Showing papers on "Lysis published in 2019"
TL;DR: Evidence is reviewed supporting a model in which the spanins function by fusing the inner membrane and outer membrane, and it is proposed that spanin function is inhibited by the meshwork of the peptidoglycan, thus coupling the spanin step to the first two steps mediated by the holin and endolysin.
Abstract: The first steps in phage lysis involve a temporally controlled permeabilization of the cytoplasmic membrane followed by enzymatic degradation of the peptidoglycan. For Caudovirales of Gram-negative hosts, there are two different systems: the holin-endolysin and pinholin-SAR endolysin pathways. In the former, lysis is initiated when the holin forms micron-scale holes in the inner membrane, releasing active endolysin into the periplasm to degrade the peptidoglycan. In the latter, lysis begins when the pinholin causes depolarization of the membrane, which activates the secreted SAR endolysin. Historically, the disruption of the first two barriers of the cell envelope was thought to be necessary and sufficient for lysis of Gram-negative hosts. However, recently a third functional class of lysis proteins, the spanins, has been shown to be required for outer membrane disruption. Spanins are so named because they form a protein bridge that connects both membranes. Most phages produce a two-component spanin complex, composed of an outer membrane lipoprotein (o-spanin) and an inner membrane protein (i-spanin) with a predominantly coiled-coil periplasmic domain. Some phages have a different type of spanin which spans the periplasm as a single molecule, by virtue of an N-terminal lipoprotein signal and a C-terminal transmembrane domain. Evidence is reviewed supporting a model in which the spanins function by fusing the inner membrane and outer membrane. Moreover, it is proposed that spanin function is inhibited by the meshwork of the peptidoglycan, thus coupling the spanin step to the first two steps mediated by the holin and endolysin.
140 citations
TL;DR: This work shows that Escherichia coli cells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG.
Abstract: Gram-negative bacteria have a tripartite cell envelope with the cytoplasmic membrane (CM), a stress-bearing peptidoglycan (PG) layer, and the asymmetric outer membrane (OM) containing lipopolysaccharide (LPS) in the outer leaflet. Cells must tightly coordinate the growth of their complex envelope to maintain cellular integrity and OM permeability barrier function. The biogenesis of PG and LPS relies on specialized macromolecular complexes that span the entire envelope. In this work, we show that Escherichia coli cells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG. This PG remodeling program relies mainly on the activities of the stress response LDT, LdtD, together with the major PG synthase PBP1B, its cognate activator LpoB, and the carboxypeptidase PBP6a. Our data support a model according to which these proteins cooperate to strengthen the PG in response to defective OM synthesis.IMPORTANCE In Gram-negative bacteria, the outer membrane protects the cell against many toxic molecules, and the peptidoglycan layer provides protection against osmotic challenges, allowing bacterial cells to survive in changing environments. Maintaining cell envelope integrity is therefore a question of life or death for a bacterial cell. Here we show that Escherichia coli cells activate the LD-transpeptidase LdtD to introduce 3-3 cross-links in the peptidoglycan layer when the integrity of the outer membrane is compromised, and this response is required to avoid cell lysis. This peptidoglycan remodeling program is a strategy to increase the overall robustness of the bacterial cell envelope in response to defects in the outer membrane.
106 citations
TL;DR: Because PHA-rich bacterial cells are resistant to osmotic imbalances, they could be utilized in in-situ bioremediation technologies or during enrichment of mixed microbial consortia in PHA producers under conditions of fluctuating salinity.
65 citations
TL;DR: In this article, seven methods were compared to extract extracellular polymeric substances (EPSs) from anaerobic ammonium oxidation (anammox) granules, and the effectiveness and cell lysis of these methods were evaluated.
60 citations
TL;DR: The reports involving passive techniques that are able to extract lipid from microalgae without significant cell wall shearing are discussed and the economics of microalgal lipid extraction using passive disruption methods are analyzed.
55 citations
TL;DR: Results indicated that lysozyme boosted the ES hydrolysis significantly with approximately 236.5 mg/L soluble chemical oxygen demand (SCOD), 58.6 gr/L polysaccharide and 662.7 mg/L protein release within 240 min at the lyso enzyme dosage of 150 MG/gSS, and fungi were barely influenced by the enzymatic catalysis.
43 citations
TL;DR: It is shown that alterations in surface polymers called teichoic acids (TAs) play a key role in penicillin-induced lysis of the Gram-positive pathogen Streptococcus pneumoniae (Sp), and changes in surface polymer assembly may underlie the mechanism of antibiotic- and/or growth phase- induced lysis for other important Gram- positive pathogens.
Abstract: Penicillin and related antibiotics disrupt cell wall synthesis to induce bacteriolysis. Lysis in response to these drugs requires the activity of cell wall hydrolases called autolysins, but how penicillins misactivate these deadly enzymes has long remained unclear. Here, we show that alterations in surface polymers called teichoic acids (TAs) play a key role in penicillin-induced lysis of the Gram-positive pathogen Streptococcus pneumoniae (Sp). We find that during exponential growth, Sp cells primarily produce lipid-anchored TAs called lipoteichoic acids (LTAs) that bind and sequester the major autolysin LytA. However, penicillin-treatment or prolonged stationary phase growth triggers the degradation of a key LTA synthase, causing a switch to the production of wall-anchored TAs (WTAs). This change allows LytA to associate with and degrade its cell wall substrate, thus promoting osmotic lysis. Similar changes in surface polymer assembly may underlie the mechanism of antibiotic- and/or growth phase-induced lysis for other important Gram-positive pathogens.
41 citations
TL;DR: The history of lysis inhibition, its laboratory manifestation, its molecular basis, how it may benefit expressing phages, and its potential ecological role are considered.
Abstract: That communication can occur between virus-infected cells has been appreciated for nearly as long as has virus molecular biology. The original virus communication process specifically was that seen with T-even bacteriophages—phages T2, T4, and T6—resulting in what was labeled as a lysis inhibition. Another proposed virus communication phenomenon, also seen with T-even phages, can be described as a phage-adsorption-induced synchronized lysis-inhibition collapse. Both are mediated by virions that were released from earlier-lysing, phage-infected bacteria. Each may represent ecological responses, in terms of phage lysis timing, to high local densities of phage-infected bacteria, but for lysis inhibition also to locally reduced densities of phage-uninfected bacteria. With lysis inhibition, the outcome is a temporary avoidance of lysis, i.e., a lysis delay, resulting in increased numbers of virions (greater burst size). Synchronized lysis-inhibition collapse, by contrast, is an accelerated lysis which is imposed upon phage-infected bacteria by virions that have been lytically released from other phage-infected bacteria. Here I consider some history of lysis inhibition, its laboratory manifestation, its molecular basis, how it may benefit expressing phages, and its potential ecological role. I discuss as well other, more recently recognized examples of virus-virus intercellular communication.
39 citations
TL;DR: The effectiveness of FAC6 against L. monocytogenes is highlighted, suggesting that FAC6 with both antioxidant and antibacterial activities can be used as an effective and safe multifunctional food additive for American (Amer) sturgeon caviar preservation.
39 citations
TL;DR: This work successfully combine thermal lysis and loop-mediated isothermal amplification (LAMP) into a single reaction step on paper, making this integrated assay safe and suitable for incorporation into diverse paperfluidic sensors for field use.
Abstract: Developing sensors for food safety, soil analysis, water quality monitoring and healthcare often requires distinguishing between different species of bacteria. The most rapid, sensitive and specific method to identify bacteria is to analyse their DNA sequence, which comprises of disinfection and lysis of bacterial cells, amplification of the isolated DNA and detection of the amplified sequence. Seamless integration of these steps on a paper substrate is a big challenge. This problem is even more complex for mycobacteria as its thick cell wall structure impedes lysis and the high GC-content of the genome requires careful optimization of enzymatic denaturation. Here we successfully combine thermal lysis and loop-mediated isothermal amplification (LAMP) into a single reaction step on paper. We demonstrate our integrated assay by amplifying DNA from 100 CFU/mL of Escherichia coli (MG1655) and Mycobacterium smegmatis (mc2155) cells in 30 min on a paper substrate. We also confirm that E. coli and M. smegmatis can be completely disinfected on paper by heating at 60 °C for 5 min and 15 min respectively, making this assay safe and suitable for incorporation into diverse paperfluidic sensors for field use.
28 citations
TL;DR: The final protocol for DNA extraction using the two ILs is very low-cost, avoids the use of hazardous chemicals and can be performed in five minutes on a simple heating block, which makes the method ideal for high sample throughput and offers the opportunity forDNA extraction from bacteria in resource-limited settings or even in the field.
Abstract: The extraction of nucleic acids from microorganisms for subsequent molecular diagnostic applications is still a tedious and time-consuming procedure. We developed a method for the rapid preparation of genomic DNA from bacteria based on hydrophilic ionic liquids (ILs). First, we tested eight ILs in different buffer systems for their inhibitory effects on quantitative PCR. The cell lysis potential of different IL/buffer combinations was assessed by application on Enterococcus faecalis as a model organism for Gram-positive bacteria. The two best ILs, choline hexanoate and 1-ethyl-3-methylimidazolium acetate, were compared with the reference enzymatic method and two commercial DNA extraction kits. All methods were evaluated on four Gram-positive and four Gram-negative bacterial species that are highly relevant for environmental, food, or clinical diagnostics. In comparison to the reference method, extraction yields of the IL-based procedure were within one order of magnitude for most of the strains. The final protocol for DNA extraction using the two ILs is very low-cost, avoids the use of hazardous chemicals and can be performed in five minutes on a simple heating block. This makes the method ideal for high sample throughput and offers the opportunity for DNA extraction from bacteria in resource-limited settings or even in the field.
TL;DR: Enzymatic pretreatment was effective at damaging microalgae cell wall, releasing organic compounds and increasing the rate and final methane yield in BMP tests, and observed a synergistic activity between the mixtures enzymes, which would depend on operational conditions used for each pretreatment.
Abstract: Anaerobic digestion of microalgal biomass for biogas production may be limited due to the cell wall resulting in an inefficient bioconversion. Enzymatic pretreatments are applied for inducing cell damage/lysis and organic matter solubilisation and this way increasing biogas production. We evaluated enzymatic pretreatments in different conditions for comparing in relation to cell wall rupture, increase of soluble material and increase in biogas production through anaerobic digestion performance in BMP assay. Chlorella sorokiniana cultures were subjected to three different enzymatic pretreatments, each under four different conditions of enzyme/substrate ratio, pH and application time. The results showed increases over 21% in biogas productions for all enzymatic pretreatments. Enzymatic pretreatment was effective at damaging microalgae cell wall, releasing organic compounds and increasing the rate and final methane yield in BMP tests. We observed a synergistic activity between the mixtures enzymes, which would depend on operational conditions used for each pretreatment.
TL;DR: Although only a few phage genomes are available to date, the ssRNA Leviviridae are a rich source of novel Sgls, which may facilitate further unraveling of bacterial cell wall biosynthesis and discovery of new antibacterial agents.
TL;DR: It is shown that bulging can be energetically favorable due to the relaxation of the entropic and stretching energies of the inner membrane, cell wall, and outer membrane and that the experimentally observed timescales are consistent with model predictions, and that swelling is mediated by the enlargement of wall defects.
TL;DR: The results indicate that the influence of anticancer drugs on cellular metabolism of live non-adherent cells can be obtained using the SCMS experiments combined with statistical data analysis.
TL;DR: Investigations of the potential mechanisms of membrane pore formation are explored by performing molecular simulations of the MU1140-lipid II complex in the bacterial membrane to provide atomistic level insight into a novel mode of action for Mutacin 1140 that can be exploited to develop optimized peptide variants with improved antimicrobial properties.
Abstract: The emergence of antibiotic-resistance is a major concern to global human health and identification of novel antibiotics is critical to mitigate the threat. Mutacin 1140 (MU1140) is a promising antimicrobial lanthipeptide and is effective against Gram-positive bacteria. Like nisin, MU1140 targets and sequesters lipid II and interferes with its function, which results in the inhibition of bacterial cell wall synthesis, and leads to bacteria cell lysis. MU1140 contains a structurally similar thioether cage for binding the lipid II pyrophosphate as for nisin. In addition to lipid II binding, nisin is known to form membrane pores. Membrane pore formation and membrane disruption is a common mode of action for many antimicrobial peptides, including gallidermin, a lantibiotic peptide with similar structural features as MU1140. However, whether and how MU1140 and its variants can form permeable membrane pores remains to be demonstrated. In this work, we explored the potential mechanisms of membrane pore formation by performing molecular simulations of the MU1140–lipid II complex in the bacterial membrane. Our results suggest that MU1140–lipid II complexes are able to form water permeating membrane pores. We find that a single chain of MU1140 complexed with lipid II in the transmembrane region can permeate water molecules across the membrane via a single-file water transport mechanism. The ordering of the water molecules in the single-file chain region as well as the diffusion behavior is similar to those observed in other biological water channels. Multiple complexes of MU1140–lipid II in the membrane showed enhanced permeability for the water molecules, as well as a noticeable membrane distortion and lipid relocation, suggesting that a higher concentration of MU1140 assembly in the membrane can cause significant disruption of the bacterial membrane. These investigations provide an atomistic level insight into a novel mode of action for MU1140 that can be exploited to develop optimized peptide variants with improved antimicrobial properties.
TL;DR: In this paper, a microalga with high self-flocculating efficiency was isolated and identified as Neocystis mucosa SX, and the efficiency of extracting extracellular polymeric substances (EPS) from the micro alga using cation exchange resin (CER), heating, EDTA, acidic treatment and alkaline treatment were compared.
Abstract: Extracellular polymeric substances (EPS) played an important role in flocculation of microalgae. Nevertheless, no universal method was reported for EPS extraction from microalgae. In the study, a microalga with high self-flocculating efficiency was isolated and identified as Neocystis mucosa SX. Then the efficiency of extracting EPS from the microalga using cation exchange resin (CER), heating, EDTA, acidic treatment and alkaline treatment were compared. Both CER and heating treatment extracted more EPS with rich chemical groups than other three methods. Moreover, it was favorable for EPS extraction by prolonging treatment time for CER method and raising temperature for heating treatment. Nevertheless, long treatment time for CER method and high temperature for heating treatment resulted in severe cell lysis through the analysis of DNA content, dehydrogenase activity (DHA) and the cell membrane integrity. Based on both EPS extraction efficiency and cell viability, the CER extraction for 6 h could be applied for the actual study.
TL;DR: The most common and effective method for removing trace to moderate amounts of DNA contamination from RNA samples is digestion with DNase I, as described here.
Abstract: RNA samples prepared using monophasic lysis reagents may contain small amounts of contaminating genomic DNA, which must be removed if the RNA will be used in subsequent analyses such as reverse transcriptase-polymerase chain reaction (RT-PCR) or quantitative real-time RT-PCR. In addition, the presence of contaminating DNA can render the quantitative determination of RNA in a sample inaccurate. The most common and effective method for removing trace to moderate amounts of DNA contamination from RNA samples is digestion with DNase I, as described here.
TL;DR: Among the tested methods, CER resulted in EPS extraction with minimal contamination and it is proposed that CER is currently the most appropriate method for extraction of EPS from Ultisols.
Abstract: Extracellular polymeric substances (EPS) have many beneficial functions in soils. Accurate quantification of EPS in soils is crucial. Here, five methods were compared for their suitability for extraction of EPS from Ultisols: hot water extractable polysaccharide (HWEP), hot dilute acid extractable polysaccharide (HDAEP), easily extractable glomalin (EEG), sodium sulfide (SS) and cation exchange resin (CER) method. Humic-acid equivalent (HAE) was used as an indicator for extracellular contamination and ATP for quantifying intracellular contamination from cell lysis. Among the tested methods, CER resulted in EPS extraction with minimal contamination. Therefore, we propose that CER is currently the most appropriate method for extraction of EPS from Ultisols.
TL;DR: The extent of DNA displacement into the depth filter was found to increase with decreasing DNA length with increasing operational parameters such as wash volume and buffer ionic strength, and it was shown that Chinese hamster ovary cellular DNA in the lysate supernatant migrates deeper into theDepth filtration than the lYSate re-suspended pellet, elucidating the role of the size of the DNA in its form as an impurity.
TL;DR: It is shown that an integration of the nanowire-mediated lysis with LAMP provides a means for a simple, rapid, one-step identification assay (just introducing a premixed solution into a device), resulting in visual chromatic identification of microbial cells.
Abstract: Researchers have demonstrated great promise for inorganic nanowire use in analyzing cells or intracellular components. Although a stealth effect of nanowires toward cell surfaces allows preservation of the living intact cells when analyzing cells, as a completely opposite approach, the applicability to analyze intracellular components through disrupting cells is also central to understanding cellular information. However, the reported lysis strategy is insufficient for microbial cell lysis due to the cell robustness and wrong approach taken so far ( i. e., nanowire penetration into a cell membrane). Here we propose a nanowire-mediated lysis method for microbial cells by introducing the rupture approach initiated by cell membrane stretching; in other words, the nanowires do not penetrate the membrane, but rather they break the membrane between the nanowires. Entangling cells with the bacteria-compatible and flexible nanowires and membrane stretching of the entangled cells, induced by the shear force, play important roles for the nanowire-mediated lysis to Gram-positive and Gram-negative bacteria and yeast cells. Additionally, the nanowire-mediated lysis is readily compatible with the loop-mediated isothermal amplification (LAMP) method because the lysis is triggered by simply introducing the microbial cells. We show that an integration of the nanowire-mediated lysis with LAMP provides a means for a simple, rapid, one-step identification assay (just introducing a premixed solution into a device), resulting in visual chromatic identification of microbial cells. This approach allows researchers to develop a microfluidic analytical platform not only for microbial cell identification including drug- and heat-resistance cells but also for on-site detection without any contamination.
TL;DR: In the authors' hands, the most efficient cell lysis and highest RNA yield from C. albicans cells stored in RNAlater was obtained by horizontal bead beating in RiboPure Lysis Buffer followed by RNA extraction with the Ribo Pure Yeast Kit.
Abstract: Obtaining sufficient RNA yield and quality for comprehensive transcriptomic studies is cumbersome for clinical samples in which RNA from the pathogen is present in low numbers relative to the nucleic acids from the host, especially for pathogens, such as yeasts, with a solid cell wall. Therefore, yeast cell lysis including cell wall disruption constitutes an essential first step to maximize RNA yield. Moreover, during the last years, different methods for RNA extraction from yeasts have been developed, ranging from classic hot phenol methods to commercially available specific kits. They offer different RNA yield and quality, also depending on the original storage medium, such as RNAlater. We observed that, for C. albicans cells stored in Tryptic Soy Broth with 15% glycerol, 10 min of bead beating in a horizontal position in RiboPure Lysis Buffer provided complete cell lysis. Cell lysis efficiency was decreased to 73.5% when cells were stored in RNAlater. In addition, the RiboPure Yeast Kit (Ambion) offered the highest RNA yield in comparison with the automated platform NucliSENS easyMAG total nucleic extraction (bioMerieux) and the RNeasy Mini Kit (Qiagen) according to NanoDrop and Fragment Analyzer. Moreover, we showed that, in spite of the decrease of cell lysis efficiency after RNAlater storage, as compared to storage in TSB + 15% glycerol, RNAlater increased RNA yield during RNA extraction with both RiboPure Yeast Kit and easyMAG, as confirmed by Fragment Analyzer analysis and by RT-qPCR of the RNA from the Internal Transcribed Spacer 2. In our hands, the most efficient cell lysis and highest RNA yield from C. albicans cells stored in RNAlater was obtained by horizontal bead beating in RiboPure Lysis Buffer followed by RNA extraction with the RiboPure Yeast Kit.
TL;DR: A mechanical cell lysis method based on a surface acoustic wave (SAW) microchip that has a high efficiency and low sample consumption and the potential application is the pretreatment of a small amount of tested sample in a hospital or biolab is proposed.
Abstract: Cell lysis is an important and crucial step for the detection of intracellular secrets. Usually, cell lysis is based on strong ultrasonic waves or toxic chemical regents, which require a large amount of cell suspension. To obtain high efficiency cell lysis for a small amount of sample, a mechanical cell lysis method based on a surface acoustic wave (SAW) microchip is proposed. The microchip simply consists of a piece of LiNbO3 crystal substrate, interdigitated transducers (IDTs) with 80 pairs of parallel electrodes and 3M Magic Tapes. The modulated input electrical signal is coupled into the substrate through IDTs, which produces an acoustic stream in the droplet on the surface of a substrate. When a biofluid droplet containing cells and microparticles is dropped on the surface of the microchip, the cells and microparticles are accelerated and collide with each other. The fluorescence staining results illustrate that the cell membrane is efficiently destroyed and that proteins as well as nucleic acids inside the cell are released. The experimental results show that this method has a high efficiency and low sample consumption. The potential application is the pretreatment of a small amount of tested sample in a hospital or biolab.
TL;DR: P porous silica monolith capillaries capable of selectively isolating bacteria directly from human whole blood, leaving the living bacteria intact for analysis are developed, allowing rapid preparation and culture-free analysis of bacteria at the point of care.
Abstract: Rapid and efficient isolation of bacteria from complex biological matrices is necessary for effective pathogen identification in emerging single-cell diagnostics. Here, we demonstrate the isolation of intact and viable bacteria from whole blood through the selective lysis of blood cells during flow through a porous silica monolith. Efficient mechanical hemolysis is achieved while providing passage of intact and viable bacteria through the monoliths, allowing size-based isolation of bacteria to be performed following selective lysis. A process for synthesizing large quantities of discrete capillary-bound monolith elements and millimeter-scale monolith bricks is described, together with the seamless integration of individual monoliths into microfluidic chips. The impact of monolith morphology, geometry, and flow conditions on cell lysis is explored, and flow regimes are identified wherein robust selective blood cell lysis and intact bacteria passage are achieved for multiple gram-negative and gram-positive bacteria. The technique is shown to enable rapid sample preparation and bacteria analysis by single-cell Raman spectrometry. The selective lysis technique presents a unique sample preparation step supporting rapid and culture-free analysis of bacteria for the point of care.
TL;DR: In this article, the antimicrobial effects of a carbon nanoparticles-polypyrrole nanocomposite (C-PPy) upon laser irradiation were investigated to destroy the pathogenic gram-negative Pseudomonas aeruginosa.
Abstract: Background: Nowadays, it is needed to explore new routes to treat infectious bacterial pathogens due to prevalence of antibiotic-resistant bacteria. Antimicrobial photothermal therapy (PTT), as a new strategy, eradicates pathogenic bacteria. Objective : In this study, the antimicrobial effects of a carbon nanoparticles-polypyrrole nanocomposite (C-PPy) upon laser irradiation were investigated to destroy the pathogenic gram-negative Pseudomonas aeruginosa . Material and Methods: In this experimental study, the bacterial cells were incubated with 50, 100 and 250 µg mL -1 concentrations of C-PPy and irradiated with a 808-nm laser at two power densities of 0.5 and 1.0 W cm -2 . CFU numbers were counted for the irradiated cells, and compared to an untreated sample (kept in dark). To explore the antibacterial properties and mechanism(s) of C-PPy, temperature increment, reactive oxygen species formation, and protein and DNA leakages were evaluated. Field emission scanning electron microscopy was also employed to investigate morphological changes in the bacterial cell structures. Results: The results showed that following C-PPy attachment to the bacteria surface, irradiation of near-infrared light resulted in a significant decrement in the bacterial cell viability due to photothermal lysis. Slightly increase in protein leakage and significantly increase intracellular reactive oxygen species (ROS) were observed in the bacteria upon treating with C-PPy. Conclusion: Photo-ablation strategy is a new minimally invasive and inexpensive method without overdose risk manner for combat with bacteria.
TL;DR: In this article, the authors identified the differential pressure of the primary depth filter as the key factor contributing to cell lysis in Chinese Hamster Ovary manufacturing process using single-use technologies.
Abstract: Monoclonal antibody interchain disulfide bond reduction was observed in a Chinese Hamster Ovary manufacturing process that used single-use technologies. A similar reduction has been reported for processes that involved high mechanical shear recovery unit operations, such as continuous flow centrifugation and when the clarified harvest was stored under low dissolved oxygen (DO) conditions (Trexler-Schmidt et al., 2010. Biotechnology and Bioengineering, 106(3), 452-461). The work described here identifies disposable depth filtration used during cell culture harvest operations as a shear-inducing unit operation causing cell lysis. As a result, reduction of antibody interchain disulfide bonds was observed through the same mechanisms described for continuous flow centrifugation. Small-scale depth-filtration models were developed, and the differential pressure (Δ P) of the primary depth filter was identified as the key factor contributing to cell lysis. Strong correlations of Δ P and cell lysis were generated by measuring the levels of lactate dehydrogenase and thiol in the filtered harvest material. A simple risk mitigation strategy was implemented during manufacturing by providing an air overlay to the headspace of a single-use storage bag to maintain sufficient DO in the clarified harvest. In addition, enzymatic characterization studies determined that thioredoxin reductase and glucose-6-phosphate dehydrogenase are critical enzymes involved in antibody reduction in a nicotinamide adenine dinucleotide phosphate (NADP + )/NADPH-dependent manner.
TL;DR: Carrier-based immobilization has been developed to improve the functional properties of enzymes and promote the employment of enzymatic lysis in downstream processing without the necessity to separate the lysis enzyme after processing.
TL;DR: The finding that lysozyme activity against Gram‐negative bacteria is enhanced in the presence of glycine and charged amino acids over a wide range of concentrations is of particular interest.
TL;DR: It is shown that CS109 cells lacking the bifunctional aPBP PBP1B (penicillin binding protein 1B) lyse during exponential growth at elevated temperature, suggesting a biological connection that could be exploited in combination therapies.
Abstract: Peptidoglycan (PG) is a highly cross-linked polysaccharide that encases bacteria, resists the effects of turgor and confers cell shape. PG precursors are translocated across the cytoplasmic membrane by the lipid carrier undecaprenyl phosphate (Und-P) where they are incorporated into the PG superstructure. Previously, we found that one of our Escherichia coli laboratory strains (CS109) harbors a missense mutation in uppS, which encodes an enzymatically defective Und-P(P) synthase. Here, we show that CS109 cells lacking the bifunctional aPBP PBP1B (penicillin binding protein 1B) lyse during exponential growth at elevated temperature. PBP1B lysis was reversed by: (i) reintroducing wild-type uppS, (ii) increasing the availability of PG precursors or (iii) overproducing PBP1A, a related bifunctional PG synthase. In addition, inhibiting the catalytic activity of PBP2 or PBP3, two monofunctional bPBPs, caused CS109 cells to lyse. Limiting the precursors required for Und-P synthesis in MG1655, which harbors a wild-type allele of uppS, also promoted lysis in mutants lacking PBP1B or bPBP activity. Thus, simultaneous inhibition of Und-P production and PG synthases provokes a synergistic response that leads to cell lysis. These findings suggest a biological connection that could be exploited in combination therapies.
TL;DR: This work methodically investigated the impact of different MATH test parameters on the calculation of the cell surface hydrophobicity of Streptococcus thermophilus, a widespread exopolysaccharide-producing lactic acid bacterium used in the production of fermented milk products.
Abstract: The microbial adhesion to hydrocarbons (MATH) test is one of the most common method to determine the hydrophobicity of cell surfaces. Despite its prevalence, no standard test parameters are used in literature, making a direct comparison of data almost impossible. Criticism also focuses on test parameters that may mask hydrophobic interactions and hence lead to erroneous test results. We methodically investigated the impact of different MATH test parameters on the calculation of the cell surface hydrophobicity of Streptococcus thermophilus, a widespread exopolysaccharide-producing lactic acid bacterium used in the production of fermented milk products. Besides composition and ionic strength of the buffer used for cell re-suspension, we observed a pronounced time dependency of the turbidity of the cell suspension during phase separation due to sedimentation and/or cell lysis. A new modification of the MATH assay was applied to enable the determination of cell surface hydrophobicity of long chain-forming bacteria. As the cell surface hydrophobicity was not altered during exponential growth phase, we assume that the cell surface and its capsular exopolysaccharide layer are not changed during cultivation.