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Team:Brasil-SP/Results/Sporulation
From 2014.igem.org
Sporulation and Germination
Who is it?
When we begin to outline our project, we first decided to use Escherichia coli as a chassis. However, looking at Bacillus subtilis more closely we concluded it would be a very better choice to what we aimed.
More than a thousand years ago humans started working with B. subtilis. Japaneses discovered that fermented soybeans could be delicious at breakfast, and for centuries Bacillus subtilis has been used to produce natto1. Nowadays, Bacillus subtilis is also used as a biological factory, to produce enzymes and fine biochemicals. It’s also an interesting host for the heterologous protein production. This Gram +ve bacteria can produce and secrete large amounts of protein to the medium. In addition, the genre Bacillus is noticeable by his high capacity of adsorbing and uptaking exogenous DNA2. This is one of the main reasons why it has been extensively used in both applied and fundamental research in the past decades.
As a model organism, there is available a lot of detailed data about it. Bacillus subtilis was the first Gram-positive bacteria to have its entire genome sequenced and annotated (strain 168)3. Following this project, all essential genes were indentified4. Currently, large-scale data such as transcriptome5, proteome6, secretome7 and metabolome8 are available.
Furthermore, its use is promoted mainly due to the non-pathogenic nature of this organism. It has been awarded the GRAS (Generally Recognized as Safe) status by the US Food and Drug Administration - FDA. In the opposite, Bacillus subtilis is close enough to other clinically relevant Gram-positive pathogens to be used as an organism relevant to research new targets to antimicrobials and anti-infectives2.
Well, Bacillus subtilis can easily uptake exogenous DNA, secrete a lot of proteins in the medium, has been used through centuries and is a safe chassis. Can it be better?
Yes it can.
We haven’t mentioned before what made us choice Bacillus subtilis as our favorite “pet”-chassis and why you should use it too: the endospores.
Bacteria's life can be stressful and complex, needing to face a lot of adversities and we can use this in our favor. In this situation, like starvation, many bacteria trigger a cellular response involving many genes. In Bacillus subtilis this process entails the activity of more than 500 genes and take approximately ten hours.9
In a sweetable growth medium, cells double in length and then divide centrally to produce two identical daughter cells. The sporulation process starts with an asymmetric division. The biggest cell (called mother) engulfs the smallest (called prespore). When this process is completed, the membrane which surrounds the cytoplasm of the prespore (now called the forespore) gets a very amorphous appearance, probably due to the absence of peptidioglycan layer to define the cell shape. Next, a modified form of cell wall, known as cortex, is synthesized between the prespore membranes, providing it with an oblong shape. Simultaneously, the proteinaceous spore coat begins to be deposited on the outside surface of the prespore. The lysis of a mother cell releases the mature spore. The spore is a resistant structure, conceived to resist to hazards like heat, radiation and toxic chemicals, remaining dormant until be exposed to mild conditions when it get germinated. As a commercial product, it’s perfect. The storage and the cells switch on is equally easy, bringing to open-and-shut product.10
The primary question is: exposing our cells to a stressful medium will lead them all to form spores? Certainly no. Sporulation is the ultimate bacteria's decision. And when they finally decide to it, always there are some of them who betray the movement.
Many of other strategies are tried before sporulation. Those include secretion of antibiotics and other chemical weapons to kill other microbes that are in the same niche; the activation of cellular motility by flagella to search for new nutrients source and the secretion of hydrolytic enzymes to remove extracellular polysacharides and proteins. Furthermore, before sporulation starts the cells check chromosome integrity and the state of chromosomal replication, so if the process begins the bacterium is sure that it will be finished.9
Once situation is extreme, cells must choice between three ways: sporulation, competence or cannibalism. Most cells make the commitment to sporulation. However, the genetic material realized by cell lysis isn’t wasted by the colony. Some cells opt for a different state of competence, triggered by ComK exceeding a certain threshold level. If a cell chose this way, it can uptake exogenous DNA resulting from lysis and use it to repair his own DNA or sometimes new genetic information. In the case of Bacillus subtilis approximately 10% of cells choose this path and after approximately 20 h they switch back to the vegetative state. In On the other hand, it has been showed that some bacteria further along to sporulation produce and secrete antibacterial factors that block sibling cells from sporulating. This makes them to lysis. Such cannibalistic cells feed on the nutrients released by the lysed cells. So, in the end, they prevent their own progression toward sporulation by satiating their feeding needs.9
But it's even more important to understand how spore's germination occur. In our device, we provide nutrients to the bacteria. Those nutrients act as germinants. The spores can recognize them receptor proteins encoded by the gerA family of operons, which includes gerA, gerB and gerK. We can induce germination providing alanine (Ala) or a mixture of asparagine, glucose, fructose and potassium ions (AGFK) for example. The first one activate the alanine receptor GerA and the second the asparagine receptor GerB. The interaction between nutrient germinants and receptors triggers the replace of spore core's huge depot of dipicolinic acid and cations by water. This effect triggers the hydrolysis of the spore's peptidoglycan cortex by one of two redundant enzymes. The completion of cortex hydrolysis and subsequent germ cell wall expansion allows spore core hydration. This results in resumption of spore metabolism and macromolecular synthesis.11 Understanding this process we can chose when and how our product will start working.
What we did
Here you can see all our microbiology protocols. Feel free to use all of them. We acknowledge to LMU-Munich team 2012 for… (hyperlink para: https://2012.igem.org/Team:LMU-Munich ). We based our protocols on their work.
In this project, it was used the 168 strain (TROCAR PELA LINHAGEM QUE USAREMOS)
First step was to grow our chassis. Bacillus subtilis grows quickly at Luria-Bertani medium (agar or broth) and it was used to propagate our cultures.
FOTO PLACA DE PETRI COM BACILLUS FORRADA APÓS 24 HORAS
Next, sporulation was induced through extensive culture (18 h) in DSM medium (incluir Fórmula). Then, it was used a proper Spore Count Protocol, resulting in the graphic below:
GRÁFICO A SER CONSTRUÍDO MOSTRANDO ESPORULAÇÃO
TABELA COM OS DADOS DO GRÁFICO, SE NECESSÁRIO
FOTO DOS ESPOROS OBSERVADOS AO MICROSCÓPIO
We could achieve a high sporulation level (XX%). This is very important from industrial outlook. However, it’s important to know spores viability. To induce germination, it was used Luria-Bertani broth. As a rich medium, it induces the receptors before mentioned. The following graphic shows the efficiency of our method:
GRÁFICO A SER CONTRSUÍDO MOSTRANDO ATIVAÇÃO
FOTO DO BACILLUS APÓS TESTE DE GRAM EM DIFERENTES ZOOMS
Our work showed the efficiency of both sporulation and germination. This is very important from industrial view. Once our circuit is finished, it will be simple and cheap to grow Bacillus subtilis cells and to sporulate them. Understanding both processes in theory and practice, we can achieve a high efficiency approaches, which could bring our device to market.
1. SCHALLMEY, M. et al Developments in the use of Bacillus species for industrial production. Canadian Journal of Microbiology, 2004, 50(1): 1-17.
2. ZWEERS, J. C. et al Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes. Microbial Cell Factories, 2008, 7:10. The electronic version of this article is the complete one and can be found online at: http://www.microbialcellfactories.com/content/7/1/10 . Acess: 09/13/2014 3. KUNST, F. et al The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature, 1997, 390:249-256. 4. KOBAYASHI, K. et al Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A, 2003, 100:4678-4683. 5. SIERRO, N. et al DBTBS: a database of transcriptional regulation in Bacillus subtilis containing upstream intergenic conservation information. Nucleic Acids Res, 2007. 6. WOLFF, S. et al Towards the entire proteome of the model bacterium Bacillus subtilis by gel-based and gel-free approaches. J Chromatogr B Analyt Technol Biomed Life Sci, 2007, 849:129-140. 7. TJALSMA, H. Proteomics of protein secretion by Bacillus subtilis: separating the "secrets" of the secretome. Microbiol Mol Biol Rev, 2004, 68:207-233. 8. FISCHER, E., SAUER, U. Large-scale in vivo flux analysis shows rigidity and suboptimal performance of Bacillus subtilis metabolism. Nat Genet, 2005, 37:636-640. 9. SCHULTZ, D. et al Deciding fate in adverse times: Sporulation and competence in Bacillus subtilis. PNAS, December 15, 2009. 106. 10. ERRINGTON, J. Bacillus subtilis Sporulation: Regulation of Gene Expression and Control of Morphogenesist. Microbiological Reviews, Mar. 1993, 57: 1-33 11. SETLOW, P. Spore germination. Current Opinion in Microbiology, December 2003, 6: 550-556.
