Lysis

About: Lysis is a research topic. Over the lifetime, 6072 publications have been published within this topic receiving 216978 citations.

Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture

Abstract: The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h−1 and by 3 orders of magnitude at a lower dilution rate (0.327 h−1). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h−1 and persisted until the end of the experiment (∼200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.

The effect of chlorhexidine on the electrophoretic mobility, cytoplasmic constituents, dehydrogenase activity and cell walls of Escherichia coli and Staphylococcus aureus

Abstract: Chlorhexidine does not cause lysis of isolated cell walls, nor does it prevent the synthesis of the mucopeptide component of the cell wall. Low concentrations of the drug stimulate dehydrogenase activity but higher concentrations inhibit the activity. Chlorhexidine reacts with and precipitates proteinaceous and pentose-containing components of a solution of cell-free cytoplasmic constituents in concentrations greater than those causing their maximum leakage. The effect of chlorhexidine concentration on the electrophoretic mobility of bacterial cells is consistent with the hypothesis that the drug accumulates in aggregates at the cell surface rather than in the form of a monolayer or multilayers of drug.

Induction of target cell DNA release by the cytotoxic T lymphocyte granule protease granzyme A.

Abstract: The rapid breakdown of target cell DNA during CTL-mediated lysis has been difficult to explain by the granule exocytosis model of cytotoxicity. The involvement of CTL granule proteases in this process was strongly suggested by experiments in which CTL were pretreated with the serine protease inhibitor PMSF, in combination with agents that raise the pH of acidic intracellular compartments. While PMSF pretreatment alone had little effect on target lysis or DNA breakdown, the combination of PMSF and NH4Cl or monensin profoundly reduced target cell DNA release, while little effect was observed on target lysis, as measured by 51Cr release. CTL granule extracts cause release of 125I-DNA from detergent-permeabilized cells. This nuclear DNA-releasing (NDR) activity is inhibited by serine esterase inhibitors that also inhibit the granule BLT-esterase activity, and is specifically immunoabsorbed by antibodies to the CTL granule protease granzyme A. The NDR activity comigrates with BLT-esterase activity during subcellular fractionation, solubilization, gel filtration, and aprotinin-Sepharose affinity chromatography. SDS-PAGE analysis of the affinity-purified product indicates a molecular mass of 60,000 daltons under non-reducing conditions, which moves to 30,000 daltons upon reduction, consistent with previously reported behavior of granzyme A. When the purified material was reduced and alkylated, both esterase and NDR activities comigrated at 30,000 daltons upon gel filtration. Although fully lytic concentrations of purified LGL granule cytolysin alone failed to induce target cell DNA release, a combination of purified granzyme A and the cytolysin induces substantial DNA release.

Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation

Abstract: All molecular analyses of soil bacterial diversity are based on the extraction of a representative fraction of cellular DNA. Methods of DNA extraction for this purpose are divided into two categories: those in which cells are lysed within the soil (direct extraction) and those in which cells are first removed from soil (cell extraction) and then lysed. The purpose of this study was to compare a method of direct extraction with a method in which cells were first separated from the soil matrix by Nycodenz gradient centrifugation in order to evaluate the effect of these different approaches on the analysis of the spectrum of diversity in a microbial community. We used a method based on polymerase chain reaction (PCR) amplification of a 16S rRNA gene fragment, followed by hybridization of the amplified fragments to a set of specific probes to assess the phylogenetic diversity of our samples. Control parameters, such as the relationship between amount of DNA template and amount of PCR product and the influence of competing DNA on PCR amplification, were first examined. Comparison between extraction methods showed that less DNA was extracted when cells were first separated from the soil matrix (0.4 microg g(-1) dry weight soil versus 38-93 microg g(-1) obtained by in situ lysis methods). However, with the exception of the gamma-subclass of Proteobacteria, there was no significant difference in the spectrum of diversity resulting from the two extraction strategies.

Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils.

Abstract: Aim: To establish a rapid, improved soil environmental DNA extraction and purification protocol. Methods and Results: Three different soil DNA isolation and four purification strategies were compared on different soil samples with variable rates of success. Bead beating extraction gave significantly higher DNA yields than microwave-based and liquid nitrogen grinding DNA extraction methods. The inclusion of soil washing prior to cell lysis decreased the amount of purification steps required. Although these soil types differed, polyvinylpolypyrrolidone (PVPP)-sepharose 2B column elution was sufficient for all three samples, yielding DNA pure enough for successful application in molecular studies. One soil sample retained 80% of the initial DNA after successful purification. Conclusions: Optimization of a purification protocol confirmed that only a combination of previously described methods proved sufficient in yielding pure environmental DNA from humic-rich soils. Total processing time for DNA extraction and subsequent purification from multiple samples was considerably more rapid than the previously described methods. Significance and Impact of the Study: This study developed a new optimized soil DNA extraction and purification protocol that is suitable for different environmental sources that are rich in humic acid content.