Team:SDU-Denmark/project-t

From 2010.igem.org

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=== Background ===  
=== Background ===  
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Motility can be a very beneficial quality for a microorganism. Evolution has therefore provided bacteria with two general means of transportation; the flagella and the pili. These qualities allow the bacteria to move away from a hostile environment and towards more favorable conditions. Flagella and pili are however viewed as a virulence factor as they also serve as an advantage in colonizing a host organism and yet they can cause a strong immune response (Josenhans). <br><br>
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Motility can be a very beneficial quality for a microorganism. Evolution has therefore provided bacteria with two general means of transportation; the flagella and the pili. These qualities allow the bacteria to move away from a hostile environment and towards more favorable conditions. Flagella and pili are however viewed as a virulence factor as they also serve as an advantage in colonizing a host organism and yet they can cause a strong immune response [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 11]]. <br><br>
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Many organisms are able to synthesize a flagellum, if the external environment calls for it. The synthesis of a flagellum is a huge and energy consuming process and is therefore tightly regulated by the bacteria’s external environment. One of the most well characterized flagellation systems is the one found in ''E. coli''. Here at least 50 genes are involved in the hierarchical synthesis and operation of the flagella. These genes are sorted into 15 operons which are expressed in a transcriptional cascade separated into three classes. Class I consists of the master operon ''flhDC''. The active FlhDC protein is a hexamer organized into an FlhD<sub>4</sub>C<sub>2</sub> complex with a computed value of 96,4kDa (Wang). The homodimeric FlhC protein is able to bind DNA, while the FlhD homodimers are not. The formation of the FlhDC complex however, stabilizes and increases the DNA binding ability (Claret). The transcription of ''flhDC'' is heavily regulated by nutritional and environmental conditions. Flagellum synthesis is inhibited at high temperatures, at high salt concentrations, at extreme pH or in the presence of carbohydrates, low molecular alcohols or DNA gyrase inhibitors, as these conditions stimulate growth as opposed to motility (Li). Because the flagellum synthesis is so energy consuming, the process is not started unless the environment calls for motility rather than growth. In fact, in situations where nutrition is plenty over a long period, the bacteria will focus on growth and over time lose the ability to synthesize the flagellum, as seen with the ''E. coli'' strain MG1655 localized in mouse intestines (Gauger).
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Many organisms are able to synthesize a flagellum, if the external environment calls for it. The synthesis of a flagellum is a huge and energy consuming process and is therefore tightly regulated by the bacteria’s external environment. One of the most well characterized flagellation systems is the one found in ''E. coli''. Here at least 50 genes are involved in the hierarchical synthesis and operation of the flagella. These genes are sorted into 15 operons which are expressed in a transcriptional cascade separated into three classes. Class I consists of the master operon ''flhDC''. The active FlhDC protein is a hexamer organized into an FlhD<sub>4</sub>C<sub>2</sub> complex with a computed value of 96,4kDa [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 12]]. The homodimeric FlhC protein is able to bind DNA, while the FlhD homodimers are not. The formation of the FlhDC complex however, stabilizes and increases the DNA binding ability [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 13]]. The transcription of ''flhDC'' is heavily regulated by nutritional and environmental conditions. Flagellum synthesis is inhibited at high temperatures, at high salt concentrations, at extreme pH or in the presence of carbohydrates, low molecular alcohols or DNA gyrase inhibitors, as these conditions stimulate growth as opposed to motility [https://2010.igem.org/Team:SDU-Denmark/project-t#References 14]. Because the flagellum synthesis is so energy consuming, the process is not started unless the environment calls for motility rather than growth. In fact, in situations where nutrition is plenty over a long period, the bacteria will focus on growth and over time lose the ability to synthesize the flagellum, as seen with the ''E. coli'' strain MG1655 localized in mouse intestines [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 15]].
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[[Image:Team-SDU-Denmark-flagella-overview-1.png|600px|'''Figure 4: Overview of the flagellum synthesis cascade.''']]
[[Image:Team-SDU-Denmark-flagella-overview-1.png|600px|'''Figure 4: Overview of the flagellum synthesis cascade.''']]
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The FlhD<sub>4</sub>C<sub>2</sub> hexamer acts as a transcription factor for the Class II genes, which encodes the basal body, that is embedded in the cell membrane as well as hook proteins, which are transported to the cell exterior through the basal body. Another Class II gene is the σ<sup>28</sup> transcription factor, which is responsible for the transcription of the Class III genes. This includes ''fliC'', which encodes the flagellin subunit that composes the flagella “tail”. To ensure that the Class III genes are not transcribed before the assembly of the basal body and the hook is complete another Class II protein FliM acts as an anti-sigma factor and bind σ<sup>28</sup>, thereby preventing the transcription of ''fliC''.<br><br>
The FlhD<sub>4</sub>C<sub>2</sub> hexamer acts as a transcription factor for the Class II genes, which encodes the basal body, that is embedded in the cell membrane as well as hook proteins, which are transported to the cell exterior through the basal body. Another Class II gene is the σ<sup>28</sup> transcription factor, which is responsible for the transcription of the Class III genes. This includes ''fliC'', which encodes the flagellin subunit that composes the flagella “tail”. To ensure that the Class III genes are not transcribed before the assembly of the basal body and the hook is complete another Class II protein FliM acts as an anti-sigma factor and bind σ<sup>28</sup>, thereby preventing the transcription of ''fliC''.<br><br>
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Several studies regarding the motility of ''E. coli'' has shown the expression of the ''flhDC'' operon to be crucial (Baker+Gauger). These focused on insertion sequence (IS) elements upstream of the ''flhDC'' regulon. IS are sequences that can be inserted randomly within the DNA and therefore serve as an important factor in the plasticity of the ''E. coli'' genome as well as in many other organisms. Generally the do not encode any genes apart from those responsible for their movement within the genome, however, they can also serve as activators of neighboring genes, by disrupting repression or by the formation of hybrid promoters (Baker). In the beforementioned studies, bacteria containing an activating IS upstrem of the ''flhDC'' operon showed an increased motility compared to bacteria without this IS. It is therefore resonable to asume that by placing a constitutive active promoter in front of the ''flhDC'' operon, hyperflagellation will be induced.
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Several studies regarding the motility of ''E. coli'' has shown the expression of the ''flhDC'' operon to be crucial [[https://2010.igem.org/Team:SDU-Denmark/project-t#References 15]][[https://2010.igem.org/Team:SDU-Denmark/project-t#References 16]]. These focused on insertion sequence (IS) elements upstream of the ''flhDC'' regulon. IS are sequences that can be inserted randomly within the DNA and therefore serve as an important factor in the plasticity of the ''E. coli'' genome as well as in many other organisms. Generally the do not encode any genes apart from those responsible for their movement within the genome, however, they can also serve as activators of neighboring genes, by disrupting repression or by the formation of hybrid promoters (Baker). In the beforementioned studies, bacteria containing an activating IS upstrem of the ''flhDC'' operon showed an increased motility compared to bacteria without this IS. It is therefore resonable to asume that by placing a constitutive active promoter in front of the ''flhDC'' operon, hyperflagellation will be induced.
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[9] Trivedi VD, Spudich JL, [http://www.ncbi.nlm.nih.gov/pubmed/14636056 Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phosphotransfer in vitro.], Biochemistry. 2003 Dec 2;42(47):13887-92.<br>
[9] Trivedi VD, Spudich JL, [http://www.ncbi.nlm.nih.gov/pubmed/14636056 Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phosphotransfer in vitro.], Biochemistry. 2003 Dec 2;42(47):13887-92.<br>
[10] Jung K-H, Spudich EN, Trivedi VD, Spudich JL, [http://jb.asm.org/cgi/content/short/183/21/6365 An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli], Journal of Bacteriology, November 2001, p. 6365-6371, Vol. 183, No. 21<br>
[10] Jung K-H, Spudich EN, Trivedi VD, Spudich JL, [http://jb.asm.org/cgi/content/short/183/21/6365 An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli], Journal of Bacteriology, November 2001, p. 6365-6371, Vol. 183, No. 21<br>
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[11] Josenhans, C. and Suerbaum, S. (2002) [http://www.ncbi.nlm.nih.gov/pubmed/12008914 The role of motility as a virulence factor in bacteria.] Int. J. Med. Microbiol. 291, 605^614 <br>
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[11] Josenhans, C. and Suerbaum, S. (2002) [http://www.ncbi.nlm.nih.gov/pubmed/12008914 The role of motility as a virulence factor in bacteria.] Int. J. Med. Microbiol. 291, 605-614 <br>
[12] Wang, S. ''et al.'' (2006) [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WK7-4HMGKJ0-D-H&_cdi=6899&_user=644074&_pii=S0022283605014063&_origin=search&_coverDate=01%2F27%2F2006&_sk=996449995&view=c&wchp=dGLzVzz-zSkzS&md5=6454221c64ea21917221df6a2bcfaaaa&ie=/sdarticle.pdf Structure of the Escherichia coli FlhDC Complex, a Prokaryotic Heteromeric Regulator of Transcription.] Journ. of mol. Biol. 355, 4, 798-808 <br>
[12] Wang, S. ''et al.'' (2006) [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WK7-4HMGKJ0-D-H&_cdi=6899&_user=644074&_pii=S0022283605014063&_origin=search&_coverDate=01%2F27%2F2006&_sk=996449995&view=c&wchp=dGLzVzz-zSkzS&md5=6454221c64ea21917221df6a2bcfaaaa&ie=/sdarticle.pdf Structure of the Escherichia coli FlhDC Complex, a Prokaryotic Heteromeric Regulator of Transcription.] Journ. of mol. Biol. 355, 4, 798-808 <br>
[13] Claret, L. and Hughes, C. (2000) [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WK7-45M7T8W-51-1&_cdi=6899&_user=644074&_pii=S0022283600941494&_origin=search&_coverDate=11%2F03%2F2000&_sk=996969995&view=c&wchp=dGLbVzW-zSkzV&md5=25cecb82828b382819b79c207eaaf63b&ie=/sdarticle.pdf Functions of the Subunits in the FlhD<sub>2</sub>C<sub>2</sub> Transcriptional Master Regulator of Bacterial Flagellum Biogenesis and Swarming.] J. Mol. Biol. 303, 467-478. <br>
[13] Claret, L. and Hughes, C. (2000) [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WK7-45M7T8W-51-1&_cdi=6899&_user=644074&_pii=S0022283600941494&_origin=search&_coverDate=11%2F03%2F2000&_sk=996969995&view=c&wchp=dGLbVzW-zSkzV&md5=25cecb82828b382819b79c207eaaf63b&ie=/sdarticle.pdf Functions of the Subunits in the FlhD<sub>2</sub>C<sub>2</sub> Transcriptional Master Regulator of Bacterial Flagellum Biogenesis and Swarming.] J. Mol. Biol. 303, 467-478. <br>

Revision as of 02:00, 27 October 2010