Chromatin Liquid–Liquid Phase Separation (LLPS) Is Regulated by Ionic Conditions and Fiber Length

The dynamic regulation of the physical states of chromatin in the cell nucleus is crucial for maintaining cellular homeostasis. Chromatin can exist in solid- or liquid-like forms depending on the surrounding ions, binding proteins, post-translational modifications and many other factors. Several recent studies suggested that chromatin undergoes liquid–liquid phase separation (LLPS) in vitro and also in vivo; yet, controversial conclusions about the nature of chromatin LLPS were also observed from the in vitro studies. These inconsistencies are partially due to deviations in the in vitro buffer conditions that induce the condensation/aggregation of chromatin as well as to differences in chromatin (nucleosome array) constructs used in the studies. In this work, we present a detailed characterization of the effects of K+, Mg2+ and nucleosome fiber length on the physical state and property of reconstituted nucleosome arrays. LLPS was generally observed for shorter nucleosome arrays (15-197-601, reconstituted from 15 repeats of the Widom 601 DNA with 197 bp nucleosome repeat length) at physiological ion concentrations. In contrast, gel- or solid-like condensates were detected for the considerably longer 62-202-601 and lambda DNA (~48.5 kbp) nucleosome arrays under the same conditions. In addition, we demonstrated that the presence of reduced BSA and acetate buffer is not essential for the chromatin LLPS process. Overall, this study provides a comprehensive understanding of several factors regarding chromatin physical states and sheds light on the mechanism and biological relevance of chromatin phase separation in vivo

Carboranyl-Chlorin e6 as a Potent Antimicrobial Photosensitizer.

Antimicrobial photodynamic inactivation is currently being widely considered as alternative to antibiotic chemotherapy of infective diseases, attracting much attention to design of novel effective photosensitizers. Carboranyl-chlorin-e6 (the conjugate of chlorin e6 with carborane), applied here for the first time for antimicrobial photodynamic inactivation, appeared to be much stronger than chlorin e6 against Gram-positive bacteria, such as Bacillus subtilis, Staphyllococcus aureus and Mycobacterium sp. Confocal fluorescence spectroscopy and membrane leakage experiments indicated that bacteria cell death upon photodynamic treatment with carboranyl-chlorin-e6 is caused by loss of cell membrane integrity. The enhanced photobactericidal activity was attributed to the increased accumulation of the conjugate by bacterial cells, as evaluated both by centrifugation and fluorescence correlation spectroscopy. Gram-negative bacteria were rather resistant to antimicrobial photodynamic inactivation mediated by carboranyl-chlorin-e6. Unlike chlorin e6, the conjugate showed higher (compared to the wild-type strain) dark toxicity with Escherichia coli ΔtolC mutant, deficient in TolC-requiring multidrug efflux transporters

Combined Impact of Magnetic Force and Spaceflight Conditions on Escherichia coli Physiology

Changes in bacterial physiology caused by the combined action of the magnetic force and microgravity were studied in Escherichia coli grown using a specially developed device aboard the International Space Station. The morphology and metabolism of E. coli grown under spaceflight (SF) or combined spaceflight and magnetic force (SF + MF) conditions were compared with ground cultivated bacteria grown under standard (control) or magnetic force (MF) conditions. SF, SF + MF, and MF conditions provided the up-regulation of Ag43 auto-transporter and cell auto-aggregation. The magnetic force caused visible clustering of non-sedimenting bacteria that formed matrix-containing aggregates under SF + MF and MF conditions. Cell auto-aggregation was accompanied by up-regulation of glyoxylate shunt enzymes and Vitamin B12 transporter BtuB. Under SF and SF + MF but not MF conditions nutrition and oxygen limitations were manifested by the down-regulation of glycolysis and TCA enzymes and the up-regulation of methylglyoxal bypass. Bacteria grown under combined SF + MF conditions demonstrated superior up-regulation of enzymes of the methylglyoxal bypass and down-regulation of glycolysis and TCA enzymes compared to SF conditions, suggesting that the magnetic force strengthened the effects of microgravity on the bacterial metabolism. This strengthening appeared to be due to magnetic force-dependent bacterial clustering within a small volume that reinforced the effects of the microgravity-driven absence of convectional flows

Potassium permeability of BACE- and chlorin e₆-treated <i>B</i>. <i>subtilis</i> (A) and <i>E</i>. <i>coli</i> (B) cells after illumination measured by a potassium-selective electrode.

<p>Cells were incubated with photosensitizers for 10 min prior to illumination (4 J/cm²). Nigericin (1 μM) was added at the end of each recording to induce full potassium efflux.</p

Uptake of BACE or chlorin e₆ by <i>B</i>. <i>subtilis</i> (A) and <i>E</i>. <i>coli</i> (B) cells.

<p>Cells were incubated for 10 min in the dark with the indicated amount of a photosensitizer. The pellet obtained after centrifugation was treated with 0.1 M NaOH / 1% SDS. The photosensitizer concentration was determined from its fluorescence by using a calibration curve for the solutions of different concentrations in 0.1 M NaOH / 1% SDS.</p

Dark toxicity of BACE and chlorin e₆ towards WT <i>Escherichia coli</i> and the <i>E</i>. <i>coli</i> Δ<i>tolC</i> mutant.

<p>Chlorin e₆ or BACE (100 nM—50 μM) were added to bacterial cultures (1–5*10⁶ cells/ml), placed in 96-well plates. Cell density was determined by absorbance at 600 nm. After that bacteria were allowed to grow in the dark within 21 hours and cell density was measured again.</p

Photodynamic action on liposomes made from <i>E</i>. <i>coli</i> total lipids.

<p>Carboxyfluorescein leakage from liposomes induced by 1-min exposure to visible light (4 J/cm²) in the presence of BACE (red curve), chlorin e₆ (blue curve), and a control without illumination and photosensitizers (green curve). The solution was 100 mM KCl, 10 mM Tris, 10 mM MES, pH 7.0. Lipid concentration was 5 μg/ml.</p

Phototoxicity and dark toxicity of BACE and chlorin e₆ towards <i>Mycobacterium sp</i>. (A), <i>Staphyllococcus aureus</i> (B), and <i>Bacillus subtilis</i> (C) evaluated by CFU counts.

<p>Phototoxicity was determined upon illumination with red light at 4 J/cm². The data shown are mean values ± standard deviations.</p

Accumulation of BACE and chlorin e₆ by <i>B</i>. <i>subtilis</i> (A) and <i>E</i>. <i>coli</i> (B) cells monitored by FCS.

<p>Fluorescence intensity traces of photosensitizers were recorded with the FCS set-up in the presence or absence of bacterial cells (10⁶ per ml). <i>Inserts</i>: Corresponding dependences of the number of peaks with the fluorescence intensity <i>F</i> exceeding the threshold <i>F</i>_<i>0</i>, <i>n(F>F</i>_<i>0</i><i>)</i>, on the value of <i>F</i>_<i>0</i>.</p