Showing papers on "Lysis published in 2009"
TL;DR: The characterisation of extracellular DNA can integrate information on the composition of the microbial community of soil and sediments obtained by analysing intracellular DNA.
Abstract: The review discusses origin, state and function of extracellular DNA in soils and sediments Extracellular DNA can be released from prokaryotic and eukaryotic cells and can be protected against nuclease degradation by its adsorption on soil colloids and sand particles Laboratory experiments have shown that DNA adsorbed by colloids and sand particles can be taken up by prokaryotic competent cells and be involved in natural transformation Most of these experiments have been carried out under artificial conditions with pure DNA molecules and pure adsorbing matrices, but in soils and sediments, pure surface-reactive colloids are not present and DNA is present with other cellular components (wall debris, proteins, lipids, RNA, etc) especially if released after cell lysis The presence of inorganic compounds and organic molecules on both soil particles and DNA molecules can influence the DNA adsorption, degradation and transformation of competent cells Extracellular DNA can be used as C, N and P sources by heterotrophic microorganisms and plays a significant role in bacterial biofilm formation The nucleotides and nucleosides originated from the degradation of extracellular DNA can be re-assimilated by soil microorganisms Extracellular DNA in soil can be leached and moved by water through the soil profile by capillarity In this way, the extracellular DNA secreted by a cell can reach a competent bacterial cell far from the donor cell Finally, the characterisation of extracellular DNA can integrate information on the composition of the microbial community of soil and sediments obtained by analysing intracellular DNA
429 citations
TL;DR: The techniques for microfluidic cell lysis and nucleic acid purification are compared based on the ease of microfabrication and integration, and sample flexibility, to help determine the appropriate sample preparation technique for generating a true lab-on-a-chip.
Abstract: Due to the lack of development in the area of sample preparation, few complete lab-on-a-chip systems have appeared in recent years that can deal with raw samples. Cell lysis and nucleic acid extraction systems are sufficiently complex even before adding the complexity of an analysis system. In this review, a variety of microfluidic sample preparation methods are discussed and evaluated. Microsystems for cell lysis are discussed by grouping them into categories based on their lysis mechanisms: mechanical, chemical, thermal or electrical. We classify the nucleic acid purification techniques according to the mechanism that links nucleic acids to substrates: silica-based surface affinity, electrostatic interaction, nanoporous membrane filtration, and functionalized microparticles. The techniques for microfluidic cell lysis and nucleic acid purification are compared based on the ease of microfabrication and integration, and sample flexibility. These assessments can help us determine the appropriate sample preparation technique for generating a true lab-on-a-chip.
281 citations
TL;DR: E. coli thus appears to rely on two distinct sets of putative PG hydrolases to promote proper cell division, and the phenotypes of mutants lacking LytM-domain factors bear a striking resemblance to those of mutants defective for the N-acetylmuramyl-l-alanine amidases.
Abstract: Bacterial cytokinesis is coupled to the localized synthesis of new peptidoglycan (PG) at the division site. This newly generated septal PG is initially shared by the daughter cells. In Escherichia coli and other gram-negative bacteria, it is split shortly after it is made to promote daughter cell separation and allow outer membrane constriction to closely follow that of the inner membrane. We have discovered that the LytM (lysostaphin)-domain containing factors of E. coli (EnvC, NlpD, YgeR, and YebA) are absolutely required for septal PG splitting and daughter cell separation. Mutants lacking all LytM factors form long cell chains with septa containing a layer of unsplit PG. Consistent with these factors playing a direct role in septal PG splitting, both EnvC-mCherry and NlpD-mCherry fusions were found to be specifically recruited to the division site. We also uncovered a role for the LytM-domain factors in the process of β-lactam-induced cell lysis. Compared to wild-type cells, mutants lacking LytM-domain factors were delayed in the onset of cell lysis after treatment with ampicillin. Moreover, rather than lysing from midcell lesions like wild-type cells, LytM− cells appeared to lyse through a gradual loss of cell shape and integrity. Overall, the phenotypes of mutants lacking LytM-domain factors bear a striking resemblance to those of mutants defective for the N-acetylmuramyl-l-alanine amidases: AmiA, AmiB, and AmiC. E. coli thus appears to rely on two distinct sets of putative PG hydrolases to promote proper cell division.
210 citations
TL;DR: A protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts is described.
Abstract: The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples. We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step. We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.
197 citations
TL;DR: A microfluidic sample preparation platform for rapid on-chip detection of infectious organisms for point-of-care diagnostics that isolated and detected both gram-negative and gram-positive bacterial genomic DNA from microliter scale spiked whole human blood samples.
Abstract: Sepsis caused by gram positive and gram negative bacteria is the leading cause of death in noncoronary ICUs and the tenth leading cause of death in the United States. We have developed a microfluidic sample preparation platform for rapid on-chip detection of infectious organisms for point-of-care diagnostics. The microfluidic chips are made of a robust thermoplastic and can be easily multiplexed for high throughput applications. Bacteria are lysed on-chip via hybrid chemical/mechanical method. Once lysed, the bacterial DNA is isolated using a microscale silica bead/polymer composite solid-phase-extraction (SPE) column. Lysis was confirmed using off-chip real time PCR. We isolated and detected both gram-negative (Escherichia coli) and gram-positive (Bacillussubtilis and Enterococcus faecalis) bacterial genomic DNA from microliter scale spiked whole human blood samples. The system performs better for gram-negative bacteria than it does for gram-positive bacteria, with limits of detection at 102CFU/ml and 103–104CFU/ml, respectively. Total extraction times are less than one hour and can be further decreased by altering the channel geometry and pumping configuration.
166 citations
TL;DR: Chitosan permeabilization of the fungal plasma membrane and its uptake into fungal cells was found to be energy dependent but not to involve endocytosis.
149 citations
TL;DR: Using chemical treatment or enzymatic treatment of biofilm samples may obtain larger amounts of eDNA than using the simple filtration method, and genomic DNA and DNA sequencing analysis revealed that eDNA originated from genomic DNA but was not structurally identical to the genomic DNA.
Abstract: The occurrence of high concentrations of extracellular DNA (eDNA) in the extracellular matrices of biofilms plays an important role in biofilm formation and development and possibly in horizontal gene transfer through natural transformation. Studies have been conducted to characterize the nature of eDNA and its potential function in biofilm development, but it is difficult to extract eDNA from the extracellular matrices of biofilms without any contamination from genomic DNA released by cell lysis during the extraction process. In this report, we compared several different extraction methods in order to obtain highly pure eDNA from different biofilm samples. After different extraction methods were explored, it was concluded that using chemical treatment or enzymatic treatment of biofilm samples may obtain larger amounts of eDNA than using the simple filtration method. There was no detectable cell lysis when the enzymatic treatment methods were used, but substantial cell lysis was observed when the chemical treatment methods were used. These data suggest that eDNA may bind to other extracellular polymers in the biofilm matrix and that enzymatic treatment methods are effective and favorable for extracting eDNA from biofilm samples. Moreover, randomly amplified polymorphic DNA analysis of eDNA in Acinetobacter sp. biofilms and Acinetobacter sp. genomic DNA and DNA sequencing analysis revealed that eDNA originated from genomic DNA but was not structurally identical to the genomic DNA.
142 citations
TL;DR: A sample preparation protocol for rapid and unbiased analysis of the membrane proteome using an alimentary canal-mimicking system in which proteases are activated in the presence of bile salts and protein abundance estimation based on the number of observed peptides per protein was applied.
136 citations
TL;DR: It is proposed that LysB facilitates lysis by compromising the integrity of the mycobacterial outer membrane linkage to the arabinogalactan–peptidoglycan layer.
Abstract: Mycobacteriophages encounter a unique problem among phages of Gram-positive bacteria, in that lysis must not only degrade the peptidoglycan layer but also circumvent a mycolic acid-rich outer membrane covalently attached to the arabinogalactan-peptidoglycan complex. Mycobacteriophages accomplish this by producing two lysis enzymes, Lysin A (LysA) that hydrolyses peptidoglycan, and Lysin B (LysB), a novel mycolylarabinogalactan esterase, that cleaves the mycolylarabinogalactan bond to release free mycolic acids. The D29 LysB structure shows an {alpha}/{beta} hydrolase organization with a catalytic triad common to cutinases, but which contains an additional four-helix domain implicated in the binding of lipid substrates. Whereas LysA is essential for mycobacterial lysis, a Giles {Delta}lysB mutant mycobacteriophage is viable, but defective in the normal timing, progression and completion of host cell lysis. We propose that LysB facilitates lysis by compromising the integrity of the mycobacterial outer membrane linkage to the arabinogalactan-peptidoglycan layer.
119 citations
TL;DR: The inducible cyanobacterial lysing system eliminates the need for mechanical or chemical cell breakage and could facilitate recovery of biofuel from cyanobacteria.
Abstract: We designed and constructed a controllable inducing lysis system in Synechocystis sp. PCC 6803 to facilitate extracting lipids for biofuel production. Several bacteriophage-derived lysis genes were integrated into the genome and placed downstream of a nickel-inducible signal transduction system. We applied 3 strategies: (i) directly using the phage lysis cassette, (ii) constitutively expressing endolysin genes while restricting holin genes, and (iii) combining lysis genes from different phages. Significant autolysis was induced in the Synechocystis sp. PCC 6803 cells with this system by the addition of NiSO4. Our inducible cyanobacterial lysing system eliminates the need for mechanical or chemical cell breakage and could facilitate recovery of biofuel from cyanobacteria.
116 citations
TL;DR: The extraction with dowex resin resulted in the second-highest concentration of carbohydrate after the water extraction and the EPS were especially rich in deoxy sugars, hence increasing the hydrophobic feature of these substances.
Abstract: There is no universal method that can be applied to extract bound extracellular polymeric substances (EPS) from benthic diatoms of intertidal sediments without causing cell lysis. Six extraction methods were tested on a diatom culture of Navicula jeffreyi to establish the best compromise between high yields of carbohydrate extraction and minimum cell lysis. Extraction with distilled water provoked cell lysis (as already known). The five other extraction methods (dowex resin, artificial seawater of half salinity and extractions after pretreatment with gluteraldehyde by three methods: water, dowex water and dowex buffer) did not provoke cell lysis as shown by transmission electronic microscopy. This result was confirmed by the minimum release of internal compounds (protein, ATP) and by the low proportions of glucose in dowex-extracted EPS compared with the water-extracted EPS, from which the high glucose content must be inferred as contamination by the chrysolaminaran. The extraction with dowex resin resulted in the second-highest concentration of carbohydrate after the water extraction and the EPS were especially rich in deoxy sugars, hence increasing the hydrophobic feature of these substances. For these reasons, we recommend extraction with dowex, which is also the best method for extracting bound EPS from other biofilms such as in activated sludges.
TL;DR: It has been demonstrated by confocal microscopy that the grafted polymers can induce a significant release of endocytosed materials into the cytoplasm of HeLa cells, which is a feature critical for drug delivery applications.
TL;DR: Variations of sample preparation of the human colon carcinoma cell line SW480 exposed in vitro to the tumor-inhibiting metal complexes cisplatin and indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(iii)] (KP1019) prior to analysis with ICP-MS) were found to be tremendously influenced by adsorption to the culture dishes.
Abstract: Cellular uptake is being widely investigated in the context of diverse biological activities of metal compounds on the cellular level. However, the applied techniques differ considerably, and a validated methodology is not at hand. Therefore, we have varied numerous aspects of sample preparation of the human colon carcinoma cell line SW480 exposed in vitro to the tumor-inhibiting metal complexes cisplatin and indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) prior to analysis with ICP-MS, and the results were found to be tremendously influenced by adsorption to the culture dishes. Adsorption to culture plates increases linearly with the concentration of KP1019, depends on the protein content of the medium, the duration of contact to protein-containing medium prior to drug addition and the hydrophilicity/lipophilicity of the compound. For varying degrees of cell confluence, adsorption of Ru hardly differs from cell-free experiments. Desorption from the plates contributes to total Ru detected in dependence on the cell harvesting method. Desorption kinetics for lysis in HNO3 and tetramethylammonium hydroxide (TMAH) are comparable, but TMAH is a more potent desorbant. Sample storage conditions prior to analysis influence significantly the recovery of analyte. Protocols using cell lysis in the culture plate without proper corrections run the risk of producing artefacts resulting from metal adsorption/desorption to an extent comparable with the actual cellular content. However, experimental protocols reported in the literature frequently do not contain information whether adsorption or blank correction were performed and should be regarded with caution, especially if lysis was performed directly in the culture dishes.
TL;DR: A simple and efficient plant DNA extraction procedure for isolation of high-quality DNA from plant tissues is presented in this paper, which requires maceration of plant tissue of about 1.0 cm(2) (e.g. of a leaf blade) in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl) using 1.5-mL microfuge tubes, followed by cell lysis with 20% SDS, and DNA extraction with phenol: chloroform: iso-amyl alcohol (25:24:
Abstract: Polymerase chain reaction has found wide applications in modern research involving transformations and other genomic studies. For reproducible PCR results, however, the quantity and quality of template DNA is of considerable importance. A simple and efficient plant DNA extraction procedure for isolation of high-quality DNA from plant tissues is presented here. It requires maceration of plant tissue of about 1.0 cm(2) (e.g. of a leaf blade) in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl) using 1.5-mL microfuge tubes, followed by cell lysis with 20% SDS, and DNA extraction with phenol: chloroform: iso-amyl alcohol (25:24:1). Hydrated ether is then used to remove polysaccharides and other contaminants from the DNA preparation. Average DNA yield is 20-30 microg cm(-2) for fresh tissues, and ratio of absorbance at 260 nm to absorbance at 280 nm is 1.5-1.8. The DNA is quite suitable for PCR using microsatellites, RAPD and specific markers for recombinant selection. Amplifications have been obtained for these markers by using template DNA extracted from fresh as well as frozen leaf tissues of various plants, including barley, oat, potato and tomato. DNA stored for more than 2 years has been successfully amplified with microsatellite markers, which shows suitability of this method after long-term storage of DNA. Besides, the ease of use and cost-effectiveness make the procedure attractive.
TL;DR: An optically induced cell lysis device that can selectively lyse a single cell within a group of cells, a function which cannot be performed using traditional tools is reported.
Abstract: This letter reports an optically induced cell lysis device that can selectively lyse a single cell within a group of cells, a function which cannot be performed using traditional tools. This chip-scale device was made of a photoconductive material, which can induce a nonuniform electric field at a specific position under illumination of a beam spot generating a transmembrane potential in the cell. With this approach, cell lysis can be performed using the optically induced electric field. Fibroblast cells and oral cancer cells were used to demonstrate the capability of the developed chip. In addition to lysing the whole cell, the developed method also allowed one to selectively disrupt the cell membrane without damaging the nucleus. Operating parameters such as illumination power density and beam spot diameter for cell lysis were systematically investigated.
TL;DR: AptaPIC, Aptamer-facilitated Protein Isolation from Cells, a technology that integrates the development of aptamers for a protein in cell lysate and the utilization of the developed aptamer for protein isolation from the cell l Lysate has the potential to considerably speed up the purification of proteins and, thus, accelerate their structural and functional studies.
Abstract: Functional genomics requires structural and functional studies of a large number of proteins. While the production of proteins through over-expression in cultured cells is a relatively routine procedure, the subsequent protein purification from the cell lysate often represents a significant challenge. The most direct way of protein purification from a cell lysate is affinity purification using an affinity probe to the target protein. It is extremely difficult to develop antibodies, classical affinity probes, for a protein in the cell lysate; their development requires a pure protein. Thus, isolating the protein from the cell lysate requires antibodies, while developing antibodies requires a pure protein. Here we resolve this loop problem. We introduce AptaPIC, Aptamer-facilitated Protein Isolation from Cells, a technology that integrates (i) the development of aptamers for a protein in cell lysate and (ii) the utilization of the developed aptamers for protein isolation from the cell lysate. Using MutS protein as a target, we demonstrate that this technology is applicable to the target protein being at an expression level as low as 0.8% of the total protein in the lysate. AptaPIC has the potential to considerably speed up the purification of proteins and, thus, accelerate their structural and functional studies.
TL;DR: Results show that SIL and SB have differential effects on membrane stability; whereas SIL shows consistently stabilizing effects, SB exacerbates hepatocellular lysis or exerts only minimal stabilization effects.
TL;DR: Cell lysis has a significant effect on cell lipid composition, making handling reproducibility an important issue in lipid analysis, and HRMAS was shown to be preferable to solution-state NMR of suspensions or supernatant, giving enhanced information on lipids and comparable resolution for smaller metabolites.
Abstract: The aim of this work was to investigate the effects of cell handling and storage on cell integrity and 1H high resolution magic angle spinning (HRMAS) NMR spectra. Three different cell types have been considered (lung tumoral, amniocytes, and MG-63 osteosarcoma cells) in order for sample-dependent effects to be identified. Cell integrity of fresh cells and cells frozen in cryopreservative solution was ∼70−80%, with the former showing higher membrane degradation, probably enzymatic, as indicated by increased phosphocholine (PC) and/or glycerophosphocholine (GPC). Unprotected freezing (either gradual or snap-freezing) was found to lyse cells completely, similar to mechanical cell lysis. Besides enhanced metabolites visibility, lysed cells showed a different lipid profile compared to intact cells, with increased choline, PC, and GPC and decreased phosphatidylcholine (PTC). Cell lysis has, therefore, a significant effect on cell lipid composition, making handling reproducibility an important issue in lipid an...
TL;DR: Both the amount and the chemical composition of EPS strongly depended on the extraction method used, and the major constituents were proteins and carbohydrates, showing the major presence of carboxylic, hydroxylic and amino groups.
TL;DR: This work has developed a two-step method to produce the grape dehydrin K(2) and YSK(2), which was shown to be more than 95% pure by reversed-phase high-performance liquid chromatography.
TL;DR: This work describes expression of peptide P11‐4 in the bacterium Escherichia coli from constructs carrying tandem repeats of the peptide coding sequence and recombinant peptide with a C‐terminal homoserine lactone formed pH dependent hydrogels.
Abstract: Peptide P(11)-4 (QQRFEWEFEQQ) was designed to self-assemble to form beta-sheets and nematic gels in the pH range 5-7 at concentrations > or =12.6 mM in water. This self-assembly is reversibly controlled by adjusting the pH of the solvent. It can also self-assemble into gels in biological media. This together with its biocompatibility and biodegradability make P(11)-4 an attractive building block for the fabrication of nanoscale materials with uses in, for example, tissue engineering. A limitation to large-scale production of such peptides is the high cost of solid phase chemical synthesis. We describe expression of peptide P(11)-4 in the bacterium Escherichia coli from constructs carrying tandem repeats of the peptide coding sequence. The vector pET31b+ was used to express P(11)-4 repeats fused to the ketosteroid isomerase protein which accumulates in easily recoverable inclusion bodies. Importantly, the use of auto-induction growth medium to enhance cell density and protein expression levels resulted in recovery of 2.5 g fusion protein/L culture in both shake flask and batch fermentation. Whole cell detergent lysis allowed recovery of inclusion bodies largely composed of the fusion protein. Cyanogen bromide cleavage followed by reverse phase HPLC allowed purification of the recombinant peptide with a C-terminal homoserine lactone (rP(11)-4(hsl)). This recombinant peptide formed pH dependent hydrogels, displayed beta-structure measured by circular dichroism and fibril formation observed by transmission electron microscopy.
TL;DR: Chitosan particles are more potent in absorption enhancement than chitosans solutions, and the smaller the particles were the higher the permeation of FD4 was.
TL;DR: Advanced bioprocessing equipment and reagents have been developed over the past twenty years to complement established disruption procedures and accomplish these tasks with even greater success, and this chapter will summarize these advances.
Abstract: There are a variety of reliable methods for cellular disintegration and extraction of proteins ranging from enzymatic digestion and osmotic shock to ultrasonication, and pressure disruption. Each method has inherent advantages and disadvantages. Generally vigorous mechanical treatments reduce extract viscosity but can result in the inactivation of labile proteins by heat or oxidation, while gentle treatments may not release the target protein from the cells, and resulting extracts are extremely viscous. Depending on the cell type selected as the source for target protein expression, cellular extracts contain large amounts of nucleic acid, ribosomal material, lipids, dispersed cell wall polysaccharide, carbohydrates, chitin, small molecules, and thousands of unwanted proteins. Isolation and recovery of a single protein from this complex mixture of macromolecules presents considerable challenges. The first and possibly most important of these challenges is generation of a cellular extract that can be efficiently manipulated in downstream processes without inactivation or degradation of labile protein targets. Cell disruption techniques must rapidly and efficiently lyse cells to extract proteins with minimal proteolysis or oxidation while reducing extract viscosity caused by cell debris and genomic DNA contamination. Advanced bioprocessing equipment and reagents have been developed over the past twenty years to complement established disruption procedures and accomplish these tasks with even greater success. This chapter will summarize these advances and describe detailed protocols for some of the most popular methods for protein extraction.
TL;DR: The presented methodology facilitates detection, separation, size characterization, and quantification of inorganic nanoparticles in biological samples, within one experimental run.
Abstract: A rapid, high-resolution methodology for characterization, separation, and quantification of unlabeled inorganic nanoparticles extracted from biological media, based on sedimentation field-flow fractionation and light scattering detection is presented. Silica nanoparticles were added to either human endothelial cell lysate or rat lung tissue homogenate and incubated. The nanoparticles were extracted by acid digestion and then separated and characterized by sedimentation field-flow fractionation. Fractions collected at the peak maxima were analyzed by transmission electron microscopy (TEM) to verify the size and shape of the isolated nanoparticles. Using the linear relationship between the particle number and the area under the fractogram, the recoveries of particles from the tissue homogenate and cell lysate were calculated as 25% and 79%, respectively. The presented methodology facilitates detection, separation, size characterization, and quantification of inorganic nanoparticles in biological samples, within one experimental run.
TL;DR: Escherichia coli is used as a test organism for a point of care thermoplastic microfluidic module designed to take in a urine sample, mix it with lysis buffer, and perform a hybrid chemical/mechanical lysis and solid phase extraction of nucleic acids from the sample.
Abstract: Silica impregnated polymer monolithic columns may provide a simple method for lysing and extracting DNA from bacteria inside of microfluidic chips. Here we use Escherichia coli as a test organism for a point of care thermoplastic microfluidic module designed to take in a urine sample, mix it with lysis buffer, and perform a hybrid chemical/mechanical lysis and solid phase extraction of nucleic acids from the sample. To demonstrate proof-of-concept, we doped human hematuric urine samples with E. coli at concentrations ranging from 10(1)-10(5) colony-forming units/mL (CFU/mL) to simulate patient samples. We then performed on-chip lysis and DNA extraction. The bacterial DNA was amplified using real-time PCR demonstrating lysis and isolation down to 10(1) CFU/mL. Results were comparable to a commercial kit at higher concentrations and performed better at recovering DNA at lower concentrations.
TL;DR: The compatibility of matrix lysis with subsequent real-time PCR analysis has been demonstrated with salmon, chicken, egg, ice cream, cheese, and blood samples that were artificially contaminated with L. monocytogenes, S. aureus, and Salmonella Typhimurium.
TL;DR: Results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.
Abstract: The aim of this study was to provide new insight into the mechanism whereby the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locates to cell walls of Lactobacillus plantarum 299v. After purification, cytosolic and cell wall GAPDH (cw-GAPDH) forms were characterized and shown to be identical homotetrameric active enzymes. GAPDH concentration on cell walls was growth-time dependent. Free GAPDH was not observed on the culture supernatant at any time during growth, and provoked cell lysis was not concomitant with any reassociation of GAPDH onto the cell surface. Hence, with the possibility of cw-GAPDH resulting from autolysis being unlikely, entrapment of intracellular GAPDH on the cell wall after a passive efflux through altered plasma membrane was investigated. Flow cytometry was used to assess L. plantarum 299v membrane permeabilization after labeling with propidium iodide (PI). By combining PI uptake and cw-GAPDH activity measurements, we demonstrate here that the increase in cw-GAPDH concentration from the early exponential phase to the late stationary phase is closely related to an increase in plasma membrane permeability during growth. Moreover, we observed that increases in both plasma membrane permeability and cw-GAPDH activity were delayed when glucose was added during L. plantarum 299v growth. Using a double labeling of L. plantarum 299v cells with anti-GAPDH antibodies and propidium iodide, we established unambiguously that cells with impaired membrane manifest five times more cw-GAPDH than unaltered cells. Our results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.
TL;DR: Results have indicated that ATPS can be successfully employed as a non-organic solvent method for the isolation of PHA from hydrolyzed bacterial cells and purified protease can be recovered as a byproduct, in a single defined experiment.
Abstract: Main objective of present work was to isolate polyhydroxyalkanoate (PHA) from cell lysate of Bacillus flexus by aqueous-aqueous two-phase system (ATPS). Selected ATPS having polyethylene glycol (12%, w/v) and potassium phosphate (9.7%, pH 8.0) containing cell lysate obtained by sonication or hypochlorite treatment of B. flexus biomass (1 g%, dry weight), was held at 28°C for 30 min, which partitioned PHA into top PEG phase and residual cell materials into bottom phase. For enzymatic cell hydrolysis, Microbispora sp. culture filtrate having protease (3 U/mL) was mixed with B. flexus biomass and ATPS, incubated at 37°C for 2 h prior to phase separation. PHA recovered by centrifugation was 19∼51% of cell dry weight, depending on the mode of cell disruption. Protease was recovered along with PHA in the PEG phase and showed 7 fold increase in activity. PHA was characterized by GC, FTIR, and 1H NMR. Results indicated that ATPS can be used for the isolation of PHA from hydrolyzed bacterial cells and purified protease can be recovered as a byproduct, in a single defined experiment. Results have indicated that ATPS can be successfully employed as a non-organic solvent method for the isolation of PHA.
TL;DR: DNA quantification results using dsDNA-binding fluorescence dyes and real-time PCR are presented, comparing the lysis of Bacillus subtilis spores using the Micro Bead-Beater with other well-known lysis techniques.
Abstract: Two mechanical lysis devices have been developed as compact, robust components to provide rapid sample preparation for nucleic acid diagnostic systems. One such component, known as the Micro Bead-Beater™ (μBB™, BBTM, Claremont BioSolutions, Upland, CA), is a compact device that is capable of ultrarapid lysis (>90% lysis in 30 s) of micro volumes (<80 μL) ofBacillus spores in a continuous-flow format or in a disposable single-tube format. The μBB is also capable of processing much larger volumes of solutions containing spores or vegetative cells using a continuous-flow mode. A second mechanical lysis device designed as a disposable component is the microfluidic bead blender, which uses a small electric motor to spin vanes within the bead-laden solution. DNA quantification results using dsDNA-binding fluorescence dyes and real-time PCR are presented, comparing the lysis of Bacillus subtilis spores using the μBB™ with other well-known lysis techniques. Nanoscale imaging results obtained using scanning electr...
Patent•
15 Jul 2009
TL;DR: In this article, the lysis agent is preloaded onto the collection device and the sample transport liquid then contains both lysis agents in solution or suspension and sample components in suspension, and the running buffer then carries the analyte, including any lysis-freed components, to the detection zone.
Abstract: Devices and methods incorporate lysis agents into a point-of-care testing device. The sample is loaded, and then the sample travels until it encounters a lysis agent. The lysis agent is preferably pre-loaded onto the collection device. In a preferred embodiment, the initially lysis agent is localized between the sample application zone and the conjugate zone. The lysis agent is preferably soluble or miscible in the sample transport liquid, and the lysis agent is solubilized and activated upon contact with the sample transport liquid. The sample transport liquid then contains both lysis agent in solution or suspension and sample components in suspension. Any lysis-susceptible components in a sample, then being exposed in suspension to the lysis agent, are themselves lysed in situ. The running buffer then carries the analyte, including any lysis-freed components, to the detection zone.