Team:Fatih Turkey/Biofilm

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BIOFILM

BIOFILM

INTRODUCTION
In our project we want to apply our proteins (Reflectin 1A and anti-LPS factor) on a surface as a coat; because our aim is to obtain an anti-gram negative bacterial surface with the help of our anti-LPS protein. We use Bacillus Subtilis as a host; because this bacteria is expected to produce biofilm containing the anti-LPS protein that can be applied on a surface and this might be the best coating material for our project. Also, by killing spores of B.subtilis on the biofilm surface with aqueous dissolved oxygen, ascorbic acid, and copper ions (link verilecek) , we will try to produce a coating material which will possess a protective feature against infectious gram negative bacteria.

BIOFILM FORMATION

Bacteria are able to grow adhered to almost every surface, forming architecturally complex communities termed biofilm. In biofilm, cells grow in multicellular aggregates that are encased in an extracellular matrix produced by the bacteria themselves (1). The extracellular polymeric matrix is an important structural component of biofilm and it plays an important role in the attachment and colonization of microorganisms on a surface also acts as a diffusion barrier to small molecules. Related to this, in biofilm the diffusion of nutrients, vitamins, or cofactors is slower resulting in a bacterial community in which some of cells are metabolically inactive. (2). Bacillus subtilis forms biofilm whose constituent cells are held together by the extracellular matrix and one of the main matrix competent is the protein TasA which is a form of amyloid fibers and binds cells together in the biofilm (3). The matrix, which is composed of polysaccharides, proteins, nucleic acids and water, enables the biofilm to attach to the surfaces. One of the most important functions of the matrix is to protect the bacteria from various stress and factors such as UV radiation, extreme pH values, osmotic pressure, dehydration and antibiotics(4).

QUORUM SENSING
In the biofilm formation bacteria can talk each other via some signal mechanisms. And those mechanisms are called “quorum sensing”.
Quorum or diffusion responses in bacteria are mediated by secreted signalling molecules that accumulate extracellularly as cultures grow to high density. The regulatory response to these signalling molecules can result in dramatic changes in gene expression. In Bacillus subtilis, a quorum response is mediated by a secreted 10-amino-acid modified peptide (ComX pheromone) that activates a receptor histidine kinase (ComP) that activates a response regulator transcription factor (ComA). ComA directly activates the srfA operon which encodes enzymes needed for the production of the lipopeptide surfactin.(5)
In Bacillus subtilis, quorum responses contribute to the induction of competence development, sporulation, degradative enzyme production and antibiotic production (6). The ComX–ComP–ComA signalling pathway is a major quorum response pathway in B. Subtilis and regulates the development of genetic competence (7).

During biofilm formation in B. subtilis, most cells produce and secrete ComX. A subset of these cells become surfactin producers and secrete surfactin and a distinct population that does not itself synthesize surfactin responds to this surfactin and generates the extracellular matrix.

Microbial Interactions: Bacteria Talk to (Some of) Their Neighbors Ishita M. Shah and Jonathan Dworkin
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA

FLAKY BIOFILM
In our project, our goal is to get an easily applied anti- gram negative coat to a surface.Nevertheless; agar does not have a vicious consistency; therefore, it cannot be easily applied on a surface. For this reason; we perform a useful method which is called "biofilm paste" (benefited from Team Groningen 2010). In this method, we use corn starch with liquid LB medium. It is seen that our wild type B. subtilis grows up very fast with the corn starch. We use 2 g corn starch with 50 ml LB medium. After 3 days waiting at 37 oC we get our flaky biofilm .

REFERENCES
-Biofilms; Daniel López, Hera Vlamakis and Roberto Kolter Cold Spring Harb Perspect Biol 2010;2:a000398 originally published online June 2, 2010
1-Branda SS, Vik S, Friedman L, Kolter R. 2005. Biofilms: The matrix revisited. Trends Microbiol 13: 20–26.
2- Anderson GG, O’Toole GA. 2008. Innate and induced resistance mechanisms of bacterial biofilms. in Bacterial Biofilms (ed. Romeo T.), pp. 85–105. Springer, Heidelberg.
3-Amyloid fibers provide structural integrity to Bacillus subtilis biofilms. Romero, Diego; Aguilar, Claudio; Losick, Richard; Kolter, Roberto Proceedings of the National Academy of Sciences of the United States of America
4-BİYOFİLMLER: YÜZEYLERDEKİ MİKROBİYAL YAŞAM; İlhan Gün*1, Fatma Yeşim Ekinci2
5-Conservation of genes and processes controlled by the quorum response in bacteria: characterization of genes controlled by the quorum-sensing transcription factor ComA in Bacillus subtilis Natalia Comella and Alan D. Grossman* Department of Biology, Building 68-530, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
6- Grossman, A.D. (1995) Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis. Ann Rev Genet 29: 477–508.
Lazazzera, B., Palmer, T., Quisel, J., and Grossman, A.D. (1999a) Cell density control of gene expression and development in Bacillus subtilis. In Cell-Cell Signaling in Bacteria. Dunny, G.M., and Winans, S.C. (eds). Washington, DC: American Society for Microbiology Press, pp. 27–46.
Msadek, T. (1999) When the going gets tough: survival strategies and environmental signaling networks in Bacillus subtilis. Trends Microbiol 7: 201–207.
Tortosa, P., and Dubnau, D. (1999) Competence for transformation: a matter of taste. Curr Opin Microbiol 2: 588–592.
7- Grossman, A.D. (1995) Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis. Ann Rev Genet 29: 477–508.
Lazazzera, B., Palmer, T., Quisel, J., and Grossman, A.D. (1999a) Cell density control of gene expression and development in Bacillus subtilis. In Cell-Cell Signaling in Bacteria. Dunny, G.M., and Winans, S.C. (eds). Washington, DC: American Society for Microbiology Press, pp. 27–46.

Dubnau, D., and Lovett, C.M.J. (2002) Transformation and recombination. In Bacillus subtilis and its Closest Relatives: From Genes to Cells. Sonenshein, A.L., Hoch, J.A., and Losick, R. (eds). Washington, DC: American Society for Microbiology Press, pp. 453–471.