffusion coefficient drops much more rapidly with volume than in E. coli. We discuss these findings in the light of differences in turgor, cell volume, crowding and cytoplasmic structure of Gram-positive and Gram-negative bacteria.

On the role of individual subunits in MscL gating: "all for one, one for all?".

The mechanosensitive channel of large conductance (MscL) is a homopentameric membrane protein that protects bacteria from hypoosmotic stress. Its mechanics are coupled to structural changes in the membrane, yet the molecular mechanism of the transition from closed to open states and the cooperation between subunits are poorly understood. To determine the early stages of channel activation, we have created a chemically addressable heteropentameric MscL, which allows us to selectively trigger only one subunit in the pentameric protein assembly. By employing a liposome leakage assay developed in house, we measured the size-exclusion limits of MscL (G22C homopentamer and WTG22C heteropentamer). Patch-clamp, single-channel conductance recordings were used to electrically characterize the various channel substates. We show that a decrease in the hydrophobicity of a pore residue in only one subunit breaks the energy barrier for gating and increases the pore diameter up to 10 Å. A further decrease on the hydrophobicity of the same pore residue in other subunits opens the channel further, up to a diameter of 25 Å. However, it is not sufficient for full opening of the channel. This suggests the presence of supplementary mechanisms other than only the hydrophobic gate for MscL opening and closing and/or insufficient expansion of the channel by hydrophobic gating in the absence of applied membrane tension.

Evaluation of pulsed-FRAP and conventional-FRAP for determination of protein mobility in prokaryotic cells.

BACKGROUND: Macromolecule mobility is often quantified with Fluorescence Recovery After Photobleaching (FRAP). Throughout literature a wide range of diffusion coefficients for GFP in the cytoplasm of Escherichia coli (3 to 14 µm²/s) is reported using FRAP-based approaches. In this study, we have evaluated two of these methods: pulsed-FRAP and "conventional"-FRAP. PRINCIPAL FINDINGS: To address the question whether the apparent discrepancy in the diffusion data stems from methodological differences or biological variation, we have implemented and compared the two techniques on bacteria grown and handled in the same way. The GFP diffusion coefficients obtained under normal osmotic conditions and upon osmotic upshift were very similar for the different techniques. CONCLUSIONS: Our analyses indicate that the wide range of values reported for the diffusion coefficient of GFP in live cells are due to experimental conditions and/or biological variation rather than methodological differences.

Light-induced disassembly of self-assembled vesicle-capped nanotubes observed in real time.

Molecular self-assembly is the basis for the formation of numerous artificial nanostructures. The self-organization of peptides, amphiphilic molecules composed of fused benzene rings and other functional molecules into nanotubes is of particular interest. However, the design of dynamic, complex self-organized systems that are responsive to external stimuli remains a significant challenge. Here, we report self-assembled, vesicle-capped nanotubes that can be selectively disassembled by irradiation. The walls of the nanotubes are 3-nm-thick bilayers and are made from amphiphilic molecules with two hydrophobic legs that interdigitate when the molecules self-assemble into bilayers. In the presence of phospholipids, a phase separation between the phospholipids and the amphiphilic molecules creates nanotubes, which are end-capped by vesicles that can be chemically altered or removed and reattached without affecting the nanotubes. The presence of a photoswitchable and fluorescent core in the amphiphilic molecules allows fast and highly controlled disassembly of the nanotubes on irradiation, and distinct disassembly processes can be observed in real time using fluorescence microscopy.

Structural basis for the enhanced activity of cyclic antimicrobial peptides: the case of BPC194.

We report the molecular basis for the differences in activity of cyclic and linear antimicrobial peptides. We iteratively performed atomistic molecular dynamics simulations and biophysical measurements to probe the interaction of a cyclic antimicrobial peptide and its inactive linear analogue with model membranes. We establish that, relative to the linear peptide, the cyclic one binds stronger to negatively charged membranes. We show that only the cyclic peptide folds at the membrane interface and adopts a ß-sheet structure characterised by two turns. Subsequently, the cyclic peptide penetrates deeper into the bilayer while the linear peptide remains essentially at the surface. Finally, based on our comparative study, we propose a model characterising the mode of action of cyclic antimicrobial peptides. The results provide a chemical rationale for enhanced activity in certain cyclic antimicrobial peptides and can be used as a guideline for design of novel antimicrobial peptides.

The molecular basis for antimicrobial activity of pore-forming cyclic peptides.

The mechanism of action of antimicrobial peptides is, to our knowledge, still poorly understood. To probe the biophysical characteristics that confer activity, we present here a molecular-dynamics and biophysical study of a cyclic antimicrobial peptide and its inactive linear analog. In the simulations, the cyclic peptide caused large perturbations in the bilayer and cooperatively opened a disordered toroidal pore, 1-2 nm in diameter. Electrophysiology measurements confirm discrete poration events of comparable size. We also show that lysine residues aligning parallel to each other in the cyclic but not linear peptide are crucial for function. By employing dual-color fluorescence burst analysis, we show that both peptides are able to fuse/aggregate liposomes but only the cyclic peptide is able to porate them. The results provide detailed insight on the molecular basis of activity of cyclic antimicrobial peptides.

Macromolecule diffusion and confinement in prokaryotic cells.

We review recent observations on the mobility of macromolecules and their spatial organization in live bacterial cells. We outline the major fluorescence microscopy-based methods to determine the mobility and thus the diffusion coefficients (D) of molecules, which is not trivial in small cells. The extremely high macromolecule crowding of prokaryotes is used to rationalize the reported lower diffusion coefficients as compared to eukaryotes, and we speculate on the nature of the barriers for diffusion observed for proteins (and mRNAs) in vivo. Building on in vitro experiments and modeling studies, we evaluate the size dependence of diffusion coefficients for macromolecules in vivo, in case of both water-soluble and integral membrane proteins. We comment on the possibilities of anomalous diffusion and provide examples where the macromolecule mobility may be limiting biological processes.

Molecular sieving properties of the cytoplasm of Escherichia coli and consequences of osmotic stress.

We determined the diffusion coefficients (D) of (macro)molecules of different sizes (from approximately 0.5 to 600 kDa) in the cytoplasm of live Escherichia coli cells under normal osmotic conditions and osmotic upshift. D values decreased with increasing molecular weight of the molecules. Upon osmotic upshift, the decrease in D of NBD-glucose was much smaller than that of macromolecules. Barriers for diffusion were found in osmotically challenged cells only for GFP and larger proteins. These barriers are likely formed by the nucleoid and crowding of the cytoplasm. The cytoplasm of E. coli appears as a meshwork allowing the free passage of small molecules while restricting the diffusion of bigger ones.

On the mechanism of pore formation by melittin.

The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.

Dual-color fluorescence-burst analysis to study pore formation and protein-protein interactions.

Dual-color fluorescence-burst analysis (DCBFA) enables to study leakage of fluorescently labeled (macro) molecules from liposomes that are labeled with a second, spectrally non-overlapping fluorophore. The fluorescent bursts that reside from the liposomes diffusing through the focal volume of a confocal microscope will coincide with those from the encapsulated size-marker molecules. The internal concentration of size-marker molecules can be quantitatively calculated from the fluorescence bursts at a single liposome level. DCFBA has been successfully used to study the effective pore-size of the mechanosensitive channel of large-conductance MscL and the pore-forming mechanism of the antimicrobial peptide melittin from bee venom. In addition, DCFBA can be used to quantitatively measure the binding of proteins to liposomes and to membrane proteins. In this paper, we provide an overview of the method and discuss the experimental details of DCFBA.

The lipid dependence of melittin action investigated by dual-color fluorescence burst analysis.

Dual-color fluorescence-burst analysis was used to study melittin-induced leakage of macromolecules from liposomes of various lipid compositions. To perform dual-color fluorescence-burst analysis, fluorescently labeled size-marker molecules were encapsulated into liposomes, labeled with a second lipid-attached fluorophore. By correlating the fluorescence bursts, resulting from the liposomes diffusing through the detection volume of a dual-color confocal microscope, the distribution of size-marker molecules over the liposomes was determined. It was found that melittin causes leakage via two different mechanisms: 1), For liposomes composed of neutral bilayer-forming lipids, low melittin concentrations induced pore formation with the pore size depending on the melittin concentration. 2), For liposomes containing anionic and/or nonbilayer forming lipids, melittin induced fusion or aggregation of liposomes accompanied by a-specific leakage. Experiments with liposomes prepared from Escherichia coli lipid extracts and intact cells of Lactococcus lactis indicate that both mechanisms are physiologically relevant.