IGEM:IMPERIAL/2007/Projects/Hrp System

Hrp Characterisation: Introduction

• Introduction
• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

Summary

In Pseudomonas syringae strains, the hrp-hrc pathogenicity island consists of an HrpL-dependent regulon that encodes a type III protein translocation complex and translocated effector proteins required for pathogenesis. HrpR and HrpS function as positive regulatory factors for the hrpL promoter, but their mechanism of action has not been established. Both HrpR and HrpS are structurally related to enhancer-binding proteins, but they lack receiver domains and do not appear to require a cognate protein kinase for activity.

Starting from the current biological knowledge gathered on the Hrp System, this project intends to explore the potential behind this genetic circuit as a synthetic biology device. What would be its function ? How to characterize it ? How modular and generic can it be ?

• References (See the System Documentation for a more complete reading list):

1. Hutcheson SW, Bretz J, Sussan T, Jin S, and Pak K. Enhancer-binding proteins HrpR and HrpS interact to regulate hrp-encoded type III protein secretion in Pseudomonas syringae strains. J Bacteriol. 2001 Oct;183(19):5589-98. DOI:10.1128/JB.183.19.5589-5598.2001 |
2. Ferreira AO, Myers CR, Gordon JS, Martin GB, Vencato M, Collmer A, Wehling MD, Alfano JR, Moreno-Hagelsieb G, Lamboy WF, DeClerck G, Schneider DJ, and Cartinhour SW. Whole-genome expression profiling defines the HrpL regulon of Pseudomonas syringae pv. tomato DC3000, allows de novo reconstruction of the Hrp cis clement, and identifies novel coregulated genes. Mol Plant Microbe Interact. 2006 Nov;19(11):1167-79. DOI:10.1094/MPMI-19-1167 |
3. Thwaites R, Spanu PD, Panopoulos NJ, Stevens C, and Mansfield JW. Transcriptional regulation of components of the type III secretion system and effectors in Pseudomonas syringae pv. phaseolicola. Mol Plant Microbe Interact. 2004 Nov;17(11):1250-8. DOI:10.1094/MPMI.2004.17.11.1250 |
4. Yahr TL and Wolfgang MC. Transcriptional regulation of the Pseudomonas aeruginosa type III secretion system. Mol Microbiol. 2006 Nov;62(3):631-40. DOI:10.1111/j.1365-2958.2006.05412.x |

Why HRP system is optimized for biobricks

• Residual activity of the system is very low, and only when the number of vectors are increased that noise can be detected.
• PsPF does not compete with HspL unless it is overexpressed. Hence, the system does not suffer from cross talk and is isloated in E.coli.
• HrpR and HrpS does not bind to any other sigmafactor other than sigma 54. Hence they are very specific.
• The system can be switched on or off very quickly (when working with E.coli as a vector), as ATP is part of the regulation and can be phosphorylated and dephosphorylated in a matter of seconds.
• There is no leaky transcription.
• The system is very sensitive.

Some problem areas to solve

• HrpR and HrpS are constitutively active.HrpV are constitutively produced as well.
• HrpV negative regulation reduces the original activity of the system to 5-10%. For full repression of the activity, we need to express a large amount of HrpV first before expressing HrpR and HrpS. Another method would be to use the Lon protease to degrade HrpR.
• The half life of HrpR and HrpS is unknown.
• Inducers used for this system are arabinose and IPTG. If we use arabinose, a high concentration of glucose would be required to completely shut down the promoter of HrpR and HrpS.
• HrpL is Pseudomonas specific, and cannot operate in E.coli.
• HrpR and HrpS will can induce the promoter indpendent of each other. However, this is only about 2.5% activation compared to when both are present.
• Need to clone 300bp upstream of the HrpL promoter to ensure we have included the HrpR and HrpS binding site.
• Need to use a strain that contains the 'Integration Host Factor' IHF, this may limit our host choice.

Some points to note about the HRP system

• HrpR and HrpS functions most optimally when both are expressed together in an operon in a continuous manner
• Transcription of HrpR and HrpS results in a continuous polycistronic mRNA. They are then translated seperatly from a signle mRNA.
• HrpV binds to the HTH motiff of HrpS, not HrpR. HrpV binds to the central region of HrpS, but how it binds is still not known.
• Lon protein can be used to degrade HrpR.
• In Pseudomonas, the optimal temperature for operation of the system is 28oC; in the E.coli vector, the temeprature used can range from 20-37oC. 25oC is the default temperature to work with.
• RT-PCR can be used to detect the level of transciption, but it takes time and only gives an estimate of the amount of mRNA instead of the amount of proteins.
• The DNA sequence that HrpR and HrpS bind is unknown. What is known is that it is located somewhere upstream of the +1 position. Tests have shown that it must be within a 150bp range from the +1 site of transcription.