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.

This paper demonstrates the photophysics of curcumin inside polymeric nanoparticles (NPs), which are being recently used as targeted drug delivery vehicles. For this purpose, we have prepared three polymeric NPs by ultrasonication method from three well-defined water-insoluble random copolymers. These copolymers having various degrees of hydrophobicity were synthesized via reversible addition-fragmentation transfer (RAFT) method using styrene and three different functional monomers, namely, 2-hydroxyethyl acrylate, 4-formylphenyl acrylate, and 4-vinylbenzyl chloride. The photophysics of the curcumin molecules inside the polymeric NPs have been monitored by applying tools like steady state and time-resolved fluorescence spectroscopy. An increase in fluorescence intensity along with an increase in the lifetime values indicated a perturbation of the excited state intramolecular proton transfer (ESIPT) process of curcumin inside the polymeric NPs.

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.

For E. 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 (k trl ) can be as fast as ~20 amino acids/s. For slower growth arising from poor nutrient quality or from higher growth osmolality, k trl decreases significantly. In earlier work from the Hwa lab, a simplified Michaelis-Menten model suggested that the decrease in k trl 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 ternary complexes under nutrient limitation or by the diffusion of free ternary complexes at high osmolality. Some other step or steps must be rate limiting for translation in slow growth.

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 cationichydrophobic 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.

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Proline-rich antimicrobial peptides (PrAMPs) are cationic AMPs 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 (PTC). 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, EF-Tu, and DNA spatial distributions and diffusive properties in intact E. 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 (Cam) and kasugamycin (Ksg) 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 vs 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 non-cognate ternary complexes (TCs) at the ribosomal A-site enhances the dissociation rate of such TCs from their L7/L12 tethers. Testing and rejection of non-cognate 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.

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