HpaR PaaX Mechanism Build Our Own Sensor! Mechanism Build Our Own Sensor!

HpaR is of 17,235 Da (149 amino acid) that belongs to MarR family ^[1]. It functions as a repressor of the hpa cluster consisting of hpaGEDFHI genes (Fig. 2), which participate in the catabolic pathway of 4-hydroxyphenylacetic acid (4HPAA) (Fig. 1). HpaR de-repress the downstream genes when exposed to ligands such as 4HPAA, 3-hydroxyphenylacetic acid (3HPAA) and 3, 4-dihydroxyphenylacetic acid (3,4-DHPAA).

hpa cluster consists of three operons. The regulator gene, hpaR, is driven by PR promoter in the direction reverse to other genes. The adjacent promoter, PG, initiates the transcription of the functional hpaGEDFHI operon. PR and PG are both regulated by HpaR and located in the intergenic region between hpaR and hpaG (Fig. 2). There are two HpaR binding sites, OPR1 and OPR2, belonging to PR and PG respectively. HpaR dimer will contacts with a palindrome sequence

in the binding site in absence of ligand and inhibit the transcription initiation. OPR1 is centered in the +2 site of PG while OPR2 is centered in the +40 site downstream of PR. It is hypothesized that HpaR's binding to OPR1 inhibits the formation of open complex whereas binding to OPR2 blocks transcription elongation(Fig. 3).

Interestingly, based on the gel retardation assays, most of the HpaR dimer still contact with the OPR1 in the presence of the ligand, which recruits the RNAP and form open-complex. In this way, HpaR can also be regarded as an activator.

The two binding sites, OPR1 and OPR2, exhibit obvious cooperative effect, i.e., binding with PG significantly improves the affinity of HpaR to PR. It is hypothesized that HpaR dimers, when bound to OPR sites, will dimerize again and generate repression loops, similar with the mechanism of AraC . Binding of ligands disrupts the dimer and consequently initiates transcription of the hpaGEDFHI cluster ^[1].

We obtained hpaR coding sequence via PCR and constructed Pg/HpaR expression system. Pc promoter J23106 is selected to drive the transcription of HpaR. However, we didn't obtain a satisfactory induction ratio. We speculated that this is because binding sites of global regulators such as CRP are located in the promoter. The main function of the pathway is to use the alternative carbon sources in the environment, so bacteria will control strictly the expression of such related genes in rich medium we used to culture the cells. We hope by changing mediums we use to characterize the biosensor, we may achieve a better performance in the near future.

PaaX is a repressor of 316-amino acid. As a member of GntR family, it contains a stretch of 25 residues that is similar with the helix-turn-helix motif functioning in DNA recognition and binding ^[3]. PaaX contacts with palindrome sequence located at its cognate promoter, Pa, inhibiting the promoter at the absence of the ligand. Unlike other sensors in E. coli, PaaX detects phenylacetic acid-CoA (PA-CoA), which is the first intermediate in the PA degradation pathway. The first step is catalyzed by PaaK ^[3][4].

There are three operons in paa clusters, paaZ, paaABCDEFGHIJK and paaXY. (Fig. 4) The promoters regulated by PaaX, PaaZ and PaaA, are located at the intergenic region between paaZ and paaA. They possess a palindromic sequence respectively for binding to the repressor. (Fig. 5)

The PaaX binding site on Pa is located around the transcriptional initiation site, which interrupts the recruit of RNAP and the formation of open complex. IHF and CRP also have binding sites within the promoter, representing the overall control of the cluster. Like hpaR cluster, it is hypothesized the two promoters may form a repression loop to inhibit the leakage transcription strictly in the absence of ligand ^[4].

We standardized the paaX genes and create Pa/PaaX expression system. We tuned the expression intensity of the repressor via selecting appropriate Pc promoter. Similar with HpaR, the expression of PA promoter is inhibited by the overall-controlling factor and we haven`t got the distinct induction effect. We would like to try more condition to improve the performance of the sensors.

Figure. 1.The degradation pathway of 3HPAA. The letters on the arrows are the names of genes in the clusters. eg. HpaB,C means the step 1 is catalyzed by the product of hpaB and hpaC.

Figure. 2. The hpa cluster map in genome. The arrows inside the squares show the transcriptional direction of genes.PR, PG, PX, PA and PBC, represent the promoter controlling cognate clusters. Addition of 3-hydroxyphenylacetic acid can derepress the promoters inhibited by HpaR (for PR and PG activation) or HpaA (for PBC activation). Finally the enzymes in these clusters will degrade the 3-HPAA to the intermediates in TCA cycle.

Figure. 3. Structure of the intergenic region between hpaR and hpaG (Galán, B. et al, 2003). The elements of the promoters are enclosed by square. Especially, two OPRs are marked. The transcription direction of HpaR and HpaG are indicated with arrows. The IHF and CRP sites are marked.

Figure. 4. Structure of the paa cluster. The arrows indicate the direction of transcription of each gene. PZ, PA, PX, the promoters controlling cognate clusters. 3-hydroxyphenylacetic will derepress the promoters, PZ and PA, repressed by PaaX. Enzymes coded by the operons catalyze the degradation of PAA to intermediates in TCA cycle.

Figure. 5. The intergenic region which contain PZ and PA. (Ferrández, A. et al, 2000) The elements of the promoters are enclosed by square. The transcription direction of paaZ and paaA are indicated with arrows. The IHF and CRP sites are marked.

Reference:
[1] Galán, B., Kolb, A., Sanz, J. M., García, J. L., & Prieto, M. A. (2003). Molecular determinants of the hpa regulatory system of Escherichia coli: the HpaR repressor. Nucleic acids research, 31(22), 6598-6609.
[2] Prieto, M. A., Diaz, E., & García, J. L. (1996). Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster. Journal of bacteriology, 178(1), 111-120.
[3] Ferrández, A., Miñambres, B., Garcı́a, B., Olivera, E. R., Luengo, J. M., Garcı́a, J. L., & Dı́az, E. (1998). Catabolism of phenylacetic acid in Escherichia coli characterization of a new aerobic hybrid pathway. Journal of Biological Chemistry, 273(40), 25974-25986.
[4] Ferrández, A., Garcı́a, J. L., & Dı́az, E. (2000). Transcriptional Regulation of the Divergent paaCatabolic Operons for Phenylacetic Acid Degradation inEscherichia coli. Journal of Biological Chemistry, 275(16), 12214-12222.