DmpR

Mechanism

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DmpR from Pseudomonas sp.CF600 ^[2][4-8] is a sigma54-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 to pyruvate and acetyl-CoA^[1].(Fig.2)

DmpR is a transcriptional activator of Po promoter which controls ON/OFF expression of dmp operon. It binds to Po promoter as tetramer on two diverted UAS sequence (Upstream Activating Sequence). The transcription initiation of dmp operon also requires IHF factor (Integration Host Factor), which has two binding sites on Po promoter and enhance the transcription efficiency. (Fig.3)

DmpR protein consists of four domains (Fig.4): A domain is the signal reception domain, which undergoes conformational change when exposed to proper inducers, including phenol, 2-chlorophenol, 2,4-dichlorophenol, methyl-phenols and other substituted phenols ^[3][5].B domain is a linker, mutations of which change the interaction between A domain and C domain, regulating the relative spatial position of them. C domain is the transcriptional activation domain. D domain contains a helix-turn-helix motif, which is capable of binding DNA sequence on Po promoter ^[2].

The mechanism of Po promoter activation consists of four steps, DmpR dimer formation, DmpR tetramer 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. Comparison of these different protocols is listed in Table 2.

The normalized fluorescence intensity (Fluorescence / OD600) of biosensor without or with inducers of the three protocols were compared (Fig.7). Results showed that protocol 3 generated the best result. It is possibly because that induction during the end of the plateau phase facilitated the stable expression of regulator DmpR and the adding of fresh LB medium was conducive to the rapid expression of sfGFP under induction.

We then tested the ON-OFF ratio of all of the 78 aromatics using the 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.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 dmpR gene product,resulting in expression of catabolic enzymes when inducer like phenol is present.

Figure 2. The catabolic pathway of phenol controlled by dmp operon.Metabolic enzymes along the pathway are represented in numbers.1 through 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. Po promoter structure. The UAS of this promoter marked in green is combined of two parts in contrast direction to which DmpR binds. The box with yellow background represents IHF binding sites. The box with pink background represents σ54 binding site with -24 region and -12 region marked in red. The G with right angle represents +1 site.

Figure 4. 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 ^[8].

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.

¤Protocol 1: Overnight culture of the biosensor DmpR were diluted 100-fold into LB medium. Add appropriate phenol derivative into 200-μL samples. Exposure to phenolic molecules was for 12h with shaking at 30℃.Then the sample was measured by Flow Cytometer and Microplate Reader.
¤¤Protocol 2: Overnight culture of the biosensor DmpR were diluted 100-fold into LB medium. When the OD 600 of the cells reached between 0.2-0.3 as measured on a spectrophotometer, add appropriate phenol derivative into 200-μL samples. Exposure to phenolic molecules was for 6h with shaking at 30℃.Then the sample was measured by Flow Cytometer and Microplate Reader.^[3]
¤¤¤Protocol 3: Overnight culture of the biosensor DmpR were diluted 100-fold into LB medium. When the OD 600 of the cells reached between 0.8-1.0 as measured on a spectrophotometer. 2mL samples were pelleted by centrifugation and immediately suspended in 2 mL of fresh LB medium containing the appropriate phenol derivative. Exposure to phenolic molecules was for 4h with shaking at 30℃.Then the sample was measured by Flow Cytometer and Microplate Reader.^[8]
* We also used M9 minimal instead of LB medium in the protocols and elongate the induction time as E.coil grows much more slowly in M9 minimal.
** For more details about these three protocols, click here.

Figure 7. The result of three protocols measured by Microplate Reader when 1000μM of phenol was added. The induction ratios of three protocols are 1.69, 1.57 and 4.41, which meant that protocol 3 generated the best result.

Figure 8. Response of DmpR biosensor to various aromatics. (For the full name of the compounds, CLICK HERE(hyperlink is needed here)). (a)The induction ratio column in the ON-OFF test. DmpR could respond to 7 out of 78 aromatics with the induction ratio over 2. (b) The detection range of DmpR biosensor is profiled in dark cyan at the aromatics spectrum. The structure formula of typical inducer is listed around the cycle spectrum, near its chemical formula.

Figure 9. Dose-response curve of DmpR biosensor. The biosensor was test under the concentration of 10μM, 30μM, 100μM, 300μM, 1000μM.

REFERENCE:
[1]. 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.
[2]. 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.
[3]. 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.
[4]. 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.
[5]. 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.
[6]. 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.
[7]．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.
[8]. 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.

Table 1 .Various analogs that activate DmpR