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.     

Changes in bacterial cells induced by high pressure at subzero temperature

The aim of this study was to examine the effect of pressure treatment at 193 MPa and −20 °C on membrane damage, changes in activity of membrane-bound ATPases and degradation of nucleic acids. The experiments were carried out with three Escherichia coli strains, in the exponential and stationary phases of growth, and differing in sensitivity to pressure. All E. coli strains subjected to pressure in the exponential phase of growth were inactivated by 6 log cycles, independently of the strain, which was accompanied by a total loss of ability to plasmolyse, an increase in irreversible membrane permeability to PI, and a reduction of cellular ATP by more than 80%. After pressure treatment of stationary phase cells, the relationship between the inactivation level and the ability to plasmolyse was not as evident as in the case of exponential phase cells. Pressure treatment of two strains of E. coli K-12 and Ec160/59 in the stationary phase that decreased viability by no more than one log cycle led only to reversible permeabilization of bacterial membranes, while irreversible permeabilization was observed in the pressure sensitive E. coli IBA72 strain phase that was inactivated by 4.6 log cycles. The reduction of ATP and changes in ATPase activity after pressure treatment of tested E. coli strains in the stationary phase of growth depended on the stage of inactivation of the particular strain. Electrophoretic analysis showed degradation of RNA isolated after pressure treatment from cells of all E. coli strains tested in the exponential phase of growth. The changes of RNA induced by pressure were not visible in the case of cells in the stationary phase. The degradation of DNA isolated from pressure treated E. coli strains from the exponential as well as from the stationary phase of growth was not observed.     

Monitoring antibacterial permeabilization in real time using time-resolved flow cytometry

Despite the intensive study of antibiotic-induced bacterial permeabilization, its kinetics and molecular mechanism remain largely elusive. A new methodology that extends the concept of the live–dead assay in flow cytometry to real time-resolved detection was used to overcome these limitations. The antimicrobial activity of pepR was monitored in time-resolved flow cytometry for three bacterial strains: Escherichia coli (ATCC 25922), E. coli K-12 (CGSC Strain 4401) and E. coli JW3596-1 (CGSC Strain 11805). The latter strain has truncated lipopolysaccharides (LPS) in the outer membrane. This new methodology provided information on the efficacy of the antibiotics and sheds light on their mode of action at membrane-level. Kinetic data regarding antibiotic binding and lytic action were retrieved. Membrane interaction and permeabilization events differ significantly among strains. The truncation of LPS moieties does not hamper AMP binding but compromises membrane disruption and bacterial killing. We demonstrated the usefulness of time-resolved flow cytometry to study antimicrobial-induced permeabilization by collecting kinetic data that contribute to characterize the action of antibiotics directly on bacteria.     

Effect of oleoyl-chitosan nanoparticles as a novel antibacterial dispersion system on viability,membrane permeability and cell morphology of Escherichia coli and Staphylococcus aureus

A novel chitosan antibacterial dispersion system was prepared by oleoyl-chitosan (O-chitosan) nanoparticles (OCNP) and the bactericidal activity against Escherichia coli and Staphylococcus aureus was evaluated by the enumeration of viable organisms at different incubation times. We further investigated the antimicrobial mode of OCNP using a combination of approaches, including cell integrity measurements, outer membrane (OM) and inner membrane (IM) permeabilization assays, SDS–PAGE and transmission electron microscopy (TEM). Results showed that when treated with OCNP, release of intracellular components quickly increased for both E. coli and S. aureus. OCNP also rapidly increased the 1-N-phenylnaphthylamine (NPN) uptake and the release of cytoplasmic β-galactosidase via increasing the permeability of OM and IM. Besides, SDS–PAGE indicated the content of cellular soluble proteins decreased significantly in OCNP-treated bacteria. TEM observations demonstrated adsorption behaviors of OCNP on bacteria and extensive cell surface alterations of OCNP-treated bacteria. OCNP has potential value in the determination of antibacterial mechanism of chitosan.     

Characterization of nucleotide pools as a function of physiological state in Escherichia coli

Using a modified method that involves minimal manipulation of cells, we report new information about nucleotide pool sizes and changes throughout the Escherichia coli growth curve. Nucleotide pool sizes are critically dependent on sample manipulation and extraction methods. Centrifugation and even short (2 min) lapses in sample preparation can dramatically affect results. The measured ATP concentration at three different growth rates is at least 3 mM, well above the 0.8 mM needed to saturate the rRNA promoter P1 in vitro. Many of the pools, including ATP, GTP, and UTP, begin to decrease while the cells are still in mid-log growth. After an almost universal drop in nucleotide concentration as the cells transition from logarithmic to stationary phase, there is a “rebound” of certain nucleotides, most notably ATP, after the cells enter stationary phase, followed by a progressive decrease. UTP, in contrast, increases as the cells transition into stationary phase. The higher UTP values might be related to elevated UDP-glucose/galactose, which was found to be at higher concentrations than expected in stationary phase. dTTP is the most abundant deoxynucleoside triphosphate (dNTP) in the cell despite the fact that its precursors, UDP and UTP, are not. All dNTPs decrease through the growth curve but do not have the abrupt drop, as seen with other nucleotides when the cells transition into stationary phase.     

Isolation and characterization of the outer membrane of Escherichia coli with autodisplayed Z-domains

“Autodisplay technology” is an expression technique used to display the various recombinant proteins on the outer membrane (OM) of Escherichia coli. The resulting autodisplayed Z-domain has been used to improve the sensitivity of immunoassays. In this work, a facile isolation method of the OM fraction of E. coli with autodisplayed Z-domains was presented using (1) an enzyme reaction for the hydrolysis of the peptidoglycan layer and (2) short centrifugation steps. The purity of the isolated OM fraction was analyzed. For the estimation of contamination with bacterial proteins from other parts of E. coli, Western blots of marker proteins for the OM (OmpA), periplasm (β-lactamase), inner membrane (SecA), and cytoplasm (β-galactosidase) were performed. Additionally, assays of marker components or enzymes from each part of E. coli were carried out including the OM (KDO), inner membrane (NADH oxidase), periplasm (β-lactamase), and cytoplasm (β-galactosidase). The yield of OM isolation using this new method was determined to be 80% of the total OM amount, with less than 1% being contaminants from other parts of E. coli.     

Damage in Escherichia coli Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature

The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the cell suspension in situ under high hydrostatic pressure (HHP) and subzero-temperature conditions made it possible to show the partial reversibility of pressure-induced nucleoid condensation. However, based on visual examination of TEM micrographs, protein aggregation did not seem to be reversible. Reversible cell membrane permeabilization was noticeable, particularly for HHP treatments at subzero temperature. A correlation between membrane permeabilization and cell inactivation was established, suggesting different mechanisms at room and subzero temperatures. We propose that the inactivation of E. coli cells under combined HHP and subzero temperature occurs mainly during their transiently permeabilized state, whereas HHP inactivation at room temperature is related to a balance of transient and permanent permeabilization. The correlation between TEM results and cell inactivation was not absolute. Further work is required to elucidate the effects of pressure-induced damage on nucleoids and proteins during cell inactivation.     

Scanning assay of β-galactosidase activity

β-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of β-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, β-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the β-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular β-galactosidase activity in E. coli Tuner (DE3) expressing β-galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of β-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.     

Immobilization of Escherichia coli Cells by Use of the Antimicrobial Peptide Cecropin P1

An immobilization scheme for bacterial cells is described, in which the antimicrobial peptide cecropin P1 was used to trap Escherichia coli K-12 and O157:H7 cells on microtiter plate well surfaces. Cecropin P1 was covalently attached to the well surfaces, and E. coli cells were allowed to bind to the peptide-coated surface. The immobilized cells were detected colorimetrically with an anti-E. coli antibody-horseradish peroxidase conjugate. Binding curves were obtained in which the signal intensities were dependent upon the cell concentration and upon the amount of peptide attached to the well surface. After normalization for the amount of peptide coupled to the surface and the relative binding affinity of the antibody for each strain, the binding data were compared, which indicated that there was a strong preference for E. coli O157:H7 over E. coli K-12. The cells could be immobilized reproducibly at pH values ranging from 5 to 10 and at ionic strengths up to 0.50 M.     

Escherichia coli Cell Surface Perturbation and Disruption Induced by Antimicrobial Peptides BP100 and pepR

The potential of antimicrobial peptides (AMPs) as an alternative to conventional therapies is well recognized. Insights into the biological and biophysical properties of AMPs are thus key to understanding their mode of action. In this study, the mechanisms adopted by two AMPs in disrupting the Gram-negative Escherichia coli bacterial envelope were explored. BP100 is a short cecropin A-melittin hybrid peptide known to inhibit the growth of phytopathogenic Gram-negative bacteria. pepR, on the other hand, is a novel AMP derived from the dengue virus capsid protein. Both BP100 and pepR were found to inhibit the growth of E. coli at micromolar concentrations. Zeta potential measurements of E. coli incubated with increasing peptide concentrations allowed for the establishment of a correlation between the minimal inhibitory concentration (MIC) of each AMP and membrane surface charge neutralization. While a neutralization-mediated killing mechanism adopted by either AMP is not necessarily implied, the hypothesis that surface neutralization occurs close to MIC values was confirmed. Atomic force microscopy (AFM) was then employed to visualize the structural effect of the interaction of each AMP with the E. coli cell envelope. At their MICs, BP100 and pepR progressively destroyed the bacterial envelope, with extensive damage already occurring 2 h after peptide addition to the bacteria. A similar effect was observed for each AMP in the concentration-dependent studies. At peptide concentrations below MIC values, only minor disruptions of the bacterial surface occurred.     

Synergistic action of rapid chilling and nisin on the inactivation of Escherichia coli

The effect of rapid and slow chilling on survival and nisin sensitivity was investigated in Escherichia coli. Membrane permeabilization induced by cold shock was assessed by uptake of the fluorescent dye 1-N-phenylnapthylamine. Slow chilling (2°C min⁻¹) did not induce transient susceptibility to nisin. Combining rapid chilling (2,000°C min⁻¹) and nisin causes a dose-dependent reduction in the population of cells in both exponential and stationary growth phases. A reduction of 6 log of exponentially growing cells was achieved with rapid chilling in the presence of 100 IU ml⁻¹ nisin. Cells were more sensitive if nisin was present during stress. Nevertheless, addition of nisin to cell suspension after the rapid chilling produced up to 5 log of cell inactivation for exponentially growing cells and 1 log for stationary growing cells. This suggests that the rapid chilling strongly damaged the cell membrane by disrupting the outer membrane barrier, allowing the sensitization of E. coli to nisin post-rapid chilling. Measurements of membrane permeabilization showed a good correlation between the membrane alteration and nisin sensitivity. Application involving the simultaneous treatment with nisin and rapid cold shock could thus be of value in controlling Gram negatives, enhancing microbiological safety and stability.     

A procedure to estimate okadaic acid in whole dinoflagellate cells using immunological techniques

A single procedure to detect and estimate okadaic acid in isolated whole cells was developed based on immunofluorescence and microscope photometry. This procedure allows the study of variations in okadaic acid concentration per cell although it is no substitute for HPLC procedures. Cells from mid-log exponential and stationary phase from two different clonal cultures of the okadaic-acid-producing dinoflagellate Prorocentrum lima (PI 5V and PI 7V) were analyzed. The results showed that: (1) cells from saturated phase cultures contain more okadaic acid than those from exponentially-growing mid-log phase; (2) genetic differences exist in okadaic acid production between the clones used; (3) okadaic acid is synthesized continuously during the whole cell cycle.     

Membrane Damage and Microbial Inactivation by Chlorine in the Absence and Presence of a Chlorine-Demanding Substrate

The relationship between cell inactivation and membrane damage was studied in two gram-positive organisms, Listeria monocytogenes and Bacillus subtilis, and two gram-negative organisms, Yersinia enterocolitica and Escherichia coli, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy broth). L. monocytogenes and B. subtilis were more resistant to chlorine in distilled water. The addition of small amounts of organic matter to the chlorination medium drastically increased the resistance of both types of microorganisms, but this effect was more marked in Y. enterocolitica and E. coli. In addition, the survival curves for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal injury was not detected under most experimental conditions, and only gram-positive cells treated in distilled water showed a relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive permeabilization of the cytoplasmic membrane, but the concentrations required were much higher than those needed to inactivate cells. Therefore, there was no relationship between the occurrence of membrane permeabilization and cell death. The addition of organic matter to the treatment medium stabilized the cytoplasmic membrane against permeabilization in both the gram-positive and gram-negative bacteria investigated. Exposure of E. coli cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine and caused the shoulders in the survival curves to disappear. Based on these observations, we propose that bacterial envelopes could play a role in cell inactivation by modulating the access of chlorine to the key targets within the cell.     

Physiology of F-Pilin Synthesis and Utilization

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to study the synthesis and turnover of F-pilin in membrane preparations of Escherichia coli K-12 under conditions which have been reported to affect the production of F-pili. Incorporation of [³⁵S]methionine into membrane F-pilin by cells in log phase was barely detectable at 25°C, but increased with temperature. The labeled pilin band was prominent in membranes from 37°C cultures and even more prominent if the growth temperature was raised to 42°C. The appearance of other tra products in the membranes was similarly temperature dependent. In cultures grown in glucose minimal medium at 37°C, the relative amount of membrane pilin and traT product synthesis remained unchanged from early log phase through early stationary phase; provision of glycerol or arabinose as a substitute carbon source had no obvious effect. Turnover of traT product and membrane F-pilin, as assessed in an Flac tra mutant strain which is incapable of elaborating pili, was not rapid. Both traT product and pilin subunits labeled in mid-log phase cells were still apparent in the membranes after growth of the cells to stationary phase. The relative amount of labeled pilin decreased with prolonged incubation in stationary phase, but the relative amount of traT product did not decrease even after the culture was incubated for 24 h. When wild-type Flac piliated cells were used, a similar result was obtained, but in this case, loss of F-pilin from the preparations could be acclerated by blending the cells. Although intermittent blending during culture growth caused a slow depletion of the labeled pilin pool, continuous blending resulted in the rapid disappearance of this pool from our preparations. Loss of other membrane polypeptides was not accelerated by our blending procedure, and blending did not affect the turnover of the pilin pool of the Flac tra mutant. Our data are consistent with a model in which pilin subunits are assembled transiently into pili, conserved by retraction, and made available for subsequent reassembly. Growth in 0.01% sodium dodecyl sulfate did not accelerate loss of pilin from the Flac strain compared with the Flac tra strain, and we suggest that in the presence of sodium dodecyl sulfate at this concentration, F-pili are not elaborated from cell surfaces.     

Dual mechanism of bacterial lethality for a cationic sequence-random copolymer that mimics host-defense antimicrobial peptides

Flexible sequence-random polymers containing cationic and lipophilic subunits that act as functional mimics of host-defense peptides have recently been reported. We used bacteria and lipid vesicles to study one such polymer, having an average length of 21 residues, that is active against both Gram-positive and Gram-negative bacteria. At low concentrations, this polymer is able to permeabilize model anionic membranes that mimic the lipid composition of Escherichia coli, Staphylococcus aureus, or Bacillus subtilis but is ineffective against model zwitterionic membranes, which explains its low hemolytic activity. The polymer is capable of binding to negatively charged vesicles, inducing segregation of anionic lipids. The appearance of anionic lipid-rich domains results in formation of phase-boundary defects through which leakage can occur. We had earlier proposed such a mechanism of membrane disruption for another antimicrobial agent. Experiments with the mutant E. coli ML-35p indicate that permeabilization is biphasic: at low concentrations, the polymer permeabilizes the outer and inner membranes; at higher polymer concentrations, permeabilization of the outer membrane is progressively diminished, while the inner membrane remains unaffected. Experiments with wild-type E. coli K12 show that the polymer blocks passage of solutes into the intermembrane space at high concentrations. Cell membrane integrity in E. coli K12 and S. aureus exhibits biphasic dependence on polymer concentration. Isothermal titration calorimetry indicates that the polymer associates with the negatively charged lipopolysaccharide of Gram-negative bacteria and with the lipoteichoic acid of Gram-positive bacteria. We propose that this polymer has two mechanisms of antibacterial action, one predominating at low concentrations of polymer and the other predominating at high concentrations.     

Co-autodisplay of Z-domains and bovine caseins on the outer membrane of E. coli

In this work, two proteins, Z-domains and bovine casein, were autodisplayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different autodisplay vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12 × 10⁵ and 1.55 × 10⁵ proteins/E. coli cell, respectively. The co-autodisplayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-autodisplayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-autodisplayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-autodisplayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with autodisplayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements.     

Genomic landscape of single-stranded DNA gapped intermediates in Escherichia coli

Single-stranded (ss) gapped regions in bacterial genomes (gDNA) are formed on W- and C-strands during replication, repair, and recombination. Using non-denaturing bisulfite treatment to convert C to U on ssDNA, combined with deep sequencing, we have mapped gDNA gap locations, sizes, and distributions in Escherichia coli for cells grown in mid-log phase in the presence and absence of UV irradiation, and in stationary phase cells. The fraction of ssDNA on gDNA is similar for W- and C-strands, ∼1.3% for log phase cells, ∼4.8% for irradiated log phase cells, and ∼8.5% for stationary phase cells. After UV irradiation, gaps increased in numbers and average lengths. A monotonic reduction in ssDNA occurred symmetrically between the DNA replication origin of (OriC) and terminus (Ter) for log phase cells with and without UV, a hallmark feature of DNA replication. Stationary phase cells showed no OriC → Ter ssDNA gradient. We have identified a spatially diverse gapped DNA landscape containing thousands of highly enriched ‘hot’ ssDNA regions along with smaller numbers of ‘cold’ regions. This analysis can be used for a wide variety of conditions to map ssDNA gaps generated when DNA metabolic pathways have been altered, and to identify proteins bound in the gaps.     

A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.     

Dependence of Single-Stranded DNA Length on Stage of Growth Cycle in Escherichia coli B/r and Effect of Irradiation

Alkaline sucrose density gradient profiles of DNA from log phase Escherichia coli B/r (CSH) show a main peak with sedimentation coefficient at approximately 130S and a shoulder or second peak at approximately 90S. Incorporation of radioactive precursors into the 90S peak precedes their appearance in the main peak. The size of the second peak appears to be directly related to the rate of replication and it is not present in profiles of nondividing stationary phase cultures. The decrease in weight average molecular weight (Mw) of DNA produced by X-rays is also directly related to the rate of replication. It is greatest in log phase E. coli B/r and least in stationary phase cells, because of the efficiency of rejoining of radiation-induced single strand breaks in DNA of the latter cells.