Team:Caltech/Project/Details

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Three quorum sensing systems were chosen to test in E. coli:

agrBCDA

The agrBCDA quorum sensing system is native to Streptococcus aureus and is composed of 4 proteins: agrB, agrD, agrC, and agrA. agrB is a membrane protein involved in the export and modification/processing of agrD, the precursor peptide that is modified into the signaling peptide. Once exported out of the cell and fully modified, the ligand then binds to agrC, a histidine kinase receptor. Upon binding the ligand, agrC then phosphorylates the response regulator agrA, which acts as a transcriptional activator for the P2 promoter, which we have placed in front of the reporter GFP. Figure 1 contains shows the reception mechanism for this system.

Figure 1 The agr quorum sensing system, like most quorum sensing systems, has 2 components involved in signal reception: a histidine kinase receptor (agrC) that detects the ligand outside the cell and a response regulator (agrA) that is phosphorylated inside the cell by the activated histidine kinase receptor.

In the endogenous system, the P2 promoter is actually found before the entire operon, so that the entire system forms a self-inducing, positive feedback loop. In the context of the agrBCDA as a quorum sensing system regulating virulence factors, it makes perfect sense as a system that will "push itself forward" and progress, once the Streptococcus reach a critical density. However, in our system, we are controlling ligand production via a pTet promoter and using the P2 promoter only to activate expression of the reporter.

lamBCDA

The lamBCDA system is a quorum sensing system native to Lactobacillus plantarum that is homologous to the agrBCDA system mentioned above. lamB is purported to be a membrane protein assisting in the export and post-translational modification of lamD, the signaling peptide precursor, while lamC is the histidine kinase membrane receptor protein responsible for detecting presence of lamD and, upon doing so, phosphorylating lamA, the response regulator, which proceeds to activate transcription of any gene downstream of the pLam promoter [is that its name?].

fsrABC

The fsrABC system is a quorum sensing system native to Enterococcus faecalis believed to regulate virulence factors involved in biofilm formation in its native host. In this system, FsrC and FsrA proteins function as histidine kinase receptor and response regulator respectively, while the FsrB protein is a membrane protein that is cleaved to form GBAP, the peptide mature signaling ligand.

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