HbpR

Mechanism

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HbpR is a 63 kDa prokaryotic transcriptional activators from NtrC family. It shares a highly conserved homology to members of the XylR/DmpR subclass. In our project, HbpR was bioinformatically mined from Pseudomonas azelaica ^[1]. Pseudomonas azelaica can use 2-hydroxybiphenyl (2-HBP) and 2, 2’-dihydroxybiphenyl as sole carbon and energy sources through enzymes encoded by hbpCAD operon in meta-cleavage pathway.

The regulatory gene hbpR encodes a transcriptional activator HbpR, which activates P_C and P_D promoters when exposed to inducers (Fig. 1). Thus three enzyme-coding genes, hbpCA and hbpD, are expressed to degrade 2-HBP (Fig. 2).

The HbpR protein is composed of four domains, namely Domain A, Domain B, Domain C and Domain D (Fig. 3) . Domain C contains an AAA+ ATPase motif ^[2]. It has the ability to hydrolyze ATP and to interact with σ⁵⁴ to recruit RNA polymerase for transcription activation. Domain D binds to DNA via a typical helix-turn-helix motif. Domain A is necessary for the recognition of aromatic effector molecules to activate transcription.

Generally, AAA+ ATPase-dependent transcriptional activators act at a distance of 100 to 200 bp from the promoter core component, which are called enhancer-like elements or upstream activating sequences (UAS) ^[3]. HbpR binds to UAS C-1 and UAS C-2. The 32-bp space sequence between the centers of UASs C-1 and C-2 is critical for the cooperative multimerization of HbpR (Fig. 4).

The transcription output from the hbpC promoter is mainly mediated by the proximal UASs C-1/C-2. However, when the UASs C-1/C-2 are deleted, the UASs C-3/C-4 still could compensate the ability of the hbpC promoter to be induced by 2-HBP, albeit at a much lower level. The presence of UAS pair C-3/C-4 mediated a higher promoter activity for transcription of hbpR ^[4].

The aromatics-sensing of HbpR regulator is quite specific. Previous studies have revealed that HbpR detects 2-hydroxybiphenyl, 2, 2-dihydroxybiphenyl and structural analogs including 2-aminobiphenl and 2-hydroxybiphenylmethane (Table 1) ^[5].

Using hbpR gene coding sequence, a gene circuit working as a biosensor for 2-HBP and 2-ABP was constructed, as described in the Biosensor Introduction section (Fig. 5a).

We first performed ON/OFF test on primary HbpR biosensor (BBa_J23106-HbpR and Pc-BBa_B0034-sfGFP) when it was exposed to various aromatic compounds following Test Protocol 1. Unfortunately, the basal expression level is considerably high and only 2-HBP and 2-ABP showed slightly induction effect.

Therefore, we used a promoter library to fine-tune the expression level of HbpR to see whether this genetic tailoring could correct its performance. The expression levels of these constitutive promoters, J23109, J23113, J23114, and J23117, are 106, 21, 256, and 162, respectively, according to the Partsregistry. Results of ON/OFF test showed that, the HbpR biosensor using a weak constitutive promoter (BBa_J23114) performed best (Fig. 5b).

On the basis of this improvement, we further exploited a library of RBS sequences to tune the expression level of sfGFP, because a strong RBS sequence might cause high leakage expression at P_C promoter. Experiment results showed that, for inducers 2-ABP and 2-HBP, RBS BBa_B0032 brought a higher induction ratio, compared with RBS BBa_B0031 or RBS BBa_B0034 (Fig. 5c, d).

After the HbpR biosensor with the best configuration was determined (using constitutive promoter BBa_J23114 and RBS BBa_B0032), subsequently it was subjected to ON/OFF test via Test Protocol 1. Results showed that, as expected, 2-HBP and 2-ABP elicited significant induction ratios compared with the other tested aromatic compounds (Fig. 6a, b).

Then we carefully examined the dose-respone curves of HbpR when it was exposed to effectors 2-HBP and 2-ABP according to the Test Protocol 1 (Fig. 7).

In summary, we have constructed and fine-tuned the biosensing circuit for 2-HBP and 2-ABP using HbpR protein. The aromatics-sensing profile of HbpR biosensor is quite narrow, making it an effective sensor for the presence of 2-HBP and 2-ABP, the chemicals that needs to be tightly monitored due to their extremely toxicity for the human health.

Figure 1. HbpR as the regulator to control the expression of hbp operon. Blue and green rectangles denote hbpCA and hbpD genes controled by P_C and P_D, respectively. The orange rectangle show the hbpR gene which encodes HbpR protein. When exposed to the effectors, such as 2-hydroxybiphenyl, HbpR will activate transcription at P_C and P_D.

Figure 2. Pathway for the primary metabolism of 2-hydroxybiphenyl and 2-propylphenol in P. azelaica HBP1. The enzymes for each step of the degradation are also indicated .

Figure 3. Schematics for the domain organization of HbpR protein. N represents the N-terminal of HbpR and C represent the C-terminal. A, B, C and D denote 4 domains of HbpR, respectively, and the numbers below them denote domain boundaries at amino-acid-sequence resolution.

Figure 4. The sequences preceding hbpC promoter contains the binding sites for HbpR (UAS, boxed in red). Sequence numbers denote the locations of UASs relative to the transcriptional start site of hbpC and hbpD (See Fig. 1 as reference).

Figure 5. Construction and optimization of the HbpR biosensor. (a) Schematics for the HbpR biosensor circuit. A library of constitutive promoters preceding the coding sequence of HbpR and a library of RBS sequences preceding sfGFP, respectively were used to fine-tune the HbpR biosensor circuit. (b) Performance of HbpR biosensor using the constitutive promoters of different strength, described with induction ratios. The effectors 2-HBP and 2-ABP are plotted in color intensities. (c) Dose-response curves of HbpR when exposed to gradient concentrations of 2-ABP. Three curves represent different HbpR biosensors where sfGFP are controlled by RBS sequences of different strength. (d) As in (c), dose-response curves of HbpR biosensors when exposed to gradient concentrations of 2-HBP. The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to object inducers by the basal fluorescence intensity of the biosensor itself.

Figure 6. ON/OFF test results for the improved HbpR biosensor. (a) ON-OFF response of HbpR biosensor to overall 78 aromatic compounds. Click Here for the full names of aromatic compounds. The biosenor showed induction ratios higher than 10 folds when exposed to 2-HBP and 2-ABP. (b) The detection profile of HbpR biosensor is highlighted in yellow in the aromatics spectrum. The structure formula of typical inducer 2-HBP and 2-ABP is shown.

Figure 7. Detailed dose-response curves of our best HbpR biosensor (BBa_J23114-HbpR and P_C-BBa_B0032-sfGFP), induced by 2-HBP and 2-ABP, respectively.

Table 1 Summary of HbpR aromatics-sensing characteristics

Reference:
[1] Jaspers, M. C., Suske, W. A., Schmid, A., Goslings, D. A., Kohler, H. P. E., & van der Meer, J. R. HbpR, a new member of the XylR/DmpR subclass within the NtrC family of bacterial transcriptional activators, regulates expression of 2-hydroxybiphenyl metabolism in Pseudomonas azelaica HBP1. Journal of bacteriology, (2000).182(2), 405-417.
[2] Neuwald AF, Aravind L, Spouge JL, Koonin EV AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res (1999)9: 27–43
[3] Pe´rez-Martı´n J, de Lorenzo. VATP binding to the s54-dependent activator XylR triggers a protein multimerization cycle catalyzed by UAS DNA. Cell (1996) 86: 331–339
[4] Jaspers, M. C., Sturme, M., & van der Meer, J. R. Unusual location of two nearby pairs of upstream activating sequences for HbpR, the main regulatory protein for the 2-hydroxybiphenyl degradation pathway of ‘Pseudomonas azelaica’HBP1. Microbiology, (2001).147(8), 2183-2194.
[5] Jaspers, M. C., Suske, W. A., Schmid, A., Goslings, D. A., Kohler, H. P. E., & van der Meer, J. R.. HbpR, a new member of the XylR/DmpR subclass within the NtrC family of bacterial transcriptional activators, regulates expression of 2-hydroxybiphenyl metabolism in Pseudomonas azelaica HBP1. Journal of bacteriology, (2000)182(2), 405-417.