Team:Hong Kong HKUST/pneumosensor/modules

Figure 1. Detection Module Diagram

CSP released by S. pneumoniae is detected by its receptor ComD, which autophosphorylates at the expenditure of ATP. ComD~P then phosphorylates ComE. ComE~P then induces the promoter P_comCDE, which drives the expression of GFP.

Transformation in Streptococcus pneumoniae, like many other species, depends on specialized state called competence. Competence is achieved during the exponential growth stage of pneumococcal culture by the secretion of competence-stimulating peptide (CSP), which is a 17- residue long pheromone that is species-specific.

Competence development controlled by a two-component regulatory system (TCS), which consists of the histidine kinase (HK) ComD and its cognate response regulator (RR) ComE. When CSP binds to it, ComD autophosphorylates to become phospho-ComD, ComD~P; at the expenditure of ATP. The cytosolic protein ComE is then phosphorylated by ComD~P through transphosphorylation reactions, producing ComE~P.

These two genes are part of the comCDE operon, which includes the comC gene (Pestova et al., 1996; Cheng et al., 1997) which encodes a prepeptide that is exported and matured by a dedicated ATP-binding cassette (ABC) transport protein, into CSP. The ABC transport protein is encoded by the comAB operon.

Interestingly, ComE~P binds to repeat sites adjacent to both comCDE and comAB (Ween et al., 1999), thereby creating a positive feedback loop, producing both pre-CSP and its required machinery for maturation and transport. The signal is thus amplified and competence is coordinated throughout the population. comD and comE genes were cloned out from the genomic DNA of the NCTC strain of S. pneumoniae. They would be contitutively expressed in our Pneumosensor.

As mentioned above, ComE~P induces several promoters, one of which is P_comCDE. In part 2 of our detecting circuit design, we make use of the inducible promoter P_comCDE. We obtain the sequence by oligos, and will characterize it with the help of green fluorescence protein (GFP) that we constructed in the downstream of the promoter by BioBrick RFC10. Rather than using ComE~P that has to be phosphorylated by ComD~P, which involves a chain of reactions, we use a phosphorylmimetic ComE mutant, ComE^D58E, gratefully shared by Bernard Martin et al. After characterization of P_comCDE, we hope to put P_comCDE together with Module 2 of our project, as the ultimate goal of our project is to make a system of automatic detection and lysis of S. pneumoniae.