DmpR

Overview

Build Our Own Sensor!

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 sensor strain 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).

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).

Fig. 3. Schematic diagram of the consensus structure of the nahR-regulated promoter nah and sal. Alignment of sal and nah promoter is shown and the consensus forward sequences are marked in color. NahR binding sequence and RNAP binding sequence are shown in green and yellow respectively.

Fig. 4. Schematic diagram of the activation of sal (or nah) promoter via NahR in presence of inducer salicylate: 1. The DNA structure of sal promoter: A,B,C and D represent the binding sites for the putative tetramer of NahR; the yellow arrow shows the direction of sal promoter. 2. RNAP and σ70 bind to the sal promoter by recognizing -35 and -10 region; 3. Transcription factor NahR tightly binds to sal promoter and forms a tetramer no matter whether there is salicylate or not; 4. When salicylate is present, NahR•DNA complex undergoes a conformational change. After the hydrolysis of ATP, DNA is opened and transcription is activated.

Fig. 5. Schematic diagram of the plasmid built for sensor strain NahR. iGEM part BBa_J61051 was ligate with reporter sfGFP in the backbone pSB1C3. Promoters are in orange, RBS in light green, CDS in dark blue and terminators in red.

Fig. 6. Response of sensor strain NahR 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. NahR could respond to 18 out of 78 aromatics with the induction ratio over 20. (b) The detection range of sensor strain NahR is profiled in green at the aromatics spectrum. The structure formula of typical inducer is listed around the cycle spectrum, near its chemical formula.

Fig. 7. Dose response curves of inducers of NahR. (a) Salicylate and its homologs and derivatives; (b) Benzoate, its derivatives and special inducers like 5-ClSaD and 2,4,6-TClPhl. For the full name of the compounds, CLICK HERE(hyperlink is needed here)).

Reference:
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[2] M. A. Schell.(1983) Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7. J. Bacteriol. 153(2):822
[3] M. A. Schell, and P. E. Wender.(1986) Identification of the nahR gene product and nucleotide sequences required for its activation of the sal operon. J. Bacteriol. 116(1):9
[4] Woojun Park, Che Ok Jeon, Eugene L. Madsen.(2002) Interaction of NahR, a LysR-type transcriptional regulator, with the K subunit of RNA polymerase in the naphthalene degrading bacterium, Pseudomonas putida NCIB 9816-4. FEMS Microbiology Letters. 213:159-165
[5] Mark A. Schell, Pamela H. Brown, and Satanaryana Raju.(1990) Use of Saturation Mutagenesis to Localize Probable Functional domains in the NahR protein, a LysR-type Transcription Activator. The Journal of Biological Chemistry. 265(7): 3384-3850.
[6] Angel Cebolla, Carolina Sousa, and Vı´ctor de Lorenzo.(1997) Effector Specificity Mutants of the Transcriptional Activator NahR of Naphthalene Degrading Pseudomonas Define Protein Sites Involved in Binding of Aromatic Inducers. The Journal of Biological Chemistry. 272(7):3986-3992
[7] M. A. Schell, and E. F. Poser.(1989) Demonstration, characterization, and mutational analysis of NahR protein binding to nah and sal promoters. J. Bacteriol. 171(2):837
[8] Jianzhong Huang and Mark A. Schell.(1991) In vivo interaction of the NahR Transcriptional Activator with its target sequences. The Journal of Biological Chemistry. 266(17):10830-10838
[9] Hoo Hwi Park, Hae Yong Lee, Woon Ki Lim, Hae Ja Shin. (2005) NahR: Effects of replacements at Asn 169 and Arg 248 on promoter binding and inducer recognition. Archives of Biochemistry and Biophysics. 434:67-74