Difference between revisions of "Part:BBa K239000"

Revision as of 00:10, 22 October 2009

DegP promoter, activated by Phosporylated CpxR and SigmaE

Sequence contains 3 phosphorylated CpxR (Conjugative Plasmid eXpression) binding sites and transcription is initiated by sigma factor E(σ24). A native sequence has been used but it has been modified to remove Spe1 restriction site.

Function: Can detect various periplasmic stresses such as Copper (4mM CuCl2), Indole (4mM) and Ethanol (5%). The DegP protein is expressed by the cell to repair damage in the periplasm. We hope that it also can be used to detect presence of relevant unfolded proteins in the periplasmic space (e.g. target pharmaceuticals), to detect shear stress or other so far undefined relevant stress.

Usage and Biology

The DegP gene and promoter

Extracytoplasmic function sigma, Sigma-E (24)

Sigma-E was first discovered as an alternative sigma factor recognised by one of the promoters for RpoH. However, it is mostly involved in transcription of genes with function in the periplasm, many of which are also part of the CpxA-CpxR regulon. The pathway of Sigma-E is activated when the periplasmic sensor protease DegS cleaves the anti-sigma factor RseA. This occurs after exposure of DegS to misfolded outer membrane proteins. Following the cleavage of RseA in the periplasm another inner-membrane bound protease YaeL (also named RseP) degrades the trans-membrane domain of RseA. The latter event releases the Sigma-E transcription factor in the cytoplasm and activates its large regulon.(Rhodius et al., 2005) However, sigma E can also be activated independently of misfolded OMPs by cytoplasmic alarmone ppGpp, which is dependent on nutrient availability. (Costanzo and Ades 2006)

CpxA-CpxR envelope stress response

The CpxA-CpxR (Conjugative Plasmid eXpression) response functions as a two-component signal transduction system. The CpxA is the system’s sensor kinase which auto-phosphorylates when proteins designated for the outer membrane or secretion (e.g. pili or curli fibers) are mounting in the periplams. The concentration of proteins can be due to problems in transport or damage to the outer membrane. (Snyder and Champess 2007) The phosphorylated CpxA transfers its phosphate to CpxR which becomes activated as a DNA binding protein activating or increasing the transcription of more than 100 different genes. The genes activated are mostly chaperones and proteases involved in the refolding or degradation of proteins in the periplasm. DiGuiseppe and Silhavy (2003) have showed that of all the genes being induced by CpxR the operon cpxP (part of the cpx operon and involved in feedback inhibition) promoter is the strongest induced of them all. The CpxA-CpxR system also regulates the pore size in the membrane by increasing the transcription of ompC and repressing the transcription ompF with the effect that fewer toxins can get in through the smaller porin OmpC. OmpC and OmpF are the two major porin proteins in E.coli and their main function is to balance the osmotic pressure of the cell. They are otherwise regulated due to changes in the osmolarity via EnvZ and OmpR. (Snyder and Champess 2007) Yamamoto and Ishihama (2005) have demonstrated that CpxAR is being activated as a response to external copper. An example of an additional protein regulating the activity of CpxA is NlpE (an outer membrane lipoprotein), which increases activity following adhesion. (Bury-Moné et al., 2009) GTAAANNNNNGTAAA has been proposed as the CpxR binding site. (Wulf et al. 2002) However, an examination of a large amount of CpxR induced operons by Price and Ravio (2009) indicates that the level of consensus with the proposed sequence or its orientation seems to play a minor role when determining the strength of induction by CpxR for a particular promoter. Of larger importance seems to be the location of the binding site.

Note: The DegP promoter is dependent of Sigma E (and hence also the growth phase) and can not be induced only by over expression of e.g. CpxR. In comparison, the related Spy promoter (BBa_K239001), which is dependent on sigma factor 70, can be induced up to 40 fold by over expression of CpxR. (Bury-Moné et al. 2009) Experiments by the The UCL_London_2009 team have also showed that the activity of the DegP promoter, compared to the spy promoter, is much more dependent of the growth phase of the bacteria.

Sequence and Features

Functional Parameters

The graph above shows the RPU strength of the DegP and Spy promoter at different OD values while growing in LB media.

The Relative Promoter Unit strength of the DegP promoter (BBa_K239000) is 0.3-0.4. However, this is only true for the exponential phase. RPU strength for DegP is increasing as the bacteria are approaching their stationary phase (Probably because of ppGpp activation of Sigma-E).

(Due to problems with the transformation, experiments were carried out in comparison the TetR promoter (BBa_R0040) instead of BBa_J23101. TetR is measured to have an RPU activity of ca 1.4)