IGEM:Imperial/2010/Detection module/2 components systems

Quorum Sensing in B.substilis

"Peptide messages of Gram(+) species are genetically encoded as precursor peptides. These bacteria encode transmembrane enzymes, which proteolytically process the precursors while extruding them into the extracellular space. These proteins thus serve the function of synthases and exporters simultaneously. Two strategies are in use for detection of peptides in the environment: receptor histidine kinases and active importers, usually from the family of ABC transporters. Once imported into the cell, the peptides are sensed by intracellular receptors preventing it from dephosphorylating a transcriptionally active response regulator."ref

Cell surface peptide display and the associated QS of Gram-positive bacteria provides a useful mechanism which we can manipulate.

The idea is to fuse a split protease onto downstream RRs of an engineered QSS artificially introduced into B.substilis.

One concern is that the extracellular proteases produced by B. subtilis may interfere with the system and give false positives. There are two ways we can overcome this problem:

1. Use a strain of B. subtilis which does not produce extracellular proteases (but these cells may not be all that healthy!)
2. Ensure that the extracellular proteases are not specific for the cleavage site on the membrane-bound protein which, when cleaved, releases the peptide that we want to detect.

The membrane-bound peptide on the surface of our bacterium would have a cleavage site that only the parasite protease (eg elastase) would recognise. The resulting cleavage peptide would bind to a receptor on the extracellular side of the membrane and trigger a response.

NB Newcastle 2008 iGEM team worked on BugBuster which provides some useful info :B

Prof Selkirk suggested using the peptide autoinducer FMLP. However, if elastase always cleaves on the C terminal side of lysine, we would probably keep ending up with an extra lysine on the peptide, meaning that the peptide may no longer fit the receptor. This review has loads of information about peptide autoinducers.

Two Component Systems

Our current idea is two engineer a hybrid 2CS, the sensor receptor will be an endogenous QSS of gram positive bacteria (not naturally occurring in B.subtilis) with the effector domains and RRs of a 2CS which homo or heterodimerize. related review We need 2 downstream interacting proteins which will bind upon external signalling. These two domains will bring together the split protease which will become functional.

Image taken from 'Adaptable Functionality of Transcriptional Feedback in Bacterial Two-Component Systems' J. Christian J. Ray, Oleg A. Igoshin*

List of TCS

NtrC and PhoB: phosphorylation induces dimerization of the receiver modules paper

Dimerization allows DNA target site recognition by the NarL response regulator paper

KEGG B.subtilis list of 2CS KEGG useful list of all E.coli 2CS

The response regulator OmpR oligomerizes via beta-sheets to form head-to-head dimers EnvZ is a promising downstream 2CS which results in OmpR dimer formation.

• RcsA & B
• PhoP & Q
• SarA & R
• NrI & II
• NtrC & B
• TraA in Agrobacterium
• ComX: linear peptide appears to contain it's activity in contrast to the AgrD linear peptide. A minimal sequence has been

      identified by Okada et al. (2009): tripeptide 
      [3-5]ComXRo-2. 

So far none of the heterodimerising networks can sense a small peptide. As the result we are looking into the feasibility of making a chimeric protein in the network to connect one that uses a signal peptide and one that produces a heterodimeric output (transcription factor)

There has been much literature looking into the feasibility of combining several two-component systems. We are in the process of reviewing past experiments and methods:

Rewiring the Specificity of Two-Component Signal Transduction Systems [1]

Rewiring Bacteria, Two Components at a Time [2]

Computational design of receptor and sensor proteins with novel functions [3]

Using Engineered Scaffold Interactions to Reshape MAP Kinase Pathway Signaling Dynamics [4]

Engineering key components in a synthetic eukaryotic signal transduction pathway [5]

Altering the specificity of the EnvZ receptor

If we could change the specificity of EnvZ by changing the extracellular domain (eg by replacing it with that of an extracellular domain of a receptor which recognises AIPs), we could still use the downstream signaling pathways of EnvZ, but we'd still be able to detect AIPs.

This paper shows that it has been possible to fuse the sensory domain of a sugar binding protein receptor to the cytoplasmic domain of EnvZ. This is because both receptors had similar transmembrane domains.

QSS 2CS

The gram positive QSS we are hoping to use is from the agr-QSS locus of S.aureus. This has been used before in iGEM by Cambridge 2008. The HK (AgrC) activates the RR (ArgA) which binds the DNA to activate transcription of downstream genes. The system relies on AIP (auto-inducing peptide) which the cell produces and utilises as an external signalling peptide. We would add our specific protease tag onto the base of the cell surface displayed AIP such that it can be cleaved off when parasitic proteases are present in the environment.

Agr-QSS

an article on AgrC: [Transmembrane topology and histidine protein kinase activity of AgrC, the agr signal receptor in Staphylococcus aureus.]

Com-QSS of E.coli is the other system that we have considered. Image taken from 'The desk encyclopedia of microbiology' By Moselio Schaechter, Joshua Lederberg

Alternative ideas

Alternative approach to fusing the split protease directly to two PPI RRs could be to use interacting proteins that have been used before see Section 2.4and fuse these onto the RRs. C1 and C2 coiled coils are an example. The advantage would be that these constructs have been made and the resulting heterodimer produces a functional protease.

Allosteric Activation of DegS, a Stress Sensor PDZ Protease paper

Evolution of the ssrA degradation tag in Mycoplasma: Specificity switch to a different protease. paper In the same way Slovenia iGEM 2008 used a split ubiquitin tagging enzyme there is a AAA+ Lon protease which would cleave at a tagged site (between two fret pairs for e.g?)

Phosphate-inducible proteases

In order to create a fast responding system we are hoping to bypass a translation step. Therefore ideally we are looking for a protease which will exist in the cell in a deactivated state. Upon our selected stimulus the protease will become activated at the end of the 2CS relay. So far alternative scenarios for fast activation include using phosphorylation. Two proteases that work by this mechanism include Caspase 9 1 and PAP 2

The problem with these two proteases include PAP being a serine protease and Caspase 9 being part of an elaborate signalling mechanism which would be hard to re-engineer. Some work on PAP is being carried out by Alia Cloutier-Bosworth (BSc student)who is attempting to characterize the protease.

References

A useful review on quorum sensing. look at the middle of the article for gram positive bacteria Quorum sensing]

Some papers on the Structural Biology of (re-engineering)2CS receptors

1. Cheung J, Bingman CA, Reyngold M, Hendrickson WA,Waldburger CD: Crystal structure of a functional dimer of the PhoQ sensor domain. J Biol Chem 2008, 283:13762-13770.

2. Cheung J, Hendrickson WA: Crystal structures of C4–dicarboxylate ligand complexes with sensor domains of histidine kinases DcuS and DctB. J Biol Chem 2008,283:30256-30265.

3. Zhou YF, Nan B, Nan J, Ma Q, Panjikar S, Liang YH, Wang Y,Su XD: C4–dicarboxylates sensing mechanism revealed by the crystal structures of DctB sensor domain. J Mol Biol 2008, 383:49-61.

4. Reinelt S, Hofmann E, Gerharz T, Bott M, Madden DR: The structure of the periplasmic ligand-binding domain of the sensor kinase CitA reveals the first extracellular PAS domain. J Biol Chem 2003, 278:39189-39196.

paper on protease assays(may be useful later)

Messing with QSSs

Design principles of the bacterial quorum sensing gene networks