DmpR

Mechanism

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DmpR bioinformatically mined from Pseudomonas sp.CF600 ^[1-6] is a σ⁵⁴-dependent transcriptional factor that tightly controls the expression of the dmp operon (dmpKLMNOPQBCDEFGHI) (Fig.1). This operon carries genes encoding enzymes for the degradation of (methyl) phenols into pyruvate and acetyl-CoA^[7] (Fig.2).

The cognate promoter of DmpR is Po promoter. The DmpR protein binds to Po promoter as hexamer on two distinct UAS (Upstream Activating Sequence). The transcription initiation of dmp operon also requires IHF (Integration Host Factor), which has two binding sites in Po promoter and enhances the transcription efficiency (Fig.3).

DmpR protein consists of four domains (Fig.4): Domain A is the effector-sensing domain, which undergoes conformational change when exposed to proper inducers, including phenol, 2-chlorophenol, 2,4-dichlorophenol, methyl-phenols and other substituted phenols ^[3][8]. Domain B is a linker domain where mutations would modulate the interactions between Domain A and Domain C. Domain C is the transcriptional activation domain. Domain D contains a helix-turn-helix motif, which is responsible for the DNA binding at Po promoter ^[1].

The mechanism of Po promoter activation consists of four steps, DmpR dimer formation, DmpR hexamer formation, DNA bending and recruit of RNAP (Fig.5). With the cooperation of IHF, transcription from Po promoter initiates.

A random mutation of DmpR A domain with capacity to detect phenolic molecules was selected. People found that the mutant Q10R strongly enhanced the response to phenol and substituted ones, and mutant D116V suggested that the aspartate at position 116 acted to restrict the effector range of wild-type DmpR.

A lot of work have been done about DmpR, but there is no general method for testing the induction ratio, and different works obtained different induction ratio. Our team obtained DmpR from Professor V. Shingler and the synthesized promotor Po sequence from GeneScript. Plasmid containing Pr-DmpR was double transformed with plasmid containing the inducible promoter Po and reporter gene sfGFP (Fig.6). Similar to other sensors, plasmid with RBS BBa_B0032 before sfGFP was chosen for its relatively higher induction ratio during primary test for RBS library (data not shown) (Fig.6).

We tested the DmpR using almost every protocol mentioned in the previous work and our general method(For more details about these three protocols, Protocol 1-3, Click Here).

We then tested the ON-OFF ratio of all of the 78 aromatics using the Test Protocol 3 . DmpR stain showed low basal expression level of sfGFP and 7 compounds showed observably induction ratio (>2) (Fig.8), namely Phl, 2-MePhl, 2-ClPhl, 3-ClPhl, Cat, 4-NtPhl and 2-APhl (To see more information of the compounds, Click Here).

After finding the compounds with showed observably induction ratio, we tested the dose response curve of each compound via Test Protocol 3 (Fig.9).

In summary, we found a robust and convenient protocol to test Dmp and DmpR functions as a robust sensor for phenol and its derivative.

Figure 1. The schematic structure of dmp operon. Dmp operon carries genes encoding enzymes for the degradation of (methyl-)phenols to pyruvate and acetyl-CoA, the intermediates of TCA Cycle. The operon is positively controlled by the dmpR gene product, resulting in expression of catabolic enzymes when inducers like phenol are present.

Figure 2. The catabolic pathway of phenol controlled by the dmp operon. Metabolic enzymes along the pathway are (from Step 1 to Step 8): 1, phenol hydroxylase (PH) ; 2, catechol 2, 3-dioxygenase (C23O); 3, 2-hydroxymuconic semialdehyde hydrolase (2HMSH); 4, 2-hydroxymuconic semialdehyde dehydrogenase (2HMSD) ;5, 4-oxalocrotonate isomerase (4OI); 6, 4-oxalocrotonate decarboxylase (4OD) ;7, 2-oxopent-4-cnoate hydeatase (OEH); 8, 4-hydroxy-2-2oxovalerate aldolase (HOA).

Figure 3. The schematic structure of Po promoter. The UASs of this promoter shaded in green are dyad sequences to which the DmpR protein binds. The yellow shaded boxes denote IHF binding sites. The pink shaded box represents σ⁵⁴ binding site with -24 region and -12 motifs highlighted in red. The highlighted nucleotide G represents the transcription start (+1) site.

Figure 4. The schematic structure of DmpR protein. From N-terminal to C-terminal are Domain A, Domain B, Domain C, Domain D.

Figure 5. Schematic mode of the activation of DmpR regulator (A) The inactive regular dimer binds to its inducer, which results in a protein conformation change. (B)Binding of ATP triggers multimerization of the dimers to a hexamer (or haptamer).(C) ATP hydrolysis coupled to correct interaction with RNA polymerase triggers transcription activation. (D) Dissociation of the hexamer to a dimer on ATP hydrolysis and dissociation of the inducer ^[6].

Figure 6. The structure of the DmpR biosensor circuit. DmpR is constitutively expressed and functions to regulate the transcription of sfGFP gene via promoter Po. As for the RBS of sfGFP, BBa_B0032 was chosen due to its better performance compared to RBS of other transcriptional strengths.

Figure 7. ON/OFF test to evaluate the induction ratios of all aromatic compounds in the aromatics spectrum. (For the full names of the compounds, Click Here). (a) The induction ratioS of various aromatic species. DmpR could respond to 7 out of 78 aromatics with the induction ratio over 2. (b) The aromatics-sensing profile of DmpR biosensor.The aromatic species that can elicit strong responses of DmpR biosensor are highlighted in cyan in the aromatics spectrum. The structure formula of typical inducer is also listed around the spectrum. 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 8. Dose response curves of DmpR biosensor. Dose response curves for phenol, its homologs and derivatives.The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to inducers by the basal fluorescence intensity of the biosensor. For the full names of the compounds,For the full names of the compounds, Click Here .

REFERENCE:
[1]. SHINGLER, V.; PAVEL, H. Direct regulation of the ATPase activity of the transcriptional activator DmpR by aromatic compounds. Molecular microbiology, (1995), 17.3: 505-513.
[2]. SHINGLER, Victoria; MOORE, Terry. Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600. Journal of bacteriology, (1994), 176.6: 1555-1560.
[3]. SZE, Chun Chau; LAURIE, Andrew D.; SHINGLER, Victoria. In Vivo and In Vitro Effects of Integration Host Factor at the DmpR-Regulated σ⁵⁴-Dependent Po Promoter. Journal of bacteriology, (2001), 183.9: 2842-2851.
[4]. SARAND, Inga, et al. Role of the DmpR-mediated regulatory circuit in bacterial biodegradation properties in methylphenol-amended soils. Applied and environmental microbiology, (2001), 67.1: 162-171.
[5]．WISE, Arlene A.; KUSKE, Cheryl R. Generation of novel bacterial regulatory proteins that detect priority pollutant phenols. Applied and environmental microbiology, (2000), 66.1: 163-169.
[6]. TROPEL, David; VAN DER MEER, Jan Roelof. Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiology and Molecular Biology Reviews, (2004), 68.3: 474-500.
[7]. SHINGLER, V.; POWLOWSKI, J.; MARKLUND, U. Nucleotide sequence and functional analysis of the complete phenol/3, 4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600. Journal of bacteriology, (1992), 174.3: 711-724.
[8]. GUPTA, Saurabh, et al. An Effective Strategy for a Whole-Cell Biosensor Based on Putative Effector Interaction Site of the Regulatory DmpR Protein. PloS one, (2012), 7.8: e43527.