Showing papers on "Lysis published in 2021"

Recovery of Polyhydroxyalkanoates From Single and Mixed Microbial Cultures: A Review

Abstract: An overview of the main polyhydroxyalkanoates (PHA) recovery methods is here reported, by considering the kind of PHA-producing bacteria (single bacterial strains or mixed microbial cultures) and the chemico-physical characteristics of the extracted polymer (molecular weight and polydispersity index). Several recovery approaches are presented and categorized in two main strategies: PHA recovery with solvents (halogenated solvents, alkanes, alcohols, esters, carbonates and ketones) and PHA recovery by cellular lysis (with oxidants, acid and alkaline compounds, surfactants and enzymes). Comparative evaluations based on the recovery, purity and molecular weight of the recovered polymers as well as on the potential sustainability of the different approaches are here presented.

Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings.

Abstract: The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In this study, we evaluated lysis buffers that are commonly used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. In addition, viral stability in cell culture media at 4 °C and on display glass and plastic surfaces used in laboratory environment was analyzed. Furthermore, we evaluated chemical and non-chemical inactivation methods including heat inactivation, UV-C light, addition of ethanol, acetone-methanol, and PFA, which might be used as a subsequent inactivation step in the case of insufficient inactivation. We infected susceptible Caco-2 and Vero cells with pre-treated SARS-CoV-2 and determined the tissue culture infection dose 50 (TCID50) using crystal violet staining and microscopy. In addition, lysates of infected cells and virus containing supernatant were subjected to RT-qPCR analysis. We have found that guanidine thiocyanate and most of the tested detergent containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate the virus. In conclusion, careful evaluation of the used inactivation methods is required especially for non-denaturing buffers. Additional inactivation steps might be necessary before removal of lysed viral samples from BSL-3.

Autolysis-mediated membrane vesicle formation in Bacillus subtilis

Abstract: It is known that Bacillus subtilis releases membrane vesicles (MVs) during the SOS response, which is associated with cell lysis triggered by the PBSX prophage-encoded cell-lytic enzymes XhlAB and XlyA. In this study, we demonstrate that MVs are released under various stress conditions: sucrose fatty acid ester (SFE; surfactant) treatment, cold shock, starvation, and oxygen deficiency. B. subtilis possesses four major host-encoded cell wall-lytic enzymes (autolysins; LytC, LytD, LytE, and LytF). Deletions of the autolysin genes abolished autolysis and the consequent MV production under these stress conditions. In contrast, deletions of xhlAB and xlyA had no effect on autolysis-triggered MV biogenesis, indicating that autolysis is a novel and prophage-independent pathway for MV production in B. subtilis. Moreover, we found that the cell lysis induced by the surfactant treatment was effectively neutralized by the addition of exogenous purified MVs. This result suggests that the MVs can serve as a decoy for the cellular membrane to protect the living cells in the culture from membrane damage by the surfactant. Our results indicate a positive effect of B. subtilis MVs on cell viability and provide new insight into the biological importance of the autolysis phenomenon in B. subtilis.

Improved degradation of azo dyes by lignin peroxidase following mutagenesis at two sites near the catalytic pocket and the application of peroxidase-coated yeast cell walls

Abstract: The enzymatic degradation of azo dyes is a promising alternative to ineffective chemical and physical remediation methods. Lignin peroxidase (LiP) from Phanerochaete chrysosporium is a heme-containing lignin-degrading oxidoreductase that catalyzes the peroxide-dependent oxidation of diverse molecules, including industrial dyes. This enzyme is therefore ideal as a starting point for protein engineering. Accordingly, we subjected two positions (165 and 264) in the environment of the catalytic Trp171 residue to saturation mutagenesis, and the resulting library of 104 independent clones was expressed on the surface of yeast cells. This yeast display library was used for the selection of variants with the ability to break down structurally-distinct azo dyes more efficiently. We identified mutants with up to 10-fold greater affinity than wild-type LiP for three diverse azo dyes (Evans blue, amido black 10B and Guinea green) and up to 13-fold higher catalytic activity. Additionally, cell wall fragments displaying mutant LiP enzymes were prepared by toluene-induced cell lysis, achieving significant increases in both enzyme activity and stability compared to a whole-cell biocatalyst. LiP-coated cell wall fragments retained their initial dye degradation activity after 10 reaction cycles each lasting 8 h. The best-performing mutants removed up to 2.5-fold more of each dye than the wild-type LiP in multiple reaction cycles.

Bacteriophage infection of Escherichia coli leads to the formation of membrane vesicles via both explosive cell lysis and membrane blebbing

Abstract: Membrane vesicles (MVs) are membrane-bound spherical nanostructures that prevail in all three domains of life. In Gram-negative bacteria, MVs are thought to be produced through blebbing of the outer membrane and are often referred to as outer membrane vesicles (OMVs). We have recently described another mechanism of MV formation in Pseudomonas aeruginosa that involves explosive cell-lysis events, which shatters cellular membranes into fragments that rapidly anneal into MVs. Interestingly, MVs are often observed within preparations of lytic bacteriophage, however the source of these MVs and their association with bacteriophage infection has not been explored. In this study we aimed to determine if MV formation is associated with lytic bacteriophage infection. Live super-resolution microscopy demonstrated that explosive cell lysis of Escherichia coli cells infected with either bacteriophage T4 or T7, resulted in the formation of MVs derived from shattered membrane fragments. Infection by either bacteriophage was also associated with the formation of membrane blebs on intact bacteria. TEM revealed multiple classes of MVs within phage lysates, consistent with multiple mechanisms of MV formation. These findings suggest that bacteriophage infection may be a major contributor to the abundance of bacterial MVs in nature.

Cavitation-microstreaming-based lysis and DNA extraction using a laser-machined polycarbonate microfluidic chip

Abstract: We for the first time present a microfluidic cavitation-microstreaming-based cell lysis and DNA extraction method. Chemical lysis and DNA extraction have been demonstrated in a microfluidic format but the performance is limited by ineffective mass transport due to low Reynolds number. Here we propose to employ cavitation microstreaming for enhancing chemical lysis and magnetic-bead-based dynamic solid phase extraction (dSPE) of DNA. Cavitation microstreaming condition is optimized by exciting a microfluidic chip at its flexural resonance frequency (fr) measured via electrical impedance spectroscopy. Strong circulatory flows around bubbles excited at fr yields vigorous agitation, allowing fast lysis, and DNA extraction and purification. The microfluidic device is rapidly fabricated using CO2-laser machining and solvent-assisted thermal bonding of polycarbonate (∼25 min). Laser cutting conditions are experimentally determined to achieve a clean sidewall for negligible nonspecific binding and minimal burrs for unobstructed bonding. Solvent exposure and thermal bonding conditions are also experimentally determined for a leakage-free device with excellent dimensional integrity. Our method, although not fully optimized, exhibits an excellent DNA extraction performance, compared to a commercial kit and previous microfluidic methods. High extraction efficiency (76.9 %) and purity (A260/A280 = 1.85) are achieved for a relatively short assay time (∼25 min). Notably, DNA from as few as 18 cells is successfully extracted even from a highly diluted cell sample (0.18 cells/μl). PCR and electrophoresis results confirm the excellent quality of the extracted DNA. Considering these notable performances, and straightforward fabrication and operation, we anticipate our DNA extraction method will be widely used in microfluidic nucleic-acid analysis devices.

Genetic targeting of Card19 is linked to disrupted NINJ1 expression, impaired cell lysis, and increased susceptibility to Yersinia infection

Abstract: Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1β release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as an independently-generated CRISPR/Cas9 Card19 knockout mouse line (Card19Null) showed no defect in macrophage cell lysis. Notably, Card19 is located on chromosome 13, immediately adjacent to Ninj1, which was recently found to regulate cell lysis downstream of GSDMD activation. RNA-seq and western blotting revealed that Card19lxcn BMDMs have significantly reduced NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored their ability to undergo cell lysis in response to caspase-dependent cell death stimuli. Card19lxcn mice exhibited increased susceptibility to Yersinia infection, whereas independently-generated Card19Null mice did not, demonstrating that cell lysis itself plays a key role in protection against bacterial infection, and that the increased infection susceptibility of Card19lxcn mice is attributable to loss of NINJ1. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent gene Ninj1, disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection.

Evaluation of an E. coli Cell Extract Prepared by Lysozyme-Assisted Sonication via Gene Expression, Phage Assembly and Proteomics.

Abstract: Over the past decades, starting from crude cell extracts, a variety of successful preparation protocols and optimized reaction conditions have been established for the production of cell-free gene expression systems. One of the crucial steps during the preparation of cell extract-based expression systems is the cell lysis procedure itself, which largely determines the quality of the active components of the extract. Here we evaluate the utility of an E. coli cell extract, which was prepared using a combination of lysozyme incubation and a gentle sonication step. As quality measure, we demonstrate the cell-free expression of YFP at concentrations up to 0.6 mg/ml. In addition, we produced and assembled T7 bacteriophages up to a titer of 10 8 PFU/ml. State-of-the-art quantitative proteomics was used to compare the produced extracts with each other and with a commercial extract. The differences in protein composition were surprisingly small between lysozyme-assisted sonication (LAS) extracts, but we observed an increase in the release of DNA-binding proteins for increasing numbers of sonication cycles. Proteins taking part in carbohydrate metabolism, glycolysis, amino acid and nucleotide related pathways were found to be more abundant in the LAS extract, while proteins related to RNA modification and processing, DNA modification and replication, transcription regulation, initiation, termination and the TCA cycle were found enriched in the commercial extract.

One-step selective affinity purification and immobilization of His-tagged enzyme by recyclable magnetic nanoparticles.

Abstract: The NiFe2O4 magnetic nanoparticles (NF-MNPs) were prepared for one-step selective affinity purification and immobilization of His-tagged recombinant glucose dehydrogenase (GluDH). The prepared nanoparticles were characterized by a Fourier-transform infrared spectrophotometer and microscopy. The immobilization and purification of His-tagged GluDH on NF-MNPs were investigated. The optimal immobilization conditions were obtained that mixed cell lysis and carriers in a ratio of 0.13 in pH 8.0 Tris-HCl buffer at 30℃ and incubated for 2 h. The highest activity recovery and protein bindings were 71.39% and 38.50 μg mg-1 support, respectively. The immobilized GluDH exhibited high thermostability, pH-stability and it can retain more than 65% of the initial enzyme after 10 cycles for the conversion of glucose to gluconolactone. Comparing with a commercial Ni-NTA resin, the NF-MNPs displayed a higher specific affinity with His-tagged recombinant GluDH.

Direct Observation of Conversion From Walled Cells to Wall-Deficient L-Form and Vice Versa in Escherichia coli Indicates the Essentiality of the Outer Membrane for Proliferation of L-Form Cells.

Abstract: Gram-negative bacteria such as Escherichia coli are surrounded by an outer membrane, which encloses a peptidoglycan layer. Even if thinner than in many Gram-positive bacteria, the peptidoglycan in E. coli allows cells to withstand turgor pressure in hypotonic medium. In hypertonic medium, E. coli treated with a cell wall synthesis inhibitor such as penicillin G form wall-deficient cells. These so-called L-form cells grow well under anaerobic conditions (i.e., in the absence of oxidative stress), becoming deformed and dividing as L-form. Upon removal of the inhibitor, they return to the walled rod-shaped state. Recently, the outer membrane was reported to provide rigidity to Gram-negative bacteria and to strengthen wall-deficient cells. However, it remains unclear why L-form cells need the outer membrane for growth. Using a microfluidic system, we found that, upon treatment with the outer membrane-disrupting drugs polymyxin B and polymyxin B nonapeptide or with the outer membrane synthesis inhibitor CHIR-090, the cells lysed during cell deformation and division, indicating that the outer membrane was important even in hypertonic medium. L-form cells could return to rod-shaped when trapped in a narrow space, but not in a wide space, likely due to insufficient physical force. Outer membrane rigidity could be compromised by lack of outer membrane proteins; Lpp, OmpA, or Pal. Deletion of lpp caused cells to lyse during cell deformation and cell division. In contrast, ompA and pal mutants could be deformed and return to small oval cells even when less physical force was exerted. These results strongly suggest that wall-deficient E. coli cells require a rigid outer membrane to survive, but not too rigid to prevent them from changing cell shape.

Understanding Beta-Lactam-Induced Lysis at the Single-Cell Level.

Abstract: Mechanical rupture, or lysis, of the cytoplasmic membrane is a common cell death pathway in bacteria occurring in response to β-lactam antibiotics. A better understanding of the cellular design principles governing the susceptibility and response of individual cells to lysis could indicate methods of potentiating β-lactam antibiotics and clarify relevant aspects of cellular physiology. Here, we take a single-cell approach to bacterial cell lysis to examine three cellular features-turgor pressure, mechanosensitive channels, and cell shape changes-that are expected to modulate lysis. We develop a mechanical model of bacterial cell lysis and experimentally analyze the dynamics of lysis in hundreds of single Escherichia coli cells. We find that turgor pressure is the only factor, of these three cellular features, which robustly modulates lysis. We show that mechanosensitive channels do not modulate lysis due to insufficiently fast solute outflow, and that cell shape changes result in more severe cellular lesions but do not influence the dynamics of lysis. These results inform a single-cell view of bacterial cell lysis and underscore approaches of combatting antibiotic tolerance to β-lactams aimed at targeting cellular turgor.

High Efficiency RNA Extraction From Sperm Cells Using Guanidinium Thiocyanate Supplemented With Tris(2-Carboxyethyl)Phosphine.

Abstract: The extraction of high-quality ribonucleic acid (RNA) from tissues and cells is a key step in many biological assays. Guanidinium thiocyanate-phenol-chloroform (AGPC) is a widely used and efficient method to obtain pure RNA from most tissues and cells. However, it is not efficient with some cells like sperm cells because they are resistant to chaotropic lysis solutions containing guanidinium thiocyanate such as Buffer RLT+ and Trizol. Here, we show that disulfide bonds are responsible for the chemical resistance of sperm cells to RNA extraction reagents. We show that while β-mercaptoethanol (βME) can increase sperm lysis in Buffer RLT+, it has no effect in Trizol and leaves sperm cells intact. We measured the reduction of disulfide bonds in 2,2'-dithiodipyridine (DTDP) and observed that βME has a pH-dependent activity in chaotropic solutions, suggesting that pH is a limiting factor. We identified tris(2-carboxyethyl)phosphine (TCEP) as an efficient lysis enhancer of AGPC solutions that can retain reducing activity even at acidic pH. Trizol supplemented with TCEP allows the complete and rapid lysis of sperm cells, increasing RNA yield by 100-fold and resulting in RNA with optimal quality for reverse transcription and polymerase chain reaction. Our findings highlight the importance of efficient cell lysis and extraction of various macromolecules for bulk and single-cell assays, and can be applied to other lysis-resistant cells and vesicles, thereby optimizing the amount of required starting material and animals.

Phage-Mediated Explosive Cell Lysis Induces the Formation of a Different Type of O-IMV in Shewanella vesiculosa M7T.

Abstract: Shewanella vesiculosa M7T is a cold-adapted Antarctic bacterium that has a great capacity to secrete membrane vesicles (MVs), making it a potentially excellent model for studying the vesiculation process. S. vesiculosa M7T undergoes a blebbing mechanism to produce different types of MVs, including outer membrane vesicles and outer-inner membrane vesicles (O-IMVs). More recently, other mechanisms have been considered that could lead to the formation of O-IMVs derived from prophage-mediated explosive cell lysis in other bacteria, but it is not clear if they are of the same type. The bacterial growth phase could also have a great impact on the type of MVs, although there are few studies on the subject. In this study, we used high-resolution flow cytometry, transmission electron microscopy, and cryo-electron microscopy (Cryo-EM) analysis to determine the amount and types of MVs S. vesiculosa M7T secreted during different growth phases. We show that MV secretion increases during the transition from the late exponential to the stationary phase. Moreover, prophage-mediated explosive cell lysis is activated in S. vesiculosa M7T, increasing the heterogeneity of both single- and double-layer MVs. The sequenced DNA fragments from the MVs covered the entire genome, confirming this explosive cell lysis mechanism. A different structure and biogenesis mechanisms for the explosive cell lysis-derived double-layered MVs was observed, and we propose to name them explosive O-IMVs, distinguishing them from the blebbing O-IMVs; their separation is a first step to elucidate their different functions. In our study, we used for the first time sorting by flow cytometry and Cryo-EM analyses to isolate bacterial MVs based on their nucleic acid content. Further improvements and implementation of bacterial MV separation techniques is essential to develop more in-depth knowledge of MVs.

Co-Cultivation of Two Bacillus Strains for Improved Cell Growth and Enzyme Production to Enhance the Degradation of Aflatoxin B1.

Abstract: Bacillus sp. H16v8 and Bacillus sp. HGD9229 were identified as Aflatoxin B1 (AFB1) degrader in nutrient broth after a 12 h incubation at 37 °C. The degradation efficiency of the two-strain supernatant on 100 μg/L AFB1 was higher than the bacterial cells and cell lysate. Moreover, degradations of AFB1 were strongly affected by the metal ions in which Cu2+ stimulated the degradation and Zn2+ inhibited the degradation. The extracellular detoxifying enzymes produced by co-cultivation of two strains were isolated and purified by ultrafiltration. The molecular weight range of the detoxifying enzymes was 20–25 kDa by SDS-PAGE. The co-culture of two strains improved the total cell growth with the enhancement of the total protein content and detoxifying enzyme production. The degradation efficiency of the supernatant from mixed cultures increased by 87.7% and 55.3% compared to Bacillus sp. H16v8 and HGD9229, individually. Moreover, after the degradation of AFB1, the four products of the lower toxicity were identified by LC-Triple TOF-MS with the two proposed hypothetical degradation pathways.

Investigation of the Effect of Channel Structure and Flow Rate on On-Chip Bacterial Lysis

Abstract: Successful lysis of cells/microorganisms is a key step in the sample preparation in fields like molecular biology, bioengineering, and biomedical engineering. This study therefore aims to investigate the lysis of bacteria on-chip and its dependence on both microfluidic channel structure and flow rate. Effects of temperature on lysis on-chip were also investigated. To perform these investigations, three different microfluidic chips were designed and produced (straight, zigzag and circular configurations), while the length of the channels were kept constant. As an exemplary case, Mycobacterium smegmatis was chosen to represent the acid-fast bacteria. Bacterial suspensions of 1.5 McFarland were injected into the chips at various flow rates (0.6- $8~\mu \text{l}$ /min) either at room temperature or 50° C. In order to understand the on-chip lysis performance fully, off-chip experiments were carried out at durations which are equal to those bacteria spent in the channel from inlet to the outlet at different flow rates. We also performed COMSOL multiphysics program simulations to evaluate further the effect of the applied parameters. As a result, we found that the structure and the flow rate do not affect lysis over all in all investigated channel types, however on-chip experiments at room temperature produced more effective lysis compared to the on-chip and the off-chip samples performed at higher temperatures. Interestingly on-chip experiments at higher tempratures do not result in effective lysis.

Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness.

Abstract: With the growing interest in continuous cultivation of Escherichia coli, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, it is shown that induced decoupling of growth and heterologous gene expression in the E. coli X-press strain (derived from BL21(DE3)) facilitates extracellular recombinant protein production. The effect of the process parameters temperature and specific glucose consumption rate (qS ) on growth, productivity, lysis and leakiness, is investigated, to find the parameter space allowing extracellular protein production. Two model proteins are used, Protein A (SpA) and a heavy-chain single-domain antibody (VHH), and performance is compared to the industrial standard strain BL21(DE3). It is shown that inducible growth repression in the X-press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and qS are used to control productivity and leakiness. In the X-press strain, extracellular SpA and VHH titer reach up to 349 and 19.6 mg g-1 , respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum. The findings demonstrate that the X-press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.

Microbial Photoinactivation by Visible Light Results in Limited Loss of Membrane Integrity

Abstract: Interest in visible light irradiation as a microbial inactivation method has widely increased due to multiple possible applications. Resistance development is considered unlikely, because of the multi-target mechanism, based on the induction of reactive oxygen species by wavelength specific photosensitizers. However, the affected targets are still not completely identified. We investigated membrane integrity with the fluorescence staining kit LIVE/DEAD® BacLight™ on a Gram positive and a Gram negative bacterial species, irradiating Staphylococcus carnosus and Pseudomonas fluorescens with 405 nm and 450 nm. To exclude the generation of viable but nonculturable (VBNC) bacterial cells, we applied an ATP test, measuring the loss of vitality. Pronounced uptake of propidium iodide was only observed in Pseudomonas fluorescens at 405 nm. Transmission electron micrographs revealed no obvious differences between irradiated samples and controls, especially no indication of an increased bacterial cell lysis could be observed. Based on our results and previous literature, we suggest that visible light photoinactivation does not lead to rapid bacterial cell lysis or disruption. However, functional loss of membrane integrity due to depolarization or inactivation of membrane proteins may occur. Decomposition of the bacterial envelope following cell death might be responsible for observations of intracellular component leakage.

Comparative Assessment of Physical and Chemical Cyanobacteria Cell Lysis Methods for Total Microcystin-LR Analysis.

Abstract: Standardization and validation of alternative cell lysis methods used for quantifying total cyanotoxins is needed to improve laboratory response time goals for total cyanotoxin analysis. In this study, five cell lysis methods (i.e., probe sonication, microwave, freeze-thaw, chemical lysis with Abraxis QuikLyseTM, and chemical lysis with copper sulfate) were assessed using laboratory-cultured Microcystis aeruginosa (M. aeruginosa) cells. Methods were evaluated for destruction of cells (as determined by optical density of the sample) and recovery of total microcystin-LR (MC-LR) using three M. aeruginosa cell densities (i.e., 1 × 105 cells/mL (low-density), 1 × 106 cells/mL (medium-density), and 1 × 107 cells/mL (high-density)). Of the physical lysis methods, both freeze-thaw (1 to 5 cycles) and pulsed probe sonication (2 to 10 min) resulted in >80% destruction of cells and consistent (>80%) release and recovery of intracellular MC-LR. Microwave (3 to 5 min) did not demonstrate the same decrease in optical density ( 80% intracellular MC-LR. Abraxis QuikLyseTM was similarly effective for intracellular MC-LR recovery across the different M. aeruginosa cell densities. Copper sulfate (up to 500 mg/L Cu2+) did not lyse cells nor release intracellular MC-LR within 20 min. None of the methods appeared to cause degradation of MC-LR. Probe sonication, microwave, and Abraxis QuikLyseTM served as rapid lysis methods (within minutes) with varying associated costs, while freeze-thaw provided a viable, low-cost alternative if time permits.

Incorporation of Plasmid DNA Into Bacterial Membrane Vesicles by Peptidoglycan Defects in Escherichia coli

Abstract: Membrane vesicles (MVs) are released by various prokaryotes and play a role in the delivery of various cell-cell interaction factors. Recent studies have determined that these vesicles are capable of functioning as mediators of horizontal gene transfer. Outer membrane vesicles (OMVs) are a type of MV that is released by Gram-negative bacteria and primarily composed of outer membrane and periplasm components; however, it remains largely unknown why DNA is contained within OMVs. Our study aimed to understand the mechanism by which DNA that is localized in the cytoplasm is incorporated into OMVs in Gram-negative bacteria. We compared DNA associated with OMVs using Escherichia coli BW25113 cells harboring the non-conjugative, non-mobilized, and high-copy plasmid pUC19 and its hypervesiculating mutants that included ΔnlpI, ΔrseA and ΔtolA. Plasmid copy per vesicle was increased in OMVs derived from ΔnlpI, in which peptidoglycan (PG) breakdown and synthesis are altered. When supplemented with 1% glycine to inhibit PG synthesis, both OMV formation and plasmid copy per vesicle were increased in the wild type. The bacterial membrane condition test indicated that membrane permeability was increased in the presence of glycine at the exponential phase, in which cell lysis did not occur. Additionally, quick-freeze deep-etch and replica electron microscopy observations revealed that outer-inner membrane vesicles (O-IMVs) are formed in the presence of glycine. Thus, two proposed routes for DNA incorporation into OMVs under PG-damaged conditions are suggested. These routes include DNA leakage due to increased membrane permeation and O-IMV formation. Additionally, our findings contribute to a greater understanding of the vesicle-mediated horizontal gene transfer that occurs in nature and the utilization of MVs for DNA cargo.

Photocatalytic Bactericidal Performance of LaFeO3 under Solar Light: Kinetics, Spectroscopic and Mechanistic Evaluation

Abstract: Lanthanum orthoferrites are a versatile class of catalysts. Here, the photocatalytic bactericidal performance of LaFeO3 (LF) to inactivate pathogenic microorganisms, i.e., Escherichia coli (E. coli), in water under simulated solar irradiation conditions was investigated. Various competing and contributing factors were covered to visualize the reaction medium consisting of E. coli K12 cells, organic sub-fractions formed by cell destruction, and LF surface. LF solar photocatalytic inactivation (SPCI) kinetics revealed the highest inactivation rate in ultrapure water as expected, followed by distilled water (DW), aqueous solution containing anions and cations (WM) and saline solution (SS). Characterization of the released organic matter was achieved by UV-vis and fluorescence spectroscopic techniques as well as organic carbon contents (DOC). Upon SPCI, significant amounts of K+ along with released protein contents were detected expressing cell wall destruction and lysis. Under the specified experimental conditions, in the presence of released intracellular organic and inorganic components via cell lysis, a significant count of E. coli was still present in SS, whereas almost all bacteria were removed in other matrices due to various challenging reasons. Based on the presented data, SPCI of E. coli using LF as a novel photocatalyst was successfully demonstrated as an alternative and promising method for disinfection purposes.

Recovery and Purification of Protein Aggregates From Cell Lysates Using Ceramic Membranes: Fouling Analysis and Modeling of Ultrafiltration

Abstract: The characterization of membrane fouling provides valuable information about the performance and operational range of filtration processes. The range of operational parameters for the purification and concentration of protein aggregates from cell lysates by ultrafiltration is determined by evaluating the filtration resistances. We therefore investigated the cross-flow ultrafiltration of ovalbumin aggregates with a mean size of 304 nm using a 50 nm cut-off ceramic membrane. We observed a 90% decline in flux within the first 10 min of filtration, demanding an in-depth analysis of membrane fouling. Resistance-in-series analysis revealed that the main filtration resistance originated from the cell lysate in the feed solution. Flux decline was monitored at different transmembrane pressures and concentrations for the most significant fouling phenomenon, indicating that the intermediate pore blocking model correlated best with the observed filtration data. The transmembrane pressure for purification and concentration was set at 1.5 bar based on the prediction of a limited, mostly pressure-independent flux of 12 L·m 2·h 1 for solutions with an ovalbumin aggregate concentration of 0.5 g·L-1. Higher pressure increased the filtration performance only slightly, but led to a linear increase in filtration resistance. A 10-fold variation in protein aggregate concentration strongly influenced filtration performance, with higher protein concentrations increasing the filtration resistance by 413% and causing an 85% decline in flux.