Team:Colombia/Project/Experiments/Aliivibrio and Streptomyces

From 2012.igem.org

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Contents

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 (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 chiS, chiP, chiA and 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 chiSR, dasABC, the genes msiK, chiC and the promoter pChitSc, which corresponds to the 60bp before chiC, previously reported in [5].

References

[1] NCBI Taxonomy Browser: Aliivibrio fischeri ES114

[2] NCBI Taxonomy Browser: Streptomyces coelicolor A3(2)

[3] 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] 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] 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.