Team:Colombia/Project/Experiments/Aliivibrio and Streptomyces

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Aim

Our system heavily relies in the ability of our bacteria to sense environmental cues belonging to the pathogens we’re particularly concerned of.

Figure 2. Model for the regulation of the chitinolytic system in S. coelicolor. (a) Repression and (b) induction of the chitinolytic system in S. coelicolor in the presence of related metabolic products of chitin. Read more in: Colson et al., 2003 [4]

Figure 1. Model for Vibrio chitin utilization. A: Negative phenotype for chitin sensing. B: Positive phenotype. Activation of chitinolytic system due to presece of dimers of N-acetylglucosamine (GlcNAc)2 Read more in: Li and Roseman, 2003 [3]

Chitin is one of the main components of fungal cell walls and, in consequence, we will use it as an indicator of fungal infection.

Vibrio fischeri ES114 ([http://ijs.sgmjournals.org/content/57/12/2823 now Aliivibrio fischeri ES114]) and Streptomyces coelicolor A3(2) are environmental bacteria with well-characterized detection and catabolic cascades for chitin use as a carbon source. The way by which each bacteria detect the chitin is through a two-component system. Figure 1 illustrates the proposed sensing model for the system in A. fischeri.

Chitin is fragmented into N-acetyl glucosamine (GlcNAc)2 dimers through the action of a chitinase (ChiA). (GlcNAc)2 passes to the periplasmic space through chitoporin (ChiP) and binds the chitin-binding protein (CBP) which leads to the signalling from ChiS histidine kinase to turn on the chitinolytic genes.

Figure 2 depicts how S. coelicolor uses a pretty similar system to the previously described. The analog channel that allows the (GlcNAc)2 to cross the plasmatic membrane. (GlcNAc)2-activated DasA serves as an activator to ChiS, which phosphorylates ChiR regulator to induce transcription of the chitin metabolism genes.

In both cases, we're working on to extract and put the genes into functional and independent biobricks available to further applications of chitin sensing and/or degradation.

Our bacteria

Aliivibrio fischeri ES114

Aliivibrio fischeri ES114
Taxonomy[1]	Superkingdom	Bacteria
	Phylum	Proteobacteria
	Class	Gammaproteobacteria
	Order	Vibrionales
	Family	Vibrionaceae
	Genus	Aliivibrio
	Species	A. fischeri
	Strain	ES114

We're extracting the whole chitin sensing system, namely [http://www.ncbi.nlm.nih.gov/gene/3279287 chiS], [http://www.ncbi.nlm.nih.gov/gene/3279194 chiP], [http://www.ncbi.nlm.nih.gov/gene/3277358 chiA] and [http://www.ncbi.nlm.nih.gov/gene/3279341 CBP]. Also, as the promoter that responses to the signalling from chiS is not known, we're also extracting the 100kb upstream region of chiP that we named pChitin.

Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2)
Taxonomy[2]	Superkingdom	Bacteria
	Phylum	Actinobacteria
	Class	Actinobacteria
	Order	Actinomycetales
	Family	Streptomycetaceae
	Genus	Streptomyces
	Species	Streptomyces coelicolor
	Strain	A3(2)

From this bacterium, we're planning the cloning of the operons [http://www.ncbi.nlm.nih.gov/gene?term=SCO5377 chiSR], [http://www.ncbi.nlm.nih.gov/gene/1100673 dasABC], the genes [http://www.ncbi.nlm.nih.gov/gene/1099680 msiK], [http://www.ncbi.nlm.nih.gov/gene?term=SCO5376 chiC] and the promoter pChitSc, which corresponds to the 60bp before chiC, previously reported in [5].

References

[1] [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=312309&lvl=3&lin=f&keep=1&srchmode=1&unlock NCBI Taxonomy Browser: Aliivibrio fischeri ES114]

[2] [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=312309&lvl=3&lin=f&keep=1&srchmode=1&unlock NCBI Taxonomy Browser: Streptomyces coelicolor A3(2)]

[3] [http://www.pnas.org/content/101/2/627 Li, X., and Roseman, S. (2004). The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase. PNAS 101, 627–631.]

[4] [http://mic.sgmjournals.org/content/154/2/373.full Colson, S., Wezel, G.P. van, Craig, M., Noens, E.E.E., Nothaft, H., Mommaas, A.M., Titgemeyer, F., Joris, B., and Rigali, S. (2008). The chitobiose-binding protein, DasA, acts as a link between chitin utilization and morphogenesis in Streptomyces coelicolor. Microbiology 154, 373–382.]

[5] [http://www.springerlink.com/content/fh6g740578v78277/ Homerová, D., Knirschová, R., and Kormanec, J. (2002). Response regulator ChiR regulates expression of chitinase gene, chiC, in Streptomyces coelicolor. Folia Microbiol. (Praha) 47, 499–505.]

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-	'''Figure 2''' depicts how ''S. coelicolor'' uses a pretty similar system to the previously described~~,in which an inactive dasA is activated by N~~-~~acetylglucosamine dimers, activating thus chiS~~, which phosphorylates ChiR regulator to induce transcription of the ~~catabolism~~ genes.	+	'''Figure 2''' depicts how ''S. coelicolor'' uses a pretty similar system to the previously described. The analog channel that allows the (GlcNAc)2 to cross the plasmatic membrane. (GlcNAc)2-activated '''DasA''' serves as an activator to '''ChiS''', which phosphorylates '''ChiR''' regulator to induce transcription of the chitin metabolism genes.

	In both cases, we're working on to extract and put the genes into functional and independent biobricks available to further applications of chitin sensing and/or degradation.		In both cases, we're working on to extract and put the genes into functional and independent biobricks available to further applications of chitin sensing and/or degradation.