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| − | <b style="font-size:16px;">Containment: </b>
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| − | <br><br>Ours project involved release of genetically modified bacteria in the cave. Lascaux cave is not a completely closed system; there is interaction with external environment due to water infiltration. The threat of releasing bacteria in the cave is the possibility of horizontal gene transfer to native bacteria. The horizontal gene transfer (or lateral gene transfer) is transmission of DNA and it is known to occur between different species. Here the aim of the containment module was to provide transfer of non natural genetic content to endogenous cave bacteria
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| − | <br><br>Paleotilis project involve more than 15 kb of gene therefore a two plasmid construction was necessary. To provide any of these plasmids dissemination a double toxin-antitoxin was designed (Figure1). One plasmid contains antitoxin 1 and toxin 2, the other contains antitoxin 2 and toxin 1. This construction divide toxin-antitoxin couples, the toxin is on a different plasmid then it corresponding antitoxin. Through this system both plasmids are compulsory for bacteria survival. If endogenous bacteria of the cave received a single plasmid, toxin expression will lead to death. In the same time this design obligates the cell to maintain both (or none) plasmids.
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| − | <br><br>The toxin –antitoxin are under the control of a constitutive promoter of Bacillus subtilis: Pveg (BBa_K143012), to assure a constant expression of the toxin (Figure2). The two pairs of Toxin-Antitoxin combinations selected for this project are MazF/MazE and Zeta/Epsilon. Both are type II Toxin-Antitoxin systems, meaning that antitoxin and toxin are proteins and the protein toxin is inhibited by the binding of a protein antitoxin (Wang et al., 2013)
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| − | <br><br>Figure 1: Design of the containment system of paleotilis
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| − | <br><br><b style="font-size:16px;">MazF /MazE toxin-antitoxin couple</b>
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| − | <br><br>The mazEF operon was the first Toxin-Antitoxin system found on the Escherichia coli chromosome. It is related to the kis/kid module on plasmid R1 (Bravo et al., 1987). MazF is an endoribonuclease which specifically cleaves mRNAs at ACA sequences (Zhang et al., 2005). In Escherichia coli mazF inhibits protein synthesis by cleaving mRNAs. The excess of MazF toxin (in case of single plasmid) provide by our system will lead to bactericidal effect.
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| − | <br><br><b style="font-size:16px;">Epsilon/Zeta toxin-antitoxin couple</b>
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| − | <br><br>The Epsilon and Zeta Toxin-antitoxin system was discovered in Streptococcus pyogenes more precisely in the low-copy-number plasmid pSM19035, its function is to stabilize plasmid segregation. The zeta toxin has shown toxic effects on Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae (Zielenkiewicz and Cegłowski, 2005). The zeta toxins phosphorylate the peptidoglycane precursor that inhibits the bacterial peptidoglycane synthesis (Mutschler et al., 2011). Without abilities to cell wall synthesis bacteria are lyses.
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| − | <br><br><b style="font-size:16px;">Cloning approach:</b>
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| − | <br><br>We synthesized our constructions thanks to IDT gblock. Each fragment contains a toxin but not its corresponding antitoxin. Therefore building our plasmids needed a strategy to avoid cell death. We chose to use theophylline sensitive riboswitch. In the absence of the ligand RBS sequence is available and RNA translation is possible. In ligand presence RNA shape change and RBS is not accessible (Topp and Gallivan, 2008). In this case theophylline riboswitch is among our toxin sequence. Theophylline presence represses the toxin expression. These conditions allow us to transform our bacteria with a single plasmid in a media containing 1mM Theophylline.
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| − | <br><br><b style="font-size:16px;">References </b>
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| − | <br><br>Bravo, A., de Torrontegui, G., and Díaz, R. (1987). Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid. Mol. Gen. Genet. MGG 210, 101–110.
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| − | <br>Mutschler, H., Gebhardt, M., Shoeman, R.L., and Meinhart, A. (2011). A novel mechanism of programmed cell death in bacteria by toxin-antitoxin systems corrupts peptidoglycan synthesis. PLoS Biol. 9, e1001033.
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| − | <br>Topp, S., and Gallivan, J.P. (2008). Riboswitches in unexpected places—A synthetic riboswitch in a protein coding region. RNA 14, 2498–2503.
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| − | <br>Wang, X., Lord, D.M., Hong, S.H., Peti, W., Benedik, M.J., Page, R., and Wood, T.K. (2013). Type II Toxin/Antitoxin MqsR/MqsA Controls Type V Toxin/Antitoxin GhoT/GhoS. Environ. Microbiol. 15, 1734–1744.
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| − | <br>Zhang, Y., Zhang, J., Hara, H., Kato, I., and Inouye, M. (2005). Insights into the mRNA Cleavage Mechanism by MazF, an mRNA Interferase. J. Biol. Chem. 280, 3143–3150.
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| − | <br>Zielenkiewicz, U., and Cegłowski, P. (2005). The Toxin-Antitoxin System of the Streptococcal Plasmid pSM19035. J. Bacteriol. 187, 6094–6105.
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| − | http://parts.igem.org/Part:BBa_K1493602
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| − | <br>http://parts.igem.org/Part:BBa_K1096001
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| − | <b>Predation module</b>
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| − | <br><br>We designed this part of the project to find a way to provide nutrients to our Paleotilis bacteria. Indeed, it is not possible to add organic matter in the cave or it would modify the microbiota in an uncontrolled manner. As <i>Bacillus subtilis</i> presents cannibalistic and predatory behaviour in specific conditions, we aimed to express it constitutively: our Paleotilis would be able to kill <i>Pseudomonas fluorescens</i> naturally present in the cave and use the nutrients released to grow.
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| − | <br>Expression of atypical operons during a step of <i>B. subtilis</i> cell cycle: the sporulation
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| − | <br>Prokaryotes are well known to have developed ingenious strategies to survive best under stressful conditions. <i>Bacillus subtilis</i> is no exception to this phenomenon: when it finds itself in a medium with very few nutrients, it enters in sporulation.
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| − | <br>Sporulation is a development process that transforms a growing cell in a dorming cell called spore or endospore. The particularity of this spore is that it can survive under very harsh conditions: no nutrient source, extreme temperatures and even radiations. It has been determined one protein has a key role in sporulation: Spo0A. It is a DNA-binding protein from the family of transcription factors able to repress or activate several genes. Spo0A has to be phosphorylated to be activated in order to do so <i>B. subtilis</i> uses a multicomponent phosphorelay composed of five histidine autokinases and two phosphorelay proteins, Spo0F and Spo0B. The kinases transfer phosphoryl groups into the phosphorelay by phosphorylating first Spo0F which gives itself the phosphoryl group to Spo0B and then Spo0B transfers it to Spo0A. But it is important to note that the phosphorylation level of Spo0A is also influenced by phosphatases that dephosphorylase both Spo0A and Spo0F.
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| − | <br>Once activated, Spo0A bounds to DNA on a specific sequence named 0A-box from where it induces or represses the genes. Before 2003 only eighteen genes were known to be regulated by Spo0A, but with the development of new technologies (microarrays, biocomputation and transcriptional profiling in particular), scientists have identified in total 121 genes organized in 30 single-gene units and 24 operons under the control of Spo0A. (Molle et al, 2003)
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| − | <br>When <i>B. subtilis</i> detects a lack of nutrients in the medium, Spo0A is activated and it enters in sporulation.
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| − | <br><br>This phenomenon consists in an asymmetric division of the cell resulting in the formation of a forespore and of a mother cell that follow different pathways. The mother cell swallows the forespore and contributes to it transformation in spore by being its nutrient source. Thus, the forespore can acquire its cortex and coat that will protect the spore. When the transformation is complete, the mother cell lyses. This process, rather complicated considering the number of genes involved, takes several hours and costs a lot of energy. Also, once the cells have finished the asymmetric division, they have to go through the all transformation to a spore even if a nutrient source is suddenly added to the medium. This last inconvenient is the reason why <i>B. subtilis</i> developed strategies to delay sporulation. (Gonzalez-Pastor, 2011)
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| − | <br>Among the Spo0A regulated genes, two operons have catch the attention because coding for proteins very alike to antibiotics. In fact <i>B. subtilis</i> is well known for its antibiotic production as 4-5% of its genome is dedicated to it. In total, twelve antibiotics have been identified, but one strain does not produce them all (Stein, 2005). The two antibiotics regulated by Spo0A are a sporulation killing factor (skf) and a sporulation delay protein (sdp). They are strongly induced at the beginning of sporulation. Without Spo0A, their expression rate is insignificant but there are fully activated with low concentrations of Spo0A. This means there are part of the low threshold genes of the Spo0A regulon, in contrast with the high threshold ones, and they are expressed in the early stage of sporulation. Also it appears that there are inhibited in presence of high levels of Spo0A. (Fujita et al, 2005)
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| − | <br><br>Structure and functions of the genes expressed in sdp and skf operons
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| − | <br>The genes coding for the killing factor and the delay protein are in fact part of two operons named skf and sdp that are constituted with several genes each one: skf A, B, C, D, E, F, G, H and sdp A, B, C, D, I, R. To determine the specific role of each genes and of the whole operons, several strategies have been used: comparison with similar operons already characterized, mutagenesis and imaging mass-spectrometry.
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| − | <br>The activity of the sdp and skf operons have been highlighted in 2003 during the study of genes regulated by Spo0Aby building mutants of these genes (Gonzalez-Pastor et al, 2003). The deletion of the two operons resulted in an acceleration of the sporulation process. When studying the gene sequences, it appeared that skfA encoded for a small peptide, probably an antibiotic. skfD contained a characteristic domain from a amino terminal protease family. Also skfE and skfF seemed to conduct to the production of an ATP-binding cassette transport complex, also named ABC transporter: their role is to export the antibiotic peptide as well as conferring resistance to it. An interesting result obtained by the authors was that in a mixed culture of <i>B. subtilis</i> Wild-Type and mutants of skf (with deletion of the operon) after onset of the sporulation, the ratio mutants to wild-type cells dropped significantly. This demonstrated one part of the operon produces an extracellular killing factor while another part confers resistance to it. Further investigations showed that skfE and skfF are responsibles of the resistance.
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| − | <br> The sdp operon was properly described later and it appeared to be composed of two convergent operons with different functions: sdpABC and sdpIR (Ellermeier et al, 2006; Gonzalez-Pastor, 2011). sdpABC is involved in the production and secretion of peptide coming from the C-terminal of sdpC. At first, sdpC was suspected to have a role in the delaying of sporulation (Gonzalez-Pastor et al, 2003) but them it was proven to be a toxin expressed at the early stage of sporulation. Thus, sdpIR produces proteins responsible for the immunity to the toxin and their transcription is altered when sdpC is not produced by the cell. SdpI correspond to membrane protein and confers immunity to SdpC while SdpR is similar to a family of regulators, the ArsR. In fact SdpR regulate the sdpIR operon by binding to the promoter and acting as an auto repressor that inhibits the transcription in absence of SdpC production.
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| − | <br><br>The sdp operon has been most studied afterward, for example, by comparison to known antibiotics, SdpC has been proven to collapse proton motive force of the cells (Lamsa et al, 2012). The reason to it is that sdp has a much greater effect than skf (Liu et al, 2010).
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| − | <br><br>To conclude, the toxins produced by sdp and skf set in motion the lysis of non-resistant cells. As the sporulation is a complex phenomenon, in a population all the individuals do not sporulate at the same time, the level of sporulation within the population is even pretty heterogenous: the sporulating ones produce SkfA and SdpC and the proteins for the resistance associated. The non-sporulating cells however do not and they will be lysed by the toxins. This way, they release nutrients in their environment that will be used by the other cells to overcome the nutrient deficiency. This cannibalism is a manner to delay the costing process of sporulation in case another source of nourishment would appear because in this case the sporulating cells would be disadvantaged comparing to cells able to resume a vegetative growth right away.
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| − | <br>The interesting thing about these toxins is that they also are efficient against other bacteria and not only against <i>B. subtilis</i>. In particular, it is effective against <i>Pseudomonas fluorescens</i> (Nandy et al, 2006) which is naturally present in the Lascaux cave (Martin-Sanchez et al, 2011). For these reasons, we thought about constitutively express these operons to enable <i>B. subtilis</i> to always predate strains like <i>P. fluorescens</i> to make it a nutrient source.
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| − | <br><br>Ellermeier, Craig D., Errett C. Hobbs, Jose E. Gonzalez-Pastor, and Richard Losick. “A Three-Protein Signaling Pathway Governing Immunity to a Bacterial Cannibalism Toxin.” Cell 124, no. 3 (February 10, 2006): 549–59. doi:10.1016/j.cell.2005.11.041.
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| − | <br>Fujita, Masaya, José Eduardo González-Pastor, and Richard Losick. “High- and Low-Threshold Genes in the Spo0A Regulon of Bacillus Subtilis.” Journal of Bacteriology 187, no. 4 (February 15, 2005): 1357–68. doi:10.1128/JB.187.4.1357-1368.2005.
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| − | <br>González-Pastor, José Eduardo. “Cannibalism: A Social Behavior in Sporulating Bacillus Subtilis.” FEMS Microbiology Reviews 35, no. 3 (May 1, 2011): 415–24. doi:10.1111/j.1574-6976.2010.00253.x.
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| − | <br>González-Pastor, José E., Errett C. Hobbs, and Richard Losick. “Cannibalism by Sporulating Bacteria.” Science (New York, N.Y.) 301, no. 5632 (July 25, 2003): 510–13. doi:10.1126/science.1086462.
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| − | <br>Lamsa, Anne, Wei-Ting Liu, Pieter C. Dorrestein, and Kit Pogliano. “The Bacillus Subtilis Cannibalism Toxin SDP Collapses the Proton Motive Force and Induces Autolysis.” Molecular Microbiology 84, no. 3 (May 2012). doi:10.1111/j.1365-2958.2012.08038.x.
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| − | <br>Liu, Wei-Ting, Yu-Liang Yang, Yuquan Xu, Anne Lamsa, Nina M. Haste, Jane Y. Yang, Julio Ng, et al. “Imaging Mass Spectrometry of Intraspecies Metabolic Exchange Revealed the Cannibalistic Factors of Bacillus Subtilis.” Proceedings of the National Academy of Sciences of the United States of America 107, no. 37 (September 14, 2010): 16286–90. doi:10.1073/pnas.1008368107.
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| − | <br>Martin-Sanchez, P. M., F. Bastian, A. Nováková, E. Porca, V. Jurado, S. Sanchez-Cortes, E. Lopez-Tobar, et al. “Écologie Microbienne de La Grotte de Lascaux,” January 1, 2011. https://www.researchgate.net/publication/257958803_Ecologie_Microbienne_de_la_Grotte_de_Lascaux.
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| − | <br>Molle, Virginie, Masaya Fujita, Shane T. Jensen, Patrick Eichenberger, José E. González-Pastor, Jun S. Liu, and Richard Losick. “The Spo0A Regulon of Bacillus Subtilis.” Molecular Microbiology 50, no. 5 (December 1, 2003): 1683–1701. doi:10.1046/j.1365-2958.2003.03818.x.
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| − | <br>Nandy, Subir Kumar, Prashant M. Bapat, and K. V. Venkatesh. “Sporulating Bacteria Prefers Predation to Cannibalism in Mixed Cultures.” FEBS Letters 581, no. 1 (January 9, 2007): 151–56. doi:10.1016/j.febslet.2006.12.011.
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| − | <br>Stein, Torsten. “Bacillus Subtilis Antibiotics: Structures, Syntheses and Specific Functions.” Molecular Microbiology 56, no. 4 (May 1, 2005): 845–57. doi:10.1111/j.1365-2958.2005.04587.x.
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| − | <br><br>Antifungal compounds
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| − | <br><br>The Paleotilis main part is the destruction of the fungi which grow on the Lascaux frescos. To achieve this idea, we design two genetics modules, Antifungal A and Antifungal B, with four different antifugal peptides. These compounds are chosen according to their activities and their different targets. We wanted too antifungals with a broad spectrum. All these criterions must permit to minimise the fungi resistance.
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| − | <br><br>D4E1 is a synthetic peptide analog to Cecropin B AMPs (AntiMicrobial Peptides) made of 17 amino acids which has been shown to have an antifungal activity by complexing with a sterol present in the conidia’s wall of numerous fungi.
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| − | <br><br>Dermaseptin-b1 is a 78 amino acids from Phyllomedusa bicolor which it has antifungal activities against filamentus fungus. This peptide is membranotropic and depolarizes the fungi plasma membrane.
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| − | <br><br>GAFP-1 (Gastrodia Anti Fungal Protein 1), also known as gastrodianin, is a mannose and chitin binding lectin originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years. GAFP-1 accumulates in nutritive corms where the fungal infection takes place, and in vitro assays demonstrated it can inhibit the growth of ascomycete and basidiomycete fungal plant pathogens.
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| − | <br><br>Metchikowin is a 26 residus prolin-rich peptid from Drosophilia melanogaster. This action way rest unknow for now but tests prooved that it is a board spectrum antifungal petide against Ascomycete and Basidiomycete. It also has gram positive antibacterial activity. During its expression, it is cleaved in the endoplasmic reticulum. We do not know if cleavage is essential for the antifungal activity so we choose to integrate two forms of this peptide, the cut one and the complet one.
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| − | <br><br>Image des constructions
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| − | <br><br>Parts : BBa_K1937007(AF_A) + celle avec pBSOK ?
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| − | <br><br>Secretion :
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| − | <br><br>For the secretion of these antifungal compounds, we put on the N-terminal end of the coding sequences a signal peptide call AmyE. This peptide is cleaved during the secretion process.
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| − | <br><br>Expression :
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| − | <br><br>We would like to express the antifungals in answer of the fungi presence. To do that, two N acetyl glucosamine sensitive promotors, PnagA and PnagP, were used because the N acetyl glucosamine is a major fungi chitin compound.
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| − | <br><br>PnagA is the NagA gene promotor. PnagP is the NagP gene promotor. These gene are expressed in the N-acetylglucosamine- 6-phosphate- deacetylasein presence in Bacillus
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| − | subtilis. These gene are from the NagR regulon, a negative transcriptional regulon of the N-acetyl-gluosamine catabolisme.
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| − | <br><br>Parts : BBa_K1937003 (pNagA), BBa_K1937005 (pNagP) + celles avec pBSOK ?
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| − | <br><br>Image des constructions
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| − | <br><br>References :
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| − | <br><br>Bertram R, Rigali S, Wood N, Lulko AT, Kuipers OP & Titgemeyer F (2011) Regulon of the N-acetylglucosamine utilization regulator NagR in Bacillus subtilis. J. Bacteriol. 193: 3525–3536
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| − | <br><br>De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE & Walsh TJ (1998) Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1. Can. J. Microbiol. 44: 514–520
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| − | <br><br>Fleury Y, Vouille V, Beven L, Amiche M, Wróblewski H, Delfour A & Nicolas P (1998) Synthesis, antimicrobial activity and gene structure of a novel member of the dermaseptin B family. Biochim. Biophys. Acta 1396: 228–236
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| − | <br><br>Levashina EA, Ohresser S, Bulet P, Reichhart J-M, Hetru C & Hoffmann JA (1995) Metchnikowin, a Novel Immune-Inducible Proline-Rich Peptide from Drosophila with Antibacterial and Antifungal Properties. Eur. J. Biochem. 233: 694–700
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| − | <br><br>Matejuk A, Leng Q, Begum MD, Woodle MC, Scaria P, Chou S-T & Mixson AJ (2010) Peptide-based Antifungal Therapies against Emerging Infections. Drugs Future 35: 197
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| − | <br><br>Wang X, Bauw G, Van Damme EJ, Peumans WJ, Chen ZL, Van Montagu M, Angenon G & Dillen W (2001) Gastrodianin-like mannose-binding proteins: a novel class of plant proteins with antifungal properties. Plant J. Cell Mol. Biol. 25: 651–661
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