Part:BBa_K239001

Spy promoter, activated by Phosporylated CpxR, BaeR and Sigma70

Sequence contains 2 phosphorylated BaeR (Bacterial Adaptative rEsponse) binding sites, 1 phosphorylated CpxR (Conjugative Plasmid eXpression) binding site and transcription is initiated by sigma factor 70. Experiments by Bury-Moné et al. (2009) indicates that also RcsB (Regulator of colanic acid Capsule Synthesis) induces expression of the Spy gene.

Function: Can detect various periplasmic stresses such as Copper (4mM CuCl2), Indole (4mM) and Ethanol (5%). Possibly it can also be used to detect presence of heterogeneous unfolded proteins in the periplasmic space (e.g. target pharmaceuticals) or to detect high levels of shear stress during cultivation in a bioreactor.

Usage and Biology

The spy gene and promoter

Spy (Spheroplast protein Y) was first discovered by Hagenmaier et al. (1997) as a protein being expressed exclusively in cells being subjected to spheroplasting stress. Raffa and Ravio (2002) demonstrated that Spy is, in addition to CpxAR, also being induced by the much smaller regulon BaeSR. BaeSR and CpxAR are also sharing some stimulon related to the periplasm, Screening of spy inducing metals by Yamamoto et al. (2008) has showed that Zn2+ is the metal causing the strongest induction of the operon over longer exposure, acting via the BaeSR system. However, the copper shock induction through CpxAR seems to mediate a quicker response. Bury-Moné et al. (2009) are showing that also RcsB (Regulator of colanic acid Capsule Synthesis) could induce expression of the Spy gene. Experiments, by Yamamoto et al. (2008), in which spy promoter fragments have been exposed to purified BaeR and acetyl phosphate have revealed a promoter-distal site between -162 and -137 and a promoter-proximal site between -109 and -79. The BaeR-box TCTNCANAA is present as a direct repeat in the promoter-distal site. The promoter-proximal site includes one single copy of the BaeR-box.

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.

BaeS-BaeR envelope stress response

The BaeSR (Bacterial Adaptative rEsponse) is another example of a two component signal transduction system and its signal pathway is the same as for CpxAR system. It was first discovered as an envelope stress signal transduction pathway in E.coli by Raffa and Ravio (2002). BaeR was discovered as an additional transcription factor of the uncharacterized Spy gene, in addition to the previously known CpxAR system. BaeSR was showed to partly share stimuli with CpxAR as the two systems both are induced by PapG overexpression, spheroplast formation and indole. Indole is regarded as a putative inducer of the BaeSR system and as such it has also been used for investigations of how the BaeSR system responds and influences gene expression. (Nishino et al., 2005) BaeSR stress induced activation in E.coli is less well characterized than the activation of the Cpx system and only 4 operons have been showed to be induced by BaeR. No negative regulation by the system has been found. BaeR is binding upstream of spy, arcD, ycaC and the mtd-bae operon. (Bury-Moné et al., 2009) Baranova and Nikaido (2002) have showed that BaeR activates transcription of yegMNOB /mdtABCD (multidrug transporters) which increases e.coli’s resistance to Novobiocin and Deoxycholate. BaeSR seem to have a conserved function in at least one more gram negative bacteria as experiments by Nishino et al. (2007) have showed that the BaeSR complex is responsible for multidrug and metal resistance in Salmonella enterica. Yamamoto et al. (2008) has identified BaeRS two component system as a fifth regulon member of the zinc-responsive stimulon (adding to previously existing Zur, ZraSR, ZntR and NhaR). Analysis of the upstream region of spy, mdtA and arcD, by Nishino et al. (2005), has led to the identification of a 18bp long binding sequence for BaeR. It is located between -156 to -54 bp before initial transcription point and reads: 5’-TTTTTCTCCATDATTGGC-3’. Where D is any nucleotide G, A or T. Experiments on the spy promoter, by Yamamoto et al. (2008), identify the sequence TCTNCANAA as the BaeR-box.

Note: The related DegP promoter (BBa_K239000) 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 Spy promoter, 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

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

The Relative Promoter Unit strength of the spy promoter (BBa_K239001) is 0.0

(Due to problem with transformations, 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)