Rigidification of the Escherichia coli cytoplasm by the human antimicrobial peptide LL-37 revealed by superresolution fluorescence microscopy.

Superresolution, single-particle tracking reveals effects of the cationic antimicrobial peptide LL-37 on the Escherichia coli cytoplasm. Seconds after LL-37 penetrates the cytoplasmic membrane, the chromosomal DNA becomes rigidified on a length scale of ∼30 nm, evidenced by the loss of jiggling motion of specific DNA markers. The diffusive motion of a subset of ribosomes is also frozen. The mean diffusion coefficients of the DNA-binding protein HU and the nonendogenous protein Kaede decrease twofold. Roughly 108 LL-37 copies flood the cell (mean concentration ∼90 mM). Much of the LL-37 remains bound within the cell after extensive rinsing with fresh growth medium. Growth never recovers. The results suggest that the high concentration of adsorbed polycationic peptides forms a dense network of noncovalent, electrostatic linkages within the chromosomal DNA and among 70S-polysomes. The bacterial cytoplasm comprises a concentrated collection of biopolymers that are predominantly polyanionic (e.g., DNA, ribosomes, RNA, and most globular proteins). In normal cells, this provides a kind of electrostatic lubrication, enabling facile diffusion despite high biopolymer volume fraction. However, this same polyanionic nature renders the cytoplasm susceptible to massive adsorption of polycationic agents once penetration of the membranes occurs. If this phenomenon proves widespread across cationic agents and bacterial species, it will help explain why resistance to antimicrobial peptides develops only slowly. The results suggest two design criteria for polycationic peptides that efficiently kill gram-negative bacteria: facile penetration of the outer membrane and the ability to alter the cytoplasm by electrostatically linking double-stranded DNA and 70S-polysomes.

Real-Time Fluorescence Microscopy on Living E. coli Sheds New Light on the Antibacterial Effects of the King Penguin ß-Defensin AvBD103b.

(1) Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. Among AMPs, the disulfide-rich ß-defensin AvBD103b, whose antibacterial activities are not inhibited by salts contrary to most other ß-defensins, is particularly appealing. Information about the mechanisms of action is mandatory for the development and approval of new drugs. However, data for non-membrane-disruptive AMPs such as ß-defensins are scarce, thus they still remain poorly understood. (2) We used single-cell fluorescence imaging to monitor the effects of a ß-defensin (namely AvBD103b) in real time, on living E. coli, and at the physiological concentration of salts. (3) We obtained key parameters to dissect the mechanism of action. The cascade of events, inferred from our precise timing of membrane permeabilization effects, associated with the timing of bacterial growth arrest, differs significantly from the other antimicrobial compounds that we previously studied in the same physiological conditions. Moreover, the AvBD103b mechanism does not involve significant stereo-selective interaction with any chiral partner, at any step of the process. (4) The results are consistent with the suggestion that after penetrating the outer membrane and the cytoplasmic membrane, AvBD103b interacts non-specifically with a variety of polyanionic targets, leading indirectly to cell death.

Local rigidification and possible coacervation of the Escherichia coli DNA by cationic nylon-3 polymers.

Synthetic, cationic random nylon-3 polymers (ß-peptides) show promise as inexpensive antimicrobial agents less susceptible to proteolysis than normal peptides. We have used superresolution, single-cell, time-lapse fluorescence microscopy to compare the effects on live Escherichia coli cells of four such polymers and the natural antimicrobial peptides LL-37 and cecropin A. The longer, densely charged monomethyl-cyclohexyl (MM-CH) copolymer and MM homopolymer rapidly traverse the outer membrane and the cytoplasmic membrane. Over the next ∼5 min, they locally rigidify the chromosomal DNA and slow the diffusive motion of ribosomal species to a degree comparable to LL-37. The shorter dimethyl-dimethylcyclopentyl (DM-DMCP) and dimethyl-dimethylcyclohexyl (DM-DMCH) copolymers, and cecropin A are significantly less effective at rigidifying DNA. Diffusion of the DNA-binding protein HU and of ribosomal species is hindered as well. The results suggest that charge density and contour length are important parameters governing these antimicrobial effects. The data corroborate a model in which agents having sufficient cationic charge distributed across molecular contour lengths comparable to local DNA-DNA interstrand spacings (∼6 nm) form a dense network of multivalent, electrostatic "pseudo-cross-links" that cause the local rigidification. In addition, at times longer than ∼30 min, we observe that the MM-CH copolymer and the MM homopolymer (but not the other four agents) cause gradual coalescence of the two nucleoid lobes into a single dense lobe localized at one end of the cell. We speculate that this process involves coacervation of the DNA by the cationic polymer, and may be related to the liquid droplet coacervates observed in eukaryotic cells.

Long-term effects of the proline-rich antimicrobial peptide Oncocin112 on the Escherichia coli translation machinery.

Proline-rich antimicrobial peptides (PrAMPs) are cationic antimicrobial peptides unusual for their ability to penetrate bacterial membranes and kill cells without causing membrane permeabilization. Structural studies show that many such PrAMPs bind deep in the peptide exit channel of the ribosome, near the peptidyl transfer center. Biochemical studies of the particular synthetic PrAMP oncocin112 (Onc112) suggest that on reaching the cytoplasm, the peptide occupies its binding site prior to the transition from initiation to the elongation phase of translation, thus blocking further initiation events. We present a superresolution fluorescence microscopy study of the long-term effects of Onc112 on ribosome, elongation factor-Tu (EF-Tu), and DNA spatial distributions and diffusive properties in intact Escherichia coli cells. The new data corroborate earlier mechanistic inferences from studies in vitro Comparisons with the diffusive behavior induced by the ribosome-binding antibiotics chloramphenicol and kasugamycin show how the specific location of each agent's ribosomal binding site affects the long-term distribution of ribosomal species between 30S and 50S subunits versus 70S polysomes. Analysis of the single-step displacements from ribosome and EF-Tu diffusive trajectories before and after Onc112 treatment suggests that the act of codon testing of noncognate ternary complexes (TCs) at the ribosomal A-site enhances the dissociation rate of such TCs from their L7/L12 tethers. Testing and rejection of noncognate TCs on a sub-ms timescale is essential to enable incorporation of the rare cognate amino acids into the growing peptide chain at a rate of ∼20 aa/s.

Functional mapping of the E. coli translational machinery using single-molecule tracking.

The organization of the chromosomal DNA and ribosomes in living Escherichia coli is compared under two growth conditions: 'fast' (50 min doubling time) and 'slow' (147 min doubling time). Superresolution fluorescence microscopy reveals strong DNA-ribosome segregation in both cases. In both fast and slow growth, free ribosomal subunits evidently must circulate between the nucleoid (where they initiate co-transcriptional translation) and ribosome-rich regions (where most translation occurs). Single-molecule diffusive behavior dissects the ribosome copies into translating 70S polysomes and free 30S subunits, providing separate spatial distributions for each. In slow growth, ~21,000 total 30S copies/cell comprise ~65% translating 70S ribosomes and ~35% free 30S subunits. The ratio of 70S ribosomes to free 30S subunits is ~2.5 outside the nucleoid and ~0.50 inside the nucleoid. This new level of quantitative detail may motivate development of comprehensive, three-dimensional reaction-diffusion models of ribosome, DNA, mRNA and RNAP spatial distributions and dynamics within the E. coli cytoplasm.

Oxidative stress induced in E. coli by the human antimicrobial peptide LL-37.

Antimicrobial peptides (AMPs) are thought to kill bacterial cells by permeabilizing their membranes. However, some antimicrobial peptides inhibit E. coli growth more efficiently in aerobic than in anaerobic conditions. In the attack of the human cathelicidin LL-37 on E. coli, real-time, single-cell fluorescence imaging reveals the timing of membrane permeabilization and the onset of oxidative stress. For cells growing aerobically, a CellROX Green assay indicates that LL-37 induces rapid formation of oxidative species after entry into the periplasm, but before permeabilization of the cytoplasmic membrane (CM). A cytoplasmic Amplex Red assay signals a subsequent burst of oxidative species, most likely hydrogen peroxide, shortly after permeabilization of the CM. These signals are much stronger in the presence of oxygen, a functional electron transport chain, and a large proton motive force (PMF). They are much weaker in cells growing anaerobically, by either fermentation or anaerobic respiration. In aerobic growth, the oxidative signals are attenuated in a cytochrome oxidase-bd deletion mutant, but not in a -bo3 deletion mutant, suggesting a specific effect of LL-37 on the electron transport chain. The AMPs melittin and LL-37 induce strong oxidative signals and exhibit O2-sensitive MICs, while the AMPs indolicidin and cecropin A do not. These results suggest that AMP activity in different tissues may be tuned according to the local oxygen level. This may be significant for control of opportunistic pathogens while enabling growth of commensal bacteria.

Melittin-Induced Permeabilization, Re-sealing, and Re-permeabilization of E. coli Membranes.

The permeabilization of model lipid bilayers by cationic peptides has been studied extensively over decades, with the bee-sting toxin melittin perhaps serving as the canonical example. However, the relevance of these studies to the permeabilization of real bacterial membranes by antimicrobial peptides remains uncertain. Here, we employ single-cell fluorescence microscopy in a detailed study of the interactions of melittin with the outer membrane (OM) and the cytoplasmic membrane (CM) of live Escherichia coli. Using periplasmic green fluorescent protein (GFP) as a probe, we find that melittin at twice the minimum inhibitory concentration first induces abrupt cell shrinkage and permeabilization of the OM to GFP. Within ∼4 s of OM permeabilization, the CM invaginates to form inward facing "periplasmic bubbles." Seconds later the bubbles begin to leak periplasmic GFP into the cytoplasm. Permeabilization is localized, consistent with possible formation of toroidal pores. Within ∼20 s, first the OM and then the CM re-seals to GFP. Some 2-20 min later, both CM and OM are re-permeabilized to GFP. We invoke a mechanism based on curvature stress concepts derived from model bilayer studies. The permeabilization and re-sealing events involve sequential, time-dependent build-up of melittin density within the outer and inner leaflets of each bilayer. We also propose a mechanical explanation for the early cell shrinkage event induced by melittin and a variety of other cationic peptides. As peptides gain access to the periplasm, they bind to the anionic peptido-crosslinks of the lipopolysaccharide layer, increasing its longitudinal elastic modulus. The cell wall shrinks because it can withstand the same turgor pressure with smaller overall extension. Shrinkage in turn induces invagination of the CM, preserving its surface area. We conclude by comparing the behavior of different peptides.

Modified Pearson correlation coefficient for two-color imaging in spherocylindrical cells.

The revolution in fluorescence microscopy enables sub-diffraction-limit ("superresolution") localization of hundreds or thousands of copies of two differently labeled proteins in the same live cell. In typical experiments, fluorescence from the entire three-dimensional (3D) cell body is projected along the z-axis of the microscope to form a 2D image at the camera plane. For imaging of two different species, here denoted "red" and "green", a significant biological question is the extent to which the red and green spatial distributions are positively correlated, anti-correlated, or uncorrelated. A commonly used statistic for assessing the degree of linear correlation between two image matrices R and G is the Pearson Correlation Coefficient (PCC). PCC should vary from - 1 (perfect anti-correlation) to 0 (no linear correlation) to + 1 (perfect positive correlation). However, in the special case of spherocylindrical bacterial cells such as E. coli or B. subtilis, we show that the PCC fails both qualitatively and quantitatively. PCC returns the same + 1 value for 2D projections of distributions that are either perfectly correlated in 3D or completely uncorrelated in 3D. The PCC also systematically underestimates the degree of anti-correlation between the projections of two perfectly anti-correlated 3D distributions. The problem is that the projection of a random spatial distribution within the 3D spherocylinder is non-random in 2D, whereas PCC compares every matrix element of R or G with the constant mean value [Formula: see text] or [Formula: see text]. We propose a modified Pearson Correlation Coefficient (MPCC) that corrects this problem for spherocylindrical cell geometry by using the proper reference matrix for comparison with R and G. Correct behavior of MPCC is confirmed for a variety of numerical simulations and on experimental distributions of HU and RNA polymerase in live E. coli cells. The MPCC concept should be generalizable to other cell shapes.

Effects of alterations of the E. coli lipopolysaccharide layer on membrane permeabilization events induced by Cecropin A.

The outermost layer of Gram negative bacteria is composed of a lipopolysaccharide (LPS) network that forms a dense protective hydrophilic barrier against entry of hydrophobic drugs. At low µM concentrations, a large family of cationic polypeptides known as antimicrobial peptides (AMPs) are able to penetrate the LPS layer and permeabilize the outer membrane (OM) and the cytoplasmic membrane (CM), causing cell death. Cecropin A is a well-studied cationic AMP from moth. Here a battery of time-resolved, single-cell microscopy experiments explores how deletion of sugar layers and/or phosphoryl negative charges from the core oligosaccharide layer (core OS) of K12 E. coli alters the timing of OM and CM permeabilization induced by Cecropin A. Deletion of sugar layers, or phosphoryl charges, or both from the core OS shortens the time to the onset of OM permeabilization to periplasmic GFP and also the lag time between OM permeabilization and CM permeabilization. Meanwhile, the 12-h minimum inhibitory concentration (MIC) changes only twofold with core OS alterations. The results suggest a two-step model in which the core oligosaccharide layers act as a kinetic barrier to penetration of Cecropin A to the lipid A outer leaflet of the OM. Once a threshold concentration has built up at the lipid A leaflet, nucleation occurs and the OM is locally permeabilized to GFP and, by inference, to Cecropin A. Whenever Cecropin A permeabilizes the OM, CM permeabilization always follows, and cell growth subsequently halts and never recovers on the 45 min observation timescale.

Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15.

Antibiotics target specific biochemical mechanisms in bacteria. In response to new drugs, pathogenic bacteria rapidly develop resistance. In contrast, antimicrobial peptides (AMPs) have retained broad spectrum antibacterial potency over millions of years. We present single-cell fluorescence assays that detect reactive oxygen species (ROS) in the Escherichia coli cytoplasm in real time. Within 30 s of permeabilization of the cytoplasmic membrane by the cationic AMP CM15 [combining residues 1-7 of cecropin A (from moth) with residues 2-9 of melittin (bee venom)], three fluorescence signals report oxidative stress in the cytoplasm, apparently involving O2 (-), H2O2, and •OH. Mechanistic studies indicate that active respiration is a prerequisite to the CM15-induced oxidative damage. In anaerobic conditions, signals from ROS are greatly diminished and the minimum inhibitory concentration increases 20-fold. Evidently the natural human AMP LL-37 also induces a burst of ROS. Oxidative stress may prove a significant bacteriostatic mechanism for a variety of cationic AMPs. If so, host organisms may use the local oxygen level to modulate AMP potency.

Single-cell, time-resolved study of the effects of the antimicrobial peptide alamethicin on Bacillus subtilis.

Alamethicin is a well-studied antimicrobial peptide (AMP) that kills Gram-positive bacteria. It forms narrow, barrel-stave pores in planar lipid bilayers. We present a detailed, time-resolved microscopy study of the sequence of events during the attack of alamethicin on individual, live Bacillus subtilis cells expressing GFP in the cytoplasm. At the minimum inhibitory concentration (MIC), the first observed symptom is the halting of growth, as judged by the plateau in measured cell length vs time. The data strongly suggest that this growth-halting event occurs prior to membrane permeabilization. Gradual degradation of the proton-motive force, inferred from a decrease in pH-dependent GFP fluorescence intensity, evidently begins minutes later and continues over about 5 min. There follows an abrupt permeabilization of the cytoplasmic membrane to the DNA stain Sytox Orange and concomitant loss of small osmolytes, causing observable cell shrinkage, presumably due to decreased turgor pressure. This permeabilization of the cytoplasmic membrane occurs uniformly across the entire membrane, not locally, on a timescale of 5s or less. GFP gradually leaks out of the cell envelope, evidently impeded by the shrunken peptidoglycan layer. Disruption of the cell envelope by alamethicin occurs in stages, with larger and larger species permeating the envelope as time evolves over tens of minutes. Some of the observed symptoms are consistent with the formation of barrel-stave pores, but the data do not rule out "chaotic pore" or "carpet" mechanisms. We contrast the effects of alamethicin and the human cathelicidin LL-37 on B. subtilis.

Effects of amino acid starvation on RelA diffusive behavior in live Escherichia coli.

During amino acid starvation, bacterial cells rapidly synthesize the nucleotides (p)ppGpp, causing a massive re-programming of the transcriptional profile known as the stringent response. The (p)ppGpp synthase RelA is activated by ribosomes harboring an uncharged tRNA at the A site. It is unclear whether synthesis occurs while RelA is bound to the ribosome or free in the cytoplasm. We present a study of three Escherichia coli strains, each expressing a different RelA-fluorescent protein (RelA-FP) construct: RelA-YFP, RelA-mEos2 and RelA-Dendra2. Single-molecule localization and tracking studies were carried out under normal growth conditions and during amino acid starvation. Study of three labeling schemes enabled us to assess potential problems with FP labeling of RelA. The diffusive trajectories and axial spatial distributions indicate that amino acid starvation induces net binding of all three RelA-FP constructs to 70S ribosomes. The data are most consistent with a model in which RelA synthesizes (p)ppGpp while bound to the 70S ribosome. We suggest a 'short hopping time' model of RelA activity during starvation. Our results contradict an earlier study of RelA-Dendra2 diffusion that inferred off-ribosome synthesis of (p)ppGpp. The reasons for the discrepancy remain unclear.

Time-dependent effects of transcription- and translation-halting drugs on the spatial distributions of the Escherichia coli chromosome and ribosomes.

Previously observed effects of rifampicin and chloramphenicol indicate that transcription and translation activity strongly affect the coarse spatial organization of the bacterial cytoplasm. Single-cell, time-resolved, quantitative imaging of chromosome and ribosome spatial distributions and ribosome diffusion in live Escherichia coli provides insight into the underlying mechanisms. Monte Carlo simulations of model DNA-ribosome mixtures support a novel nucleoid-ribosome mixing hypothesis. In normal conditions, 70S-polysomes and the chromosomal DNA segregate, while 30S and 50S ribosomal subunits are able to penetrate the nucleoids. Growth conditions and drug treatments determine the partitioning of ribosomes into 70S-polysomes versus free 30S and 50S subunits. Entropic and excluded volume effects then dictate the resulting chromosome and ribosome spatial distributions. Direct observation of radial contraction of the nucleoids 0-5 min after treatment with either transcription- or translation-halting drugs supports the hypothesis that simultaneous transcription, translation, and insertion of proteins into the membrane ('transertion') exerts an expanding force on the chromosomal DNA. Breaking of the DNA-RNA polymerase-mRNA-ribosome-membrane chain in either of two ways causes similar nucleoid contraction on a similar timescale. We suggest that chromosomal expansion due to transertion enables co-transcriptional translation throughout the nucleoids.

Real-time attack of LL-37 on single Bacillus subtilis cells.

Time-lapse fluorescence microscopy of single, growing Bacillus subtilis cells with 2-12s time resolution reveals the mechanisms of antimicrobial peptide (AMP) action on a Gram-positive species with unprecedented detail. For the human cathelicidin LL-37 attacking B. subtilis, the symptoms of antimicrobial stress differ dramatically depending on the bulk AMP concentration. At 2µM LL-37, the mean single-cell growth rate decreases, but membrane permeabilization does not occur. At 4µM LL-37, cells abruptly shrink in size at the same time that Sytox Green enters the cytoplasm and stains the nucleoids. We interpret the shrinkage event as loss of turgor pressure (and presumably the membrane potential) due to permeabilization of the membrane. Movies of Sytox Green staining at 0.5frame/s show that nucleoid staining is initially local, more consistent with pore formation than with global permeabilization models. In a novel "growth recovery" assay, cells are incubated with LL-37 for a variable period and then rinsed with fresh growth medium lacking LL-37. The growth rate attenuation observed at 2µM LL-37 is a recoverable symptom, while the abrupt cell shrinkage observed at 4µM LL-37 is not.

Nonperturbative imaging of nucleoid morphology in live bacterial cells during an antimicrobial peptide attack.

Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of "dead-cell stains" (SYTOX orange and SYTOX green) and "live-cell stains" (DRAQ5 and SYTO 61) and also 4',6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live Escherichia coli, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative E. coli and the Gram-positive Bacillus subtilis. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the E. coli cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested.

Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37.

Natural antimicrobial peptides (AMPs) provide prototypes for the design of unconventional antimicrobial agents. Existing bulk assays measure AMP activity but do not provide details of the growth-halting mechanism. We use fluorescence microscopy to directly observe the attack of the human antimicrobial peptide LL-37 on single Escherichia coli cells in real time. Our findings strongly suggest that disruption of the cytoplasmic membrane is not the growth-halting mechanism. At 8 µM, LL-37 binding saturates the outer membrane (OM) within 1 min. Translocation across the OM and access to the periplasmic space (5-25 min later) correlates in time with the halting of growth. Septating cells are attacked more readily than nonseptating cells. The halting of growth may occur because of LL-37 interference with cell wall biogenesis. Only well after growth halts does the peptide permeabilize the cytoplasmic membrane to GFP and the small dye Sytox Green. The assay enables dissection of antimicrobial design criteria into two parts: translocation across the OM and the subsequent halting of growth.

Partitioning of RNA polymerase activity in live Escherichia coli from analysis of single-molecule diffusive trajectories.

Superresolution fluorescence microscopy is used to locate single copies of RNA polymerase (RNAP) in live Escherichia coli and track their diffusive motion. On a timescale of 0.1-1 s, most copies separate remarkably cleanly into two diffusive states. The "slow" RNAPs, which move indistinguishably from DNA loci, are assigned to specifically bound copies (with fractional population ftrxn) that are initiating transcription, elongating, pausing, or awaiting termination. The "mixed-state" RNAP copies, with effective diffusion constant Dmixed = 0.21 µm(2) s(-1), are assigned as a rapidly exchanging mixture of nonspecifically bound copies (fns) and copies undergoing free, three-dimensional diffusion within the nucleoids (ffree). Longer trajectories of 7-s duration reveal transitions between the slow and mixed states, corroborating the assignments. Short trajectories of 20-ms duration enable direct observation of the freely diffusing RNAP copies, yielding Dfree = 0.7 µm(2) s(-1). Analysis of single-particle trajectories provides quantitative estimates of the partitioning of RNAP into different states of activity: ftrxn = 0.54 ± 0.07, fns = 0.28 ± 0.05, ffree = 0.12 ± 0.03, and fnb = 0.06 ± 0.05 (fraction unable to bind to DNA on a 1-s timescale). These fractions disagree with earlier estimates.

Localized permeabilization of E. coli membranes by the antimicrobial peptide Cecropin A.

Fluorescence microscopy enables detailed observation of the effects of the antimicrobial peptide Cecropin A on the outer membrane (OM) and cytoplasmic membrane (CM) of single E. coli cells with subsecond time resolution. Fluorescence from periplasmic GFP decays and cell growth halts when the OM is permeabilized. Fluorescence from the DNA stain Sytox Green rises when the CM is permeabilized and the stain enters the cytoplasm. The initial membrane disruptions are localized and stable. Septating cells are attacked earlier than nonseptating cells, and curved membrane surfaces are attacked in preference to cylindrical surfaces. Below a threshold bulk Cecropin A concentration, permeabilization is not observed over 30 min. Above this threshold, we observe a lag time of several minutes between Cecropin A addition and OM permeabilization and ∼30 s between OM and CM permeabilization. The long lag times and the existence of a threshold concentration for permeabilization suggest a nucleation mechanism. However, the roughly linear dependence of mean lag time on bulk peptide concentration is not easily reconciled with a nucleation step involving simultaneous insertion of multiple peptides into the bilayer. Monte Carlo simulations suggest that within seconds, the OM permeability becomes comparable to that of a pore of 100 nm diameter or of numerous small pores distributed over a similarly large area.

Superresolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells.

Quantitative spatial distributions of ribosomes (S2-YFP) and RNA polymerase (RNAP; ß'-yGFP) in live Escherichia coli are measured by superresolution fluorescence microscopy. In moderate growth conditions, nucleoid-ribosome segregation is strong, and RNAP localizes to the nucleoid lobes. The mean copy numbers per cell are 4600 RNAPs and 55,000 ribosomes. Only 10-15% of the ribosomes lie within the densest part of the nucleoid lobes, and at most 4% of the RNAPs lie in the two ribosome-rich endcaps. The predominant observed diffusion coefficient of ribosomes is D(ribo) = 0.04 µm(2) s(-1), attributed to free mRNA being translated by one or more 70S ribosomes. We find no clear evidence of subdiffusion, as would arise from tethering of ribosomes to the DNA. The degree of DNA-ribosome segregation strongly suggests that in E. coli most translation occurs on free mRNA transcripts that have diffused into the ribosome-rich regions. Both RNAP and ribosome radial distributions extend to the cytoplasmic membrane, consistent with the transertion hypothesis. However, few if any RNAP copies lie near the membrane of the endcaps. This suggests that if transertion occurs, it exerts a direct radially expanding force on the nucleoid, but not a direct axially expanding force.

Subdiffraction-limit study of Kaede diffusion and spatial distribution in live Escherichia coli.

Photoactivation localization microscopy (PALM) is used to study the spatial distribution and diffusion of single copies of the protein Kaede in the cytoplasm of live Escherichia coli under moderate growth conditions (67 min doubling time). The spatial distribution of Kaede is uniform within the cytoplasm. The cytoplasmic radius of 380 ± 30 nm varies little from cell to cell. Single-particle tracking using 4 ms exposure times reveals negatively curved plots of mean-square displacement versus time. A detailed comparison with Monte Carlo simulations in a spherocylindrical volume shows that the curvature can be quantitatively understood in terms of free diffusion within a confining volume. The mean diffusion coefficient across cells is = 7.3 ± 1.1 µm(2)·s(-1), consistent with a homotetrameric form of Kaede. The distribution of squared displacements along the long axis for individual Kaede molecules is consistent with homogeneous diffusion. However, for longer cells, a spatial map of one-step estimates of the diffusion coefficient along x suggests that diffusion is ∼20-40% faster within nucleoids than in the ribosome-rich region lying between nucleoid lobes at the cell mid-plane. Fluorescence recovery after photobleaching yielded = 8.3 ± 1.6 µm(2)·s(-1), in agreement with the single-particle tracking results.