Showing papers on "Lysis published in 2012"

Enterococcus faecalis Produces Abundant Extracellular Structures Containing DNA in the Absence of Cell Lysis during Early Biofilm Formation

Abstract: Enterococcus faecalis is a common Gram-positive commensal bacterium of the metazoan gastrointestinal tract capable of biofilm formation and an opportunistic pathogen of increasing clinical concern. Dogma has held that biofilms are slow-growing structures, often taking days to form mature microcolonies. Here we report that extracellular DNA (eDNA) is an integral structural component of early E. faecalis biofilms (≤4 h postinoculation). Combining cationic dye-based biofilm matrix stabilization techniques with correlative immuno-scanning electron microscopy (SEM) and fluorescent techniques, we demonstrate that—in early E. faecalis biofilms—eDNA localizes to previously undescribed intercellular filamentous structures, as well as to thick mats of extruded extracellular matrix material. Both of these results are consistent with previous reports that early biofilms are exquisitely sensitive to exogenous DNase treatment. High-resolution SEM demonstrates a punctate labeling pattern in both structures, suggesting the presence of an additional, non-DNA constituent. Notably, the previously described fratricidal or lytic mechanism reported as the source of eDNA in older (≥24 h) E. faecalis biofilms does not appear to be at work under these conditions; extensive visual examination by SEM revealed a striking lack of lysed cells, and bulk biochemical assays also support an absence of significant lysis at these early time points. In addition, some cells demonstrated eDNA labeling localized at the septum, suggesting the possibility of DNA secretion from metabolically active cells. Overall, these data are consistent with a model in which a subpopulation of viable E. faecalis cells secrete or extrude DNA into the extracellular matrix. IMPORTANCE This paper reports the production of extracellular DNA during early biofilm formation in Enterococcus faecalis. The work is significant because the mechanism of eDNA (extracellular DNA) production is independent of cell lysis and the DNA is confined to well-defined structures, suggesting a novel form of DNA secretion by viable cells. Previous models of biofilm formation in enterococci and related species propose cell lysis as the mechanism of DNA release.

Host cell protein dynamics in the supernatant of a mAb producing CHO cell line.

Abstract: The characterization of host cell protein (HCP) content during the production of therapeutic recombinant proteins is an important aspect in the drug development process. Despite this, key components of the HCP profile and how this changes with processing has not been fully investigated. Here we have investigated the supernatant HCP profile at different times throughout culture of a null and model GS-CHO monoclonal antibody producing mammalian cell line grown in fed-batch mode. Using 2D-PAGE and LC-MS/MS we identify a number of intracellular proteins (e.g., protein disulfide isomerise; elongation factor 2; calreticulin) that show a significant change in abundance relative to the general increase in HCP concentration observed with progression of culture. Those HCPs that showed a significant change in abundance across the culture above the general increase were dependent on the cell line examined. Further, our data suggests that the majority of HCPs in the supernatant of the cell lines investigated here arise through lysis or breakage of cells, associated with loss in viability, and are not present due to the secretion of protein material from within the cell. SELDI-TOF and principal components analysis were also investigated to enable rapid monitoring of changes in the HCP profile. SELDI-TOF analysis showed the same trends in the HCP profile as observed by 2D-PAGE analysis and highlighted biomarkers that could be used for process monitoring. These data further our understanding of the relationship between the HCP profile and cell viability and may ultimately enable a more directed development of purification strategies and the development of cell lines based upon their HCP profile.

The Spanin Complex Is Essential for Lambda Lysis

Abstract: Phage lysis is a ubiquitous biological process, the most frequent cytocidal event in the biosphere. Lysis of Gram-negative hosts has been shown to require holins and endolysins, which attack the cytoplasmic membrane and peptidoglycan, respectively. Recently, a third class of lysis proteins, the spanins, was identified. The first spanins to be characterized were λ Rz and Rz1, an integral cytoplasmic membrane protein and an outer membrane lipoprotein, respectively. Previous work has shown that Rz and Rz1 form complexes that span the entire periplasm. Phase-contrast video microscopy was used to record the morphological changes involved in the lysis of induced λ lysogens carrying prophages with either the λ canonical holin-endolysin system or the phage 21 pinholin-signal anchor release (SAR) endolysin system. In the former, rod morphology persisted until the instant of an explosive polar rupture, immediately emptying the cell of its contents. In contrast, in pinholin-SAR endolysin lysis, the cell began to shorten and thicken uniformly, with the resultant rounded cell finally bursting. In both cases, lysis failed to occur in inductions of isogenic prophages carrying null mutations in the spanin genes. In both systems, instead of an envelope rupture, the induced cells were converted from a rod shape to a spherical form. A functional GFPΦRz chimera was shown to exhibit a punctate distribution when coexpressed with Rz1, despite the absence of endolysin function. A model is proposed in which the spanins carry out the essential step of disrupting the outer membrane, in a manner regulated by the state of the peptidoglycan layer.

Isolation of high quality RNA from cereal seeds containing high levels of starch.

Abstract: Introduction Cereals are an important source of food, feed and fuel with a rapidly increasing global demand. However, cereal seeds contain high levels of starch and polysaccharides, making the isolation of high quality RNA extremely difficult. Objective To develop a novel method for extracting high quality total RNA from various starch- and polysaccharides-rich cereal seeds, such as maize, rice, sorghum and wheat. Methodology We developed a modified sodium dodecyl sulphate (SDS)/TRIzol method. The combined use of a Tris buffer (pH 9.0) and SDS before TRIzol extraction effectively resolved the problem of seed homogenate solidification in such a buffer. A high concentration of SDS was used separately, not only to promote cell lysis but also to effectively dissolve seed sample containing high levels of starch. Moreover, acid phenol saturated with 0.1 m citrate buffer (pH 4.3) was used to separate RNA from DNAs, proteins and high levels of starch. This rapid protocol was compared with other RNA isolation methods preferentially used for plants rich in polysaccharides and secondary metabolites. Results Gel electrophoresis analysis indicated that the extracted total RNA had good integrity without apparent DNA contamination. Furthermore, an A260/280 ratio of approximately 2.0, an A260/230 ratio of more than 2.0 and RIN values of more than 8.6 indicated that the isolated RNA was of high purity. The isolated RNA was suitable for subsequent molecular manipulations, such as reverse-transcription polymerase chain reaction (PCR), rapid amplification of cDNA ends (RACE) and real-time PCR. Conclusion The study has described an easy, efficient and highly reproducible method for RNA isolation from various cereal seeds. Copyright © 2011 John Wiley & Sons, Ltd.

Streamlined extract preparation for Escherichia coli-based cell-free protein synthesis by sonication or bead vortex mixing.

Abstract: Escherichia coli-based cell extract is a vital component of inexpensive and high-yielding cell-free protein synthesis reactions However, effective preparation of E coli cell extract is limited to high-pressure (French press-style or impinge-style) or bead mill homogenizers, which all require a significant capital investment Here we report the viability of E coli cell extract prepared using equipment that is both common to biotechnology laboratories and able to process small volume samples Specifically, we assessed the low-capital-cost lysis techniques of: (i) sonication, (ii) bead vortex mixing, (iii) freeze-thaw cycling, and (iv) lysozyme incubation to prepare E coli cell extract for cell-free protein synthesis (CFPS) We also used simple shake flask fermentations with a commercially available E coli strain In addition, RNA polymerase was overexpressed in the E coli cells prior to lysis, thus eliminating the need to add independently purified RNA polymerase to the CFPS reaction As a result, high-yielding E coli-based extract was prepared using equipment requiring a reduced capital investment and common to biotechnology laboratories To our knowledge, this is the first successful prokaryote-based CFPS reaction to be carried out with extract prepared by sonication or bead vortex mixing

Stabilizing Additives Added during Cell Lysis Aid in the Solubilization of Recombinant Proteins

Abstract: Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli) appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl(2), proline, xylitol, NDSB 201, CTAB and K(2)PO(4)) solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40%) were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.

Antimicrobial effect and mode of action of terpeneless cold‐pressed Valencia orange essential oil on methicillin‐resistant Staphylococcus aureus

Abstract: Aims: The objectives of this study were to evaluate the antistaphylococcal effect and elucidate the mechanism of action of orange essential oil against antibiotic-resistant Staphylococcus aureus strains. Methods and Results: The inhibitory effect of commercial orange essential oil (EO) against six Staph. aureus strains was tested using disc diffusion and agar dilution methods. The mechanism of EO action on MRSA was analysed by transcriptional profiling. Morphological changes of EO-treated Staph. aureus were examined using transmission electron microscopy. Results showed that 0·1% of terpeneless cold-pressed Valencia orange oil (CPV) induced the cell wall stress stimulon consistent with the inhibition of cell wall synthesis. Transmission electron microscopic observation revealed cell lysis and suggested a cell wall lysis–related mechanism of CPV. Conclusions: CPV inhibits the growth of Staph. aureus, causes gene expression changes consistent with the inhibition of cell wall synthesis, and triggers cell lysis. Significance and Impact of the Study: Multiple antibiotics resistance is becoming a serious problem in the management of Staph. aureus infections. In this study, the altered expression of cell wall-associated genes and subsequent cell lysis in MRSA caused by CPV suggest that it may be a potential antimicrobial agent to control antibiotic–resistant Staph. aureus.

Nanowire-integrated microfluidic devices for facile and reagent-free mechanical cell lysis.

Abstract: Cell lysis is an essential task for the detection of intracellular components. In this work, we introduce novel microfluidic devices integrated with patterned one-dimensional nanostructure arrays for facile and high-throughput mechanical cell lysis. The geometry of the hydrothermally grown ZnO nanowires, characterised by sharp tips and high aspect ratios, aids in anchoring the cell and tearing the plasma membrane, enabling simple and highly efficient extraction of cellular proteins and nucleic acids. This method lyses cells more effectively than conventional chemical lysis methods with simpler equipment and a shorter processing time.

An integrated PCR microfluidic chip incorporating aseptic electrochemical cell lysis and capillary electrophoresis amperometric DNA detection for rapid and quantitative genetic analysis

Abstract: A fully integrated microchip for performing cell lysis, polymerase chain reaction (PCR) and quantitative analysis of DNA amplicons in a single step is described herein. The chip was built on glass substrate using an indium-tin-oxide (ITO) microheater and PDMS engraved microchannels, which integrated an electrochemical cell lysis zone, a continuous flow PCR module and capillary electrophoresis amperometric detection (CE-AD) system. The total length of the microchannel was 4625 mm for performing 25 cycles of flow-through PCR and was laid on a handheld form factor of 96 × 96 mm2 area. The key to the fabrication of such a device lies in the use of a single medium to carry out different kinds of biochemical reactions and hence, a reagentless electrochemical cell lysis protocol was integrated on the microchip which was capable of lysing most cell types, including difficult to lyse gram positive bacteria. The lysate contained genomic DNA from a sample which was proven to be suitable for PCR reactions. Genetic analysis was successfully performed on the microchip with purified lambda phage genomic DNA and various cell types, including non-tumorigenic MCF-10A and tumorigenic MCF-7 human cell lines, gram negative bacteria Escherichia coli O157:H7, and gram positive bacteria Bacillus subtilis, at an optimized flow rate of 5 μl min−1. For the detection of amplicon DNA, a CE-AD system was used, with semisolid alkaline agarose within the capillary microchannel to minimize interference from cell debris and for efficient resolution of DNA fragments. High signal to noise ratio during amperometric detection and the use of online FFT filtering protocol enhanced the limit of detection of DNA amplicons. Therefore, with a combination of portability, cost-effectiveness and performance, the proposed integrated PCR microchip can be used for one step genetic analysis of most of the cell types and will enable more accessible healthcare.

Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells

Abstract: We describe a microfluidic device for the extraction, purification and stretching of human chromosomal DNA from single cells. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane channel was designed to capture a single human cell. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions. Intact chromosomal DNA is entangled in the array, while other cellular components are washed from the channel. To demonstrate the entrapment principle, a single chromosome was hybridized to whole chromosome paints, and imaged by fluorescence microscopy. DNA extracted from a single cell and small cell populations (less than 100) was released from the device by restriction endonuclease digestion under continuous flow and collected for off-chip analysis. Quantification of the extracted material reveals that the microdevice efficiently extracts essentially all chromosomal DNA. The device described represents a novel platform to perform a variety of analyses on chromosomal DNA at the single cell level.

Mechanism of photocatalytic bacterial inactivation on TiO2 films involving cell-wall damage and lysis

Abstract: This article addresses the cell wall damage of Escherichia coil (from now on E. coil) by TiO2 suspensions. The dynamics of TiO2 photocatalysis by thin films layers is described. The films were characterized by FTIR spectroscopy and atomic force microscopy (AFM). The E coil complete inactivation is shown to be due to the partial damage of the cell-wall components (peroxidation). A small increase in the cell wall disorder concomitant with a decrease of the cell wall functional groups leads to higher cell wall fluidity as the precursor step preceding cell lysis. (C) 2012 Elsevier B.V. All rights reserved.

Sonolysis of Escherichia coli and Pichia pastoris in microfluidics

Abstract: We report on an efficient ultrasound based technique for lysing Escherichia coli and Pichia pastoris with oscillating cavitation bubbles in an integrated microfluidic system. The system consists of a meandering microfluidic channel and four piezoelectric transducers mounted on a glass substrate, with the ultrasound exposure and gas pressure regulated by an automatic control system. Controlled lysis of bacterial and yeast cells expressing green fluorescence protein (GFP) is studied with high-speed photography and fluorescence microscopy, and quantified with real-time polymerase chain reaction (qRT-PCR) and fluorescence intensity. The effectiveness of cell lysis correlates with the duration of ultrasound exposure. Complete lysis can be achieved within one second of ultrasound exposure with a temperature increase of less than 3.3 °C. The rod-shaped E. coli bacteria are disrupted into small fragments in less than 0.4 seconds, while the more robust elliptical P. pastoris yeast cells require around 1.0 second for complete lysis. Fluorescence intensity measurements and qRT-PCR analysis show that functionality of GFP and genomic DNA for downstream analytical assays is maintained.

Screening of antibiotic susceptibility to β-lactam-induced elongation of Gram-negative bacteria based on dielectrophoresis.

Abstract: We demonstrate a rapid antibiotic susceptibility test (AST) based on the changes in dielectrophoretic (DEP) behaviors related to the β-lactam-induced elongation of Gram-negative bacteria (GNB) on a quadruple electrode array (QEA) The minimum inhibitory concentration (MIC) can be determined within 2 h by observing the changes in the positive-DEP frequency (pdf) and cell length of GNB under the cefazolin (CEZ) treatment Escherichia coli and Klebsiella pneumoniae and the CEZ are used as the sample bacteria and antibiotic respectively The bacteria became filamentous due to the inhibition of cell wall synthesis and cell division and cell lysis occurred for the higher antibiotic dose According to the results, the pdfs of wild type bacteria decrease to hundreds of kHz and the cell length is more than 10 μm when the bacterial growth is inhibited by the CEZ treatment In addition, the growth of wild type bacteria and drug resistant bacteria differ significantly There is an obvious decrease in the number of wild type bacteria but not in the number of drug resistant bacteria Thus, the drug resistance of GNB to β-lactam antibiotics can be rapidly assessed Furthermore, the MIC determined using dielectrophoresis-based AST (d-AST) was consistent with the results of the broth dilution method Utilizing this approach could reduce the time needed for bacteria growth from days to hours, help physicians to administer appropriate antibiotic dosages, and reduce the possibility of the occurrence of multidrug resistant (MDR) bacteria

Selective lysis of cells by ionic surfactants

Abstract: The present invention discloses methods, kits-of-parts, and devices for the selective lysis of eukaryotic cells in a sample comprising micro-organisms such as bacteria unicellular fungi. The selective lysis is obtained by incubating the sample in an ionic surfactant under alkaline conditions.

Genomic DNA Extraction from Cells by Electroporation on an Integrated Microfluidic Platform

Abstract: The vast majority of genetic analysis of cells involves chemical lysis for release of DNA molecules. However, chemical reagents required in the lysis interfere with downstream molecular biology and often require removal after the step. Electrical lysis based on irreversible electroporation is a promising technique to prepare samples for genetic analysis due to its purely physical nature, fast speed, and simple operation. However, there has been no experimental confirmation on whether electrical lysis extracts genomic DNA from cells in a reproducible and efficient fashion in comparison to chemical lysis, especially for eukaryotic cells that have most of the DNA enclosed in the nucleus. In this work, we construct an integrated microfluidic chip that physically traps a low number of cells, lyses the cells using electrical pulses rapidly, then purifies and concentrates genomic DNA. We demonstrate that electrical lysis offers high efficiency for DNA extraction from both eukaryotic cells (up to ∼36% for Chinese hamster ovary cells) and bacterial cells (up to ∼45% for Salmonella typhimurium) that is comparable to the widely used chemical lysis. The DNA extraction efficiency has dependence on both the electric parameters and relative amount of beads used for DNA adsorption. We envision that electroporation-based DNA extraction will find use in ultrasensitive assays that benefit from minimal dilution and simple procedures.

Characterization and determination of holin protein of Streptococcus suis bacteriophage SMP in heterologous host

Abstract: Holins are a group of phage-encoded membrane proteins that control access of phage-encoded endolysins to the peptidoglycan, and thereby trigger the lysis process at a precise time point as the 'lysis clock'. SMP is an isolated and characterized Streptococcus suis lytic phage. The aims of this study were to determine the holin gene, HolSMP, in the genome of SMP, and characterized the function of holin, HolSMP, in phage infection. HolSMP was predicted to encode a small membrane protein with three hydrophobic transmembrane helices. During SMP infections, HolSMP was transcribed as a late gene and HolSMP accumulated harmlessly in the cell membrane before host cell lysis. Expression of HolSMP in Escherichia coli induced an increase in cytoplasmic membrane permeability, an inhibition of host cell growth and significant cell lysis in the presence of LySMP, the endolysin of phage SMP. HolSMP was prematurely triggered by the addition of energy poison to the medium. HolSMP complemented the defective λ S allele in a non-suppressing Escherichia coli strain to produce phage plaques. Our results suggest that HolSMP is the holin protein of phage SMP and a two-step lysis system exists in SMP.

Protein determinants of phage T4 lysis inhibition.

Abstract: Genetic studies have established that lysis inhibition in bacteriophage T4 infections occurs when the RI antiholin inhibits the lethal hole-forming function of the T holin. The T-holin is composed of a single N-terminal transmembrane domain and a ~20 kDa periplasmic domain. It accumulates harmlessly throughout the bacteriophage infection cycle until suddenly causing permeabilization of the inner membrane, thereby initiating lysis. The RI antiholin has a SAR domain that directs its secretion to the periplasm, where it can either be inactivated and degraded or be activated as a specific inhibitor of T. Previously, it was shown that the interaction of the soluble domains of these two proteins within the periplasm was necessary for lysis inhibition. We have purified and characterized the periplasmic domains of both T and RI. Both proteins were purified in a modified host that allows disulfide bond formation in the cytoplasm, due to the functional requirement of conserved disulfide bonds. Analytical centrifugation and circular dichroism spectroscopy showed that RI was monomeric and exhibited ~80% alpha-helical content. In contrast, T exhibited a propensity to oligomerize and precipitate at high concentrations. Incubation of RI with T inhibits this aggregation and results in a complex of equimolar T and RI content. Although gel filtration analysis indicated a complex mass of 45 kDa, intermediate between the predicted 30 kDa heterodimer and 60 kDa heterotetramer, sedimentation velocity analysis indicated that the predominant species is the former. These results suggest that RI binding to T is necessary and sufficient for lysis inhibition.

Release of intracellular proteins by electroporation with preserved cell viability.

Abstract: Extraction of intracellular proteins from cells is often an important first step for conducting molecular biology and proteomics studies. Although ultrasensitive detection and analytical technology at the single molecule level is becoming routine, protein extraction techniques have not followed suit and still call for complete lysis that leads to cell death. In principle, with refined extraction techniques, intracellular proteins can potentially be extracted without killing the cell. In this Letter, we demonstrate that electroporation is capable of releasing intracellular proteins from adherent Chinese hamster ovary cells while preserving the cell viability. By tuning the duration and intensity of an electric pulse, we were able to control the average amount of protein release and the percentage of viable cells after the operation. Our results indicate that a substantial fraction of the cell population was able to release proteins under electroporation and survive the procedure. Interestingly, at the sing...

Preparation of genomic DNA from mammalian sperm.

Abstract: Semen consists of spermatozoa and the seminal fluid, also called seminal plasma This fluid is important for the survival of the spermatozoa, but may decrease the purity and thus quality of the DNA due to its fructose and protein content In this protocol, spermatozoa are washed with ethanol to remove the fluid The spermatozoa themselves are protected by a membrane rich in disulfide bonds, which impede cell lysis and thus hamper DNA isolation To break disulfide bonds a strong antioxidant, such as dithiothreitol (DDT), is necessary Similar to other protocols for DNA isolation from other mammalian tissues, proteinase K and SDS are included in the lysis reaction After lysis of the sperm cells, the DNA is precipitated using ethanol and then redissolved in TE-buffer or ddH(2)O