HbpR

Mechanism

Build Our Own Sensor!

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 PC and PD 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 A-domain, B-domain C-domain and D-domain (Fig. 3) . C-domain 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. D-domain binds to DNA via a typical helix-turn-helix motif. A-domain 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 cooperative multimerization of HbpR (Fig. 4) .

Most if not all of the transcriptional outputs from the hbpC promoter are mediated by the proximal UASs C-1/C-2. However, when the UASs C-1/C-2 are deleted and UASs C-3/C-4 are placed in an appropriate position with respect to the promoter region, the hbpC promoter is still inducible with 2-HBP, albeit at a lower level. The presence of UAS pair C-3/C-4 mediated a higher promoter activity for transcription of hbpR ^[4].

HbpR detects a limited range of effectors. Previous studies have revealed that HbpR detects 2-hydroxybiphenyl, 2, 2-dihydroxybiphenyl and structural analogs including 2-aminobiphenl and 2-hydroxybiphenylmethane (TABLE 1) ^[5].

We used PCR to get hbpR gene from bacterial strain and Pc was synthesized by Genscript Company. We construct a gene circuit working as biosensors for 2-HBP and 2-ABP. The hbpR was controlled by a constitutive promoter on plasmid pSB4K5. Another plasmid pUC57 containing Pc-RBS-sfGFP was double transformed with pSB4K5 to construct a biosensor for substituted biphenyls (Fig. 5a). Cells were cultured to appropriate OD600 and stored at -80℃ in 20% glycerol for induction test.

We firstly tested the on/off response of sensor cell HbpR (BBa_J23106-HbpR and Pc-BBa_B0034-sfGFP) when it was exposed to other aromatic compounds following test protocol 1. Unfortunately, the basal expression level is relatively high and only 2-HBP and 2-ABP showed slightly induction effect (Data not shown) possibly because the HbpR protein originally exists in Pseudomonas azelaica instead of E.coli.

Therefore, we then constructed a promoter library to fine-tune the expression intensity. The expression strength of these constitutive promoters, J23109, J23113, J23114, J23117, is 106, 21, 256, 162, respectively, according to the Partsregistry. Results of the sensor strains with different initiation strength showed that BBa_J23114 performed better in this construction (Fig. 5b). We chose BBa_J23114 rather than BBa_J23113 because of the relatively higher fluorescence intensity of cells containing the former promoter. We also created a library for the RBS of sfGFP to figure out which kind of Pc-RBS-sfGFP is optimal for our biosensor system. Experiment results showed that for inducers 2-ABP and 2-HBP, RBS BBa_B0032 had a higher induction ratio compared with RBS BBa_B0031 and RBS BBa_B0034. (Fig. 6c,d)

After we had determined the most appropriate expression level of HbpR regulator, the best HbpR sensor cell was subjected to ON-OFF test via protocol 1. Response result showed that 2-HBP and 2-ABP had significant induction ratios compared with the other tested aromatic compounds (Fig. 6a,b).

We then tested a dose-response curve 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, which serves as a practical biosensor for synthetic biology application.

Figure 1. HbpR as the regulator to control the expression of hbp operon. Blue and green rectangles denote hbpCA and hbpD genes controled by PC and PD, 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 PC and PD.

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. There is a library for the constitutive promoter before HbpR and the RBS before sfGFP, respectively, both of which function to fine-tune the expression level of HbpR. (b) Induction ratio of HbpR controlled by promoters with different expression intensity. The expression strength of these constitutive promoters, J23109, J23113, J23114, J23117, is 106, 21, 256, 162, respectively, according to the Partsregistry. The effectors 2-HBP and 2-ABP are plotted in different colors. Data were collected via Microplate Reader. (c) Induction ratio of HbpR when exposed to a series of concentration of 2-ABP. The reporter system includes Pc-RBS-sfGFP. Three lines represent sfGFP controlled by different RBS. Fluorescence intensity of sfGFP is detected and calculated to plot induction ratio. (D) Induction ratio of HbpR when exposed to a series of concentration of 2-HBP.

Figure 6. ON-OFF test results for HbpR biosensor . (a) ON-OFF response of HbpR biosensor to 78 aromatic compounds. (For the full name of the compounds, CLICK HERE(hyperlink is needed here)). The biosenor showed induction ratio more 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 showed near its chemical formula.

Figure 7. Dose response curves of the best-performed HbpR biosensor (BBa_J23114-HbpR and Pc-BBa_B0032-sfGFP), induced by 2-HBP and 2-ABP, respectively.

TABLE 1 TABLE 1 Effectors of HbpR, containing their full name, logogram and structural formula.

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.
[6] 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.