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Escherichia coli play an essential role in the circulation of materials in the nature, especially for aromatic compounds. The gene clusters related to the aromatic compounds mainly include hca (for 3-phenylpropionic acid and cinnamic acid), mhp (for 3-hydroxyphenylpropionate and phenylpropionate), paa (for phenylacetic acid) and hpa (for 4-hydroxyphenylacetic acid). All of them have the regulators to control the expression of corresponding genes, according to which we could design biosensors detecting aromatic compounds.

HcaR is a 32,838 Da (296 amino acids) protein, which belongs to LysR family. Its’ N-terminal domain functions in DNA binding via a helix-turn-helix motif, while C-terminal domain functions in multimerization. As an activator, HcaR activates the expression of hca cluster at the presence of ligands. It detects limited range of ligands, including 3-phenylpropionic acid (PPA) and cinnamic acid (CnA) [1]

MhpR is a 31,767 Da (281 amino acids) protein. It belongs to IclR family, which forms helix-turn-helix motif at N-terminal. MhpR behaves as an activator to initiate the expression of mhp cluster when contacts with its ligands, 3-hydroxyphenylpropionate (3-HPPA), 3-hydoxycinnamate (3-HCnA) and 3-(2, 3-dihydroxyphenyl) propionic acid (2,3-DHPPA). [2]

hca and mhp clusters are involved in the catabolism of PPA and CnA in E. coli (Fig. 1). The enzymes encoded by hca cluster degrade PPA and CnA to 2,3-DHPPA and 2,3-DHCnA respectively, which serve as the substrates of the mhp cluster. The enzymes in mhp cluster function in the cleavage of aromatic ring.

Compared with the sole 2,3-DHPPA, the special induction effect of PPA and 2,3-DHPPA is obtained, although PPA don’t behave as ligand alone. Based on the result and the observation of different binding site of PPA with MhpR, it is deduced that PPA and 2,3-DHPPA have synergistic effect to the activation of MhpR expression [3]. (That is to say, PPA enhances the activation effect as a cooperator of 2,3-DHPPA instead of a ligand.) The same effect is observed in 3-HPPA along with PPA.

The synergistic effect seems to be explained by pre-activation mechanism. It is that 2,3-DHPPA is a product of PPA degradation by hca cluster, and it will accumulate before activating the expression of the downstream mhp cluster. 2,3-DHPPA has cytotoxicity to the bacteria. The pre-activation mechanism activates the downstream cluster at low ligand concentration so that bacteria consume it to prevent accumulation of toxicity. The mechanism reflects the precise control across several pathways in bacteria, and also contributes to the sensor application [3].

Based on the information, our team constructed the Ph/HcaR expression system. The coding sequence of HcaR was obtained from the genome of E. coli K12 via PCR. Constitutive Pc promoters are used to initiate the expression of hcaR on pSB4K5, and sfGFP, as a reporter gene, is under the control of Ph, the cognate promoter of HcaR.

We also created a Pc library to obtain the optical performance of this expression system which gets the best induction ratio. The library consists of a series of Pc promoters with different expression intensity, including BBa_J23113, J23109, J23114 and J23106. Primary test following protocol 1 showed that HcaR performed best under the control of BBa_J23106. Then the best performed expression system is subjected to the On-Off test about 78 aromatics according to protocol 1. Results showed that HcaR worked as a specific sensor to PPA (Fig. 2).

HpaR is of 17,235 Da (149 amino acid) that belongs to MarR family [4]. It performs as a repressor of the hpa cluster consisting of hpaGEDFHI genes (Fig. 4), which participates in the catabolic pathway of 4-hydroxyphenylacetic acid (4HPAA) (Fig. 3). HpaR derepress the downstream genes when contacting with ligands, including 4HPAA, 3-hydroxyphenylacetic acid (3HPAA) and 3, 4-dihydroxyphenylacetic acid (3,4-DHPAA).

hpa cluster consists of three operons. The regulator gene, hpaR, is transcribed in the divert direction to other genes under PR promoter. 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 the hpaR and hpaG (Fig. 4). There are two HpaR binding sites, OPR1 and OPR2, belonging to PR and PG respectively. Each binding site contains palindrome sequence

which contacts with HpaR dimer in absence of ligand, inhibiting the transcription initiation. OPR1 is centered in the +2 site of PG. OPR2, however, is centered in the +40 site downstream of PR. It is hypothesized that HpaR binding to OPR1 inhibits the formation of open complex while binding to OPR2 blocks the elongation step (Fig. 5).

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 be regarded as an activator.

The two binding site, OPR1 and OPR2, perform obvious synergistic effect, i.e., binding with PG obviously improve the affinity of HpaR to PR. It is hypothesized that HpaR dimer binding to one OPR get dimerized again and generates a repression loop, similar with the AraC and PBAD. Contact with ligand disrupts the dimerization of dimer and consequently initiates transcription of the hpaGEDFHI cluster. [4]

We obtained hpaR coding sequence via PCR and constructed Pg/HpaR expression system. Pc promoter J23106 is selected to initiate the transcription of hpaR. However, we haven`t got the obvious induction ratio. It is hypothesized that several overall-controlling sites are located in the promoter, i.e., IHF and CRP. The main function of the pathway is to use the complementary carbon source in the environment, so bacteria will control strictly the expression of the relative genes in rich condition.

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 [6]. 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 [6], [7].

There are three operons in paa clusters, paaZ, paaABCDEFGHIJK and paaXY. (Fig. 6) The promoters regulated by PaaX, PZ and PA, are located at the intergenic region between paaZ and paaA. They possess a palindromic sequence respectively for binding to the repressor. (Fig. 7)

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.

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.

Fig. 1.The degradation pathway of PPA and CnA

Fig. 2. Results of On-Off test about biosensor HcaR. HcaR specifically responds to PPA (1000 μM) with the induction ration over 2.

Fig. 3.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.

Fig. 4. 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.

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.

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.

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] Díaz, E., Ferrández, A., & García, J. L. (1998). Characterization of the hca Cluster Encoding the Dioxygenolytic Pathway for Initial Catabolism of 3-Phenylpropionic Acid in Escherichia coliK-12. Journal of bacteriology, 180(11), 2915-2923.
[2] Ferrández, A., García, J. L., & Díaz, E. (1997). Genetic characterization and expression in heterologous hosts of the 3-(3-hydroxyphenyl) propionate catabolic pathway of Escherichia coli K-12. Journal of bacteriology, 179(8), 2573-2581.
[3] Manso, I., Torres, B., Andreu, J. M., Menéndez, M., Rivas, G., Alfonso, C., ... & Galán, B. (2009). 3-Hydroxyphenylpropionate and phenylpropionate are synergistic activators of the MhpR transcriptional regulator from Escherichia coli. Journal of Biological Chemistry, 284(32), 21218-21228.
[4] 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.
[5] 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.
[6] 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.
[7] 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.