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.

Diverse Impacts on Prokaryotic and Eukaryotic Membrane Activities from Hydrophobic Subunit Variation Among Nylon-3 Copolymers.

Synthetic, sequence-random polymers that feature a wide range of backbone and side chain structures have been reported to function as mimics of natural host-defense peptides, inhibiting bacterial growth while exerting little or no toxicity toward eukaryotic cells. The common themes among these materials are net positive charge, which is thought to confer preferential action toward prokaryotic vs eukaryotic cells, and the presence of hydrophobic components, which are thought to mediate membrane disruption. This study is based on a set of new binary cationic-hydrophobic nylon-3 copolymers that was designed to ask whether factors beyond net charge and net hydrophobicity influence the biological activity profile. In previous work, we found that nonpolar subunits preorganized by a ring led to copolymers with a diminished tendency to disrupt human cell membranes (as measured via lysis of red blood cells) relative to copolymers containing more flexible nonpolar subunits. An alternative mode of conformational restriction, involving geminal substitution, also minimized hemolysis. Here, we asked whether combining a cyclic constraint and geminal substitution would be synergistic; the combination was achieved by introducing backbone methyl groups to previously described cyclopentyl and cyclohexyl subunits. The new cyclic subunits containing two quaternary backbone carbons (i.e, two sites of geminal substitution) were comparable or slightly superior in terms of antibacterial potency but markedly superior in terms of low hemolytic activity, relative to cyclic subunits lacking the quaternary carbons. However, new cyclic units containing only one quaternary carbon were very hemolytic, which was unanticipated. Variations in net hydrophobicity cannot explain the trend in hemolysis, in contrast to the standard perspective in this field. The impact of each new polymer on live E. coli cells was evaluated via fluorescence microscopy. All new polymers moved rapidly across the outer membrane without large-scale disruption of barrier function. Increasing the number of quaternary carbons in the nonpolar subunit correlated with an increased propensity to permeabilize the cytoplasmic membrane of E. coli cells. Collectively, these findings show that relationships between nonpolar subunit identity and biological activity are influenced by factors in addition to hydrophobicity and charge. We propose that the variation of subunit conformational properties may be one such factor.

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.

Biophysical Properties of Escherichia coli Cytoplasm in Stationary Phase by Superresolution Fluorescence Microscopy.

In nature, bacteria must survive long periods of nutrient deprivation while maintaining the ability to recover and grow when conditions improve. This quiescent state is called stationary phase. The biochemistry of Escherichia coli in stationary phase is reasonably well understood. Much less is known about the biophysical state of the cytoplasm. Earlier studies of harvested nucleoids concluded that the stationary-phase nucleoid is "compacted" or "supercompacted," and there are suggestions that the cytoplasm is "glass-like." Nevertheless, stationary-phase bacteria support active transcription and translation. Here, we present results of a quantitative superresolution fluorescence study comparing the spatial distributions and diffusive properties of key components of the transcription-translation machinery in intact E. coli cells that were either maintained in 2-day stationary phase or undergoing moderately fast exponential growth. Stationary-phase cells are shorter and exhibit strong heterogeneity in cell length, nucleoid volume, and biopolymer diffusive properties. As in exponential growth, the nucleoid and ribosomes are strongly segregated. The chromosomal DNA is locally more rigid in stationary phase. The population-weighted average of diffusion coefficients estimated from mean-square displacement plots is 2-fold higher in stationary phase for both RNA polymerase (RNAP) and ribosomal species. The average DNA density is roughly twice as high as that in cells undergoing slow exponential growth. The data indicate that the stationary-phase nucleoid is permeable to RNAP and suggest that it is permeable to ribosomal subunits. There appears to be no need to postulate migration of actively transcribed genes to the nucleoid periphery.IMPORTANCE Bacteria in nature usually lack sufficient nutrients to enable growth and replication. Such starved bacteria adapt into a quiescent state known as the stationary phase. The chromosomal DNA is protected against oxidative damage, and ribosomes are stored in a dimeric structure impervious to digestion. Stationary-phase bacteria can recover and grow quickly when better nutrient conditions arise. The biochemistry of stationary-phase E. coli is reasonably well understood. Here, we present results from a study of the biophysical state of starved E. coli Superresolution fluorescence microscopy enables high-resolution location and tracking of a DNA locus and of single copies of RNA polymerase (the transcription machine) and ribosomes (the translation machine) in intact E. coli cells maintained in stationary phase. Evidently, the chromosomal DNA remains sufficiently permeable to enable transcription and translation to occur. This description contrasts with the usual picture of a rigid stationary-phase cytoplasm with highly condensed DNA.

Resistance of early stationary phase E. coli to membrane permeabilization by the antimicrobial peptide Cecropin A.

Antimicrobial peptides (AMPs) cause bacterial membrane permeabilization and ultimately cell death at low µM concentrations. The membrane permeabilization action of a moth derived AMP Cecropin A on E. coli cells in exponential growth (mid-log phase) is well studied. At 1× MIC concentration, Cecropin A penetrates the lipopolysaccharide (LPS) barrier and causes outer membrane (OM) and cytoplasmic membrane (CM) permeabilization. For non-septating cells, permeabilization of both membranes begins at one pole. For septating cells, OM permeabilization begins at the septal region and CM permeabilization begins at one pole. However, in nature bacteria are frequently found in nutrient-starved conditions. Here we extend our single-cell microscopy assays to the attack of Cecropin A on E. coli cells in early stationary phase. Stationary phase E. coli is much more resistant to membrane permeabilization by Cecropin A than mid-log phase E. coli. A tenfold higher concentration of Cecropin A is required to observe CM permeabilization in the majority of stationary phase cells, and even then permeabilization proceeds more slowly. In addition, the spatial pattern of initial CM permeabilization changes from localized at one pole to global. Studies of lipid mutant strains suggest that a sufficient localized concentration of the anionic phospholipid phosphatidylglycerol (PG) guides the position of initial attack of the cationic AMP Cecropin A on the CM.

Near Saturation of Ribosomal L7/L12 Binding Sites with Ternary Complexes in Slowly Growing E. coli.

For Escherichia coli growing rapidly in rich medium at 37 °C, the doubling time can be as short as ~20 min and the average rate of translation (ktrl) can be as fast as ~20 amino acids/s. For slower growth arising from poor nutrient quality or from higher growth osmolality, ktrl decreases significantly. In earlier work from the Hwa lab, a simplified Michaelis-Menten model suggested that the decrease in ktrl arises from a shortage of ternary complexes (TCs) under nutrient limitation and from slower diffusion of TCs under high growth osmolality. Here we present a single-molecule tracking study of the diffusion of EF-Tu in E. coli growing with doubling times in the range 62-190 min at 37 °C due to nutrient limitation, high growth osmolality, or both. The diffusive properties of EF-Tu remain quantitatively indistinguishable across all growth conditions studied. Dissection of the total population into ribosome-bound and free sub-populations, combined with copy number estimates for EF-Tu and ribosomes, indicates that in all cases ~3.7 EF-Tu copies are bound on average to each translating 70S ribosome. Thus, the four L7/L12 binding sites adjacent to the ribosomal A-site in E. coli are essentially saturated with TCs in all conditions, facilitating rapid testing of aminoacyl-tRNAs for a codon match. Evidently, the average translation rate is not limited by either the supply of cognate TCs under nutrient limitation or by the diffusion of free TCs at high osmolality. Some other step or steps must be rate limiting for translation in slow growth.

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.

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.

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.

HaloTag Assay Suggests Common Mechanism of E. coli Membrane Permeabilization Induced by Cationic Peptides.

Permeabilization of the Gram-negative bacterial outer membrane (OM) by antimicrobial peptides (AMPs) is the initial step enabling access of the AMP to the cytoplasmic membrane. We present a new single-cell, time-resolved fluorescence microscopy assay that reports on the permeabilization of the E. coli OM to small molecules with a time resolution of 3 s or better. When profluorophore JF646 (702 Da) crosses the outer membrane (OM) and gains access to the periplasm, it binds to the localized HaloTag protein (34 kDa) and fluoresces in a characteristic hollow spatial pattern. Previous work used the much larger periplasmic GFP (27 kDa) probe, which reports on OM permeabilization to globular proteins. We test the assay on three cationic agents: Gellman random ß-peptide copolymer MM63:CHx37, human AMP LL-37, and synthetic hybrid AMP CM15. These results combined with the previous work suggest a unifying sequence of OM and cytoplasmic membrane (CM) events that may prove commonplace in the attack of cationic peptides on Gram-negative bacteria. The peptide initially induces gradual OM permeabilization to small molecules, likely including the peptide itself. After a lag time, abrupt permeabilization of the OM, abrupt resealing of the OM, and abrupt permeabilization of the CM (all to globular proteins) occur in rapid sequence. We propose a mechanism based on membrane curvature stress induced by the time-dependent differential binding of peptide to the outer leaflet of the OM and CM. The results provide fresh insight into the critical OM-permeabilization step leading to a variety of damaging downstream events.

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.

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.

Simultaneous Binding of Multiple EF-Tu Copies to Translating Ribosomes in Live Escherichia coli.

In bacteria, elongation factor Tu is a translational cofactor that forms ternary complexes with aminoacyl-tRNA (aa-tRNA) and GTP. Binding of a ternary complex to one of four flexible L7/L12 units on the ribosome tethers a charged tRNA in close proximity to the ribosomal A site. Two sequential tests for a match between the aa-tRNA anticodon and the current mRNA codon then follow. Because one elongation cycle can occur in as little as 50 ms and the vast majority of aa-tRNA copies are not cognate with the current mRNA codon, this testing must occur rapidly. We present a single-molecule localization and tracking study of fluorescently labeled EF-Tu in live Escherichia coli Imaging at 2 ms/frame distinguishes 60% slowly diffusing EF-Tu copies (assigned as transiently bound to translating ribosome) from 40% rapidly diffusing copies (assigned as a mixture of free ternary complexes and free EF-Tu). Combining these percentages with copy number estimates, we infer that the four L7/L12 sites are essentially saturated with ternary complexes in vivo. The results corroborate an earlier inference that all four sites can simultaneously tether ternary complexes near the A site, creating a high local concentration that may greatly enhance the rate of testing of aa-tRNAs. Our data and a combinatorial argument both suggest that the initial recognition test for a codon-anticodon match occurs in less than 1 to 2 ms per aa-tRNA copy. The results refute a recent study (A. Plochowietz, I. Farrell, Z. Smilansky, B. S. Cooperman, and A. N. Kapanidis, Nucleic Acids Res 45:926-937, 2016, https://doi.org/10.1093/nar/gkw787) of tRNA diffusion in E. coli that inferred that aa-tRNAs arrive at the ribosomal A site as bare monomers, not as ternary complexes.IMPORTANCE Ribosomes catalyze translation of the mRNA codon sequence into the corresponding sequence of amino acids within the nascent polypeptide chain. Polypeptide elongation can be as fast as 50 ms per added amino acid. Each amino acid arrives at the ribosome as a ternary complex comprising an aminoacyl-tRNA (aa-tRNA), an elongation factor called EF-Tu, and GTP. There are 43 different aa-tRNAs in use, only one of which typically matches the current mRNA codon. Thus, ternary complexes must be tested very rapidly. Here we use fluorescence-based single-molecule methods that locate and track single EF-Tu copies in E. coli Fast and slow diffusive behavior determines the fraction of EF-Tu copies that are ribosome bound. We infer simultaneous tethering of ~4 ternary complexes to the ribosome, which may facilitate rapid initial testing for codon matching on a time scale of less than 1 to 2 ms per aa-tRNA.

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.

Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live Escherichia coli.

In vitro assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues. Ribosome profiling shows that stalling at Pro-Pro-X triplets is especially severe but is largely alleviated in Escherichia coli by the action of elongation factor EF-P. EF-P and its eukaryotic/archaeal homolog IF5A enhance the peptidyl transfer step of elongation. Here, a superresolution fluorescence localization and tracking study of EF-P-mEos2 in live E. coli provides the first in vivo information about the spatial distribution and on-off binding kinetics of EF-P. Fast imaging at 2 ms/frame helps to distinguish ribosome-bound (slowly diffusing) EF-P from free (rapidly diffusing) EF-P. Wild-type EF-P exhibits a three-peaked axial spatial distribution similar to that of ribosomes, indicating substantial binding. The mutant EF-PK34A exhibits a homogeneous distribution, indicating little or no binding. Some 30% of EF-P copies are bound to ribosomes at a given time. Two-state modeling and copy number estimates indicate that EF-P binds to 70S ribosomes during 25 to 100% of translation cycles. The timescale of the typical diffusive search by free EF-P for a ribosome-binding site is τfree ≈ 16 ms. The typical residence time of an EF-P on the ribosome is very short, τbound ≈ 7 ms. Evidently, EF-P binds to ribosomes during many or most elongation cycles, much more often than the frequency of Pro-Pro motifs. Emptying of the E site during part of the cycle is consistent with recent in vitro experiments indicating dissociation of the deacylated tRNA upon translocation.IMPORTANCE Ribosomes translate the codon sequence within mRNA into the corresponding sequence of amino acids within the nascent polypeptide chain, which in turn ultimately folds into functional protein. At each codon, bacterial ribosomes are assisted by two well-known elongation factors: EF-Tu, which aids binding of the correct aminoacyl-tRNA to the ribosome, and EF-G, which promotes tRNA translocation after formation of the new peptide bond. A third factor, EF-P, has been shown to alleviate ribosomal pausing at rare Pro-Pro motifs, which are translated very slowly without EF-P. Here, we use superresolution fluorescence imaging to study the spatial distribution and ribosome-binding dynamics of EF-P in live E. coli cells. We were surprised to learn that EF-P binds to and unbinds from translating ribosomes during at least 25% of all elongation events; it may bind during every elongation cycle.

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.

Lights, Camera, Action! Antimicrobial Peptide Mechanisms Imaged in Space and Time.

Deeper understanding of the bacteriostatic and bactericidal mechanisms of antimicrobial peptides (AMPs) should help in the design of new antibacterial agents. Over several decades, a variety of biochemical assays have been applied to bulk bacterial cultures. While some of these bulk assays provide time resolution of the order of 1min, they do not capture faster mechanistic events. Nor can they provide subcellular spatial information or discern cell-to-cell heterogeneity within the bacterial population. Single-cell, time-resolved imaging assays bring a completely new spatiotemporal dimension to AMP mechanistic studies. We review recent work that provides new insights into the timing, sequence, and spatial distribution of AMP-induced effects on bacterial cells.

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.

Single-Cell, Time-Resolved Antimicrobial Effects of a Highly Cationic, Random Nylon-3 Copolymer on Live Escherichia coli.

Synthetic random copolymers based on the nylon-3 (ß-peptide) backbone show promise as inexpensive antimicrobial agents resistant to proteolysis. We present a time-resolved observational study of the attack of a particular copolymer MM63:CHx37 on single, live Escherichia coli cells. The composition and chain length of MM63:CHx37 (63% cationic subunits, 37% hydrophobic subunits, 35-subunit average length) were optimized to enhance antibacterial activity while minimizing lysis of human red blood cells. For E. coli cells that export GFP to the periplasm, we obtain alternating phase-contrast and green fluorescence images with a time resolution of 12 s over 60 min following initiation of copolymer flow. Within seconds, cells shrink and exhibit the same plasmolysis spaces that occur following abrupt external osmotic upshift. The osmoprotection machinery attempts to replenish cytoplasmic water, but recovery is interrupted by permeabilization of the cytoplasmic membrane (CM) to GFP. Evidently, the highly cationic copolymer and its counterions rapidly translocate across the outer membrane without permeabilizing it to GFP. The CM permeabilization event is spatially localized. Cells whose CM has been permeabilized never recover growth. The minimum inhibitory concentration (MIC) for cells lacking the osmolyte importer ProP is 4-fold smaller than for normal cells, suggesting that osmoprotection is an important survival strategy. In addition, at the time of CM permeabilization, we observe evidence of oxidative stress. The MIC under anaerobic conditions is at least 8-fold larger than under aerobic conditions, further implicating oxidative damage as an important bacteriostatic effect. Once the copolymer reaches the periplasm, multiple growth-halting mechanisms proceed in parallel.