IGEM:IMPERIAL/2009/Encapsulation/cell death

For more on cell death see here

Summary
'Table 1. Summary of different Cell death mechanisms'

Methods
Escherichia coli (paper |here)
 * Title: From damaged genome to cell surface: transcriptome changes during bacterial cell death triggered by loss of a restriction–modification gene complex #celldeath1
 * Abstract: Genetically programmed cell deaths play important roles in unicellular prokaryotes. In postsegregational killing, loss of a gene complex from a cell leads to its descendants’ deaths. With type II restriction–modification gene complexes, such death is triggered by restriction endonuclease's attacks on under-methylated chromosomes. Here, we examined how the Escherichia coli transcriptome changes after loss of PaeR7I gene complex. At earlier time points, activation of SOS genes and E-regulon was noticeable. With time, more SOS genes, stress-response genes (including  S-regulon, osmotic-, oxidative- and periplasmic-stress genes), biofilm-related genes, and many hitherto uncharacterized genes were induced, and genes for energy metabolism, motility and outer membrane biogenesis were repressed. As expected from the activation of  E-regulon, the death was accompanied by cell lysis and release of cellular proteins. Expression of several  E-regulon genes indeed led to cell lysis. We hypothesize that some signal was transduced, among multiple genes involved, from the damaged genome to the cell surface and led to its disintegration. These results are discussed in comparison with other forms of programmed deaths in bacteria and eukaryotes.
 * Problem: We dont want to use restriction enzymes and it involves cutting out a gene complex. *Nuri Purswani 10:31, 22 July 2009 (EDT):
 * Other:
 * Title: Bacterial Programmed Cell Death and Multicellular Behavior in Bacteria #celldeath2
 * Abstract: Traditionally, programmed cell death (PCD) is associated with eukaryotic multicellular organisms. However, recently, PCD systems have also been observed in bacteria. Here we review recent research on two kinds of genetic programs that promote bacterial cell death. The first is mediated by mazEF, a toxin–antitoxin module found in the chromosomes of many kinds of bacteria, and mainly studied in Escherichia coli. The second program is found in Bacillus subtilis, in which the skf and sdp operons mediate the death of a subpopulation of sporulating bacterial cells. We relate these two bacterial PCD systems to the ways in which bacterial populations resemble multicellular organisms.
 * Problem: The mazEF system involves having toxins in the stomach.
 * Other: B.subtilis skf/sdp system is an interesting possibility to explore. *Nuri Purswani 10:31, 22 July 2009 (EDT):
 * Title: Enterohemorrhagic Escherichia coli Induces Apoptosis Which Augments Bacterial Binding and Phosphatidylethanolamine Exposure on the Plasma Membrane Outer Leaflet #celldeath3
 * Abstract: Enterohemorrhagic Escherichia coli (EHEC) is a gastrointestinal pathogen that causes watery diarrhea and hemorrhagic colitis and can lead to serious and even fatal complications such as hemolytic uremic syndrome. We investigated the ability of EHEC to kill host cells using three human epithelial cell lines. Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA and morphological changes detected by electron microscopy changes revealed evidence of apoptotic cell death. The rates and extents of cell death were similar for both verotoxin-producing and nonproducing strains of EHEC as well as for a related gastrointestinal pathogen, enteropathogenic E. coli (EPEC). The induction of apoptosis by bacterial attachment was independent of verotoxin production and greater than that produced by a similar treatment with verotoxin alone. Expression of phosphatidylethanolamine, previously reported to bind EHEC and EPEC, was also increased on apoptotic cells but with little correlation to phosphatidylserine expression. Phosphatidylethanolamine levels but not phosphatidylserine levels on dying cells correlated with EHEC binding. Cells treated with phosphatidylethanolamine-containing liposomes also showed increased EHEC binding. These results suggest that bacterial induction of apoptosis offers an advantage for bacterial attachment by augmenting outer leaflet levels of the phosphatidylethanolamine receptor.
 * Problem: I believe this is talking about how E. coli cells induce apoptosis in host cells; not in themselves. ~ Tom Adie 15:49, 23 July 2009 (EDT)
 * Other: *Nuri Purswani 10:31, 22 July 2009 (EDT):
 * Title: The Extra-cellular Death Factor (EDF): Physiological and genetic factors influencing its production and response in
 * Abstract: Gene pairs specifying for a toxin and its antitoxin (TA) are called toxinantitoxin modules and are found on the chromosomes of many bacteria. The most studied of these modules is Escherichia coli mazEF in which mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. In a previous report from this laboratory it was shown that mazEF-mediated cell death is a population phenomenon requiring a quorum sensing peptide that we call the Extracellular Death Factor (EDF). EDF is the linear penta-peptide NNWNN (31). Here we further confirmed that EDF is a signal molecule in a mixed population. In addition, we characterized some physiological conditions and genes required for EDF production and response. Furthermore, stress response and the gene specifying for MazEF, Zwf (Glucose-6-phosphate dehydrogenase) and the protease ClpXP are critical in EDF production. Significant strain difference in EDF production and response explain variation in the induction of mazEF-mediated cell death
 * David Roche 11:01, 22 July 2009 (EDT):
 * Title: MazF-Mediated Cell Death in Escherichia coli: a Point of No Return #celldeath4
 * Abstract: mazEF is a stress-induced toxin-antitoxin module, located on the chromosome of Escherichia coli, that we have previously described to be responsible for programmed cell death in E. coli. mazF specifies a stable toxin, and mazE specifies a labile antitoxin. Recently, it was reported that inhibition of translation and cell growth by ectopic overexpression of the toxin MazF can be reversed by the action of the antitoxin MazE ectopically overexpressed at a later time. Based on these results, it was suggested that rather than inducing cell death, mazF induces a state of reversible bacteriostasis (K. Pederson, S. K. Christensen, and K. Gerdes, Mol. Microbiol. 45:501-510, 2002). Using a similar ectopic overexpression system, we show here that overexpression of MazE could reverse MazF lethality only over a short window of time. The size of that window depended on the nature of the medium in which MazF was overexpressed. Thus, we found "a point of no return," which occurred sooner in minimal M9 medium than it did in the rich Luria-Bertani medium. We also describe a state in which the effect of MazF on translation could be separated from its effect on cell death: MazE overproduction could completely reverse the inhibitory effect of MazF on translation, while not affecting the bacteriocidic effect of MazF at all. Our results reported here support our view that the mazEF module mediates cell death and is part of a programmed cell death network. **Problem: See table
 * Nuri Purswani 04:30, 23 July 2009 (EDT):
 * Title: The restriction–modification genes of Escherichia coli K-12 may not be selfish: They do not resist loss and are readily replaced by alleles conferring different specificities #celldeath5


 * Abstract: Type II restriction and modification (R-M) genes have been described as selfish because they have been shown to impose selection for the maintenance of the plasmid that encodes them. In our experiments, the type I R-M system EcoKI does not behave in the same way. The genes specifying EcoKI are, however, normally residents of the chromosome and therefore our analyses were extended to monitor the deletion of chromosomal genes rather than loss of plasmid vector. If EcoKI were to behave in the same way as the plasmid-encoded type II R-M systems, the loss of the relevant chromosomal genes by mutation or recombination should lead to cell death because the cell would become deficient in modification enzyme and the bacterial chromosome would be vulnerable to the restriction endonuclease. Our data contradict this prediction; they reveal that functional type I R-M genes in the chromosome are readily replaced by mutant alleles and by alleles encoding a type I R-M system of different specificity. The acquisition of allelic genes conferring a new sequence specificity, but not the loss of the resident genes, is dependent on the product of an unlinked gene, one predicted [Prakash-Cheng, A., Chung, S. S. & Ryu, J. (1993) Mol. Gen. Genet. 241, 491–496] to be relevant to control of expression of the genes that encode EcoKI. Our evidence suggests that not all R-M systems are evolving as “selfish” units; rather, the diversity and distribution of the family of type I enzymes we have investigated require an alternative selective pressure.
 * Nuri Purswani 04:42, 23 July 2009 (EDT):

Biobricks
Helpful papers: RE1, RE2
 * Restriction_enzymes
 * Useful resource
 * Requirements:
 * 1) Use a 4-cutter (enzyme that cleaves short sequences) with tight regulation
 * 2) Flank genes with cleavage sites; include cleavage sites within the genes and other components of the construct
 * 3) Use a bistable switch to control basal levels of restriction enzyme gene expression??
 * 4) Use REs that produce blunt ends to reduce likelihood of re-ligation
 * Potential restriction enzymes:

Papers

 * 1) celldeath1 pmid=19304752
 * 2) celldeath2 pmid=17069462
 * 3) celldeath3 pmid=10816451
 * 4) celldeath4 pmid=15576778
 * 5) celldeath5 The restriction–modification genes of Escherichia coli K-12 may not be selfish: They do not resist loss and are readily replaced by alleles conferring different specificities,Mary O’Neill, Angela Chen, and Noreen E. Murray
 * 6) RE1 pmid=1920265
 * 7) RE2 pmid=16408021