NahR

Mechanism

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The nahR gene was mined from the 83 kb naphthalene degradation plasmid NAH7 of Pseudomonas putida, encoding a 34 kDa protein which binds to nah and sal promoters to activate transcription in response to the inducer salicylate. This plasmid encodes enzymes for the metabolism of naphthalene or salicylate as the sole carbon and energy source (Fig. 1a) ^[1]. The 14 genes encoding the enzymes for this metabolism are organized in two operons: nah (nahA-F), encoding six enzymes required for metabolizing naphthalene into salicylate and pyruvate, and sal (nahG-M), encoding eight enzymes which metabolize salicylate into intermediates of TCA cycle (Fig. 1b) ^[2].

Previous work have confirmed that the cloned nah, sal, and nahR genes can be expressed and normally function in the heterologous host, Escherichia coli ^[3]. NahR is a member of LysR-family transcriptional factors, featured by a conserved N-terminal domain that contains a helix-turn-helix DNA-binding motif. It is σ70-dependent and functions via contacting the α-unit of RNAP ^[4]. Mutagenesis experiments revealed the organization of functional domains of NahR protein ^[5,6]. N terminal fragment (residues 23-45) accounts for binding DNA. Interestingly, the discovery of C terminal (residues 239-291) mutants unable to bind DNA suggested that the DNA binding requires the multimerization by C-terminal domain^[6]. Additionally, mutations at residues 140-200 and 207-266 largely affected the binding specificity of inducers, indicating that those residues might serve as a ligand-binding pocket (Fig. 2) ^[6].

As for the promoters NahR regulates, the -82 to -47 regions of nal and sal promoters are highly conserved, which suggests a consensus NahR-binding site (Fig. 3) ^[7].

Several experiments conformed that NahR tightly binds to DNA in vivo in the presence or absence of salicylate. Either the amount or the affinity of NahR binding to DNA will be affected by salicylate in both E. coli and its native host Pseudomonas putida [7]. This along with the evidence from methylation protection experiments suggested a conformational change in the NahR•DNA complex before transcription activation (Fig. 4)^[8].

Wild-type NahR responds to its authentic inducer salicylate with the induction ratio higher than 20 ^[7]. NahR has been artificially evolved by mutagenesis to respond to new aromatic inducers such as substituted salicylates and substituted benzoates ^[6,9]. The novel aromatics-sensing characteristics obtained from mutagenesis have been summarized in Table 1.

We placed the Standard Biological Part BBa_J61051 which contains the constitutively expressed NahR and sal promoter in front of the reporter gene sfGFP (Fig. 5) via standard assembly. The plasmid verified by Beijing Genomics Institute was transformed into E. coli (TOP10, TransGen Biotech). Single clone of bacteria was picked and grown in rich LB medium added chloromycetin (170 μg/ml) overnight and stored at -80℃ in 20% glycerol, waiting for induction test.

ON-OFF test were first carried out for NahR biosensor following test protocol 1. NahR biosensor exposed to no inducer showed low basal expression of sfGFP and 18 compounds showed apparent activation effect with the induction ratios over 20.(Induction ratio is calculated by dividing the fluorescence intensity of biosensor exposed to inducers by the basal fluorescence intensity of the biosensor) (Fig. 6). They are listed as follows: SaA, 2-ABzO, 3-MeSaA, 4-MeSaA, 4-ClSaA, 5-ClSaA, AsPR, 2,4,6-TClPhl, 3-IBzO, 2-MeBzO, 3-MeBzO, 4-FBzO, 3-ClBzO, 3-MeOBzO, 3-HSaA, 4-HSaA, 5-ClSaD, 4-ClBzO(For the full name, CHICK HERE).

Besides salicylate derivatives, NahR biosensor specially responded to 2,4,6-TClPhl (a kind of polychlorinated phenol (short for PCP)), which is of significant hazard to water environment and human health.

One step further, NahR biosensor was subjected to dose-reponse-curve experiments with concentration of inducer ranging from 0.03 μM to 1 or 3 mM. Dose-response curves of inducers listed above are obtained according to test protocol 1 (Fig. 7). Hundreds fold of induction can be reached at micro-molar concentration for SaA and its derivatives. Substituted benzoate also functions to activate NahR but with slightly lower induction ratio.As for 2,4,6-TClPhl, apparent response could be elicited by it at concentration of 10 μM.

In summary, NahR biosensor works as a highly-sensitive and robust sensing device for salicylates homologs, benzoate derivatives and water-hazardous 2,4,6-TClPhl. We have also provided sal promoter in the Standard Biologocal Part BBa_J61051 with detailed characterization data using sfGFP as the reporter.

Figure. 1. Degradation pathway of naphthalene and the corresponding gene cluster in Pseudomonas putida. (a) Gene cluster on the NAH7 plasmid that degrades naphthalene: Naphthalene is degraded into salicylate by enzymes encoded by the "upper operon"; salicylate is further degraded to enter TCA cycle via the gene products of the "lower operon". Both of the operons are regulated by the transcription factor NahR in response to salicylate, the metabolite intermediate in the pathway. (b) Degradation pathway of naphthalene: Naphthalene is degraded by a series of enzymatic reactions, each catalyzed by a specific nah gene product represented by a capital letter. A through M: A, Naphthalene dioxygenase; B, cis-dihydroxy-naphthalene dioxygenase; D, 2-hydroxychromene-2-carboxylate isomerase; E, 2-hydroxybenzalpyruvate aldolase; F, salicylaldehyde dehydrogenase; G, salicylate hydroxylase; H, catechol 2,3-dioxygenase; I, 2-hydroxymuconate semialdehyde dehydrogenase; J, 2-hydroxymuconate tautomerase; K, 4-oxalcrotonate decarboxylase; L, 2-oxo-4-pentenoate hydratase; M, 2-oxo-4-hydroxypentanoate aldolase.

Figure. 2. The organization of NahR protein domains. The domain marked by green near the N terminal accounts for DNA binding, which contains a typical helix-turn-helix motif; red domains function to bind inducer, while the orange domain is putatively involved in multimerization of NahR during the transcription activation.

Figure. 3. Schematic diagram for the NahR-regulated promoters, nah and sal. Alignment of sal and nah promoter is shown and the consensus sequence motifs are highlighted in color. NahR binding sequence and RNAP binding sequence are boxed in green and yellow, respectively.

Figure. 4. Schematic diagram for the transcription activation at sal (or nah) promoter by NahR in the presence of inducer salicylate. 1. The DNA structure of sal promoter: A, B, C and D represent the binding sites for the 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 boxes; 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.

Figure. 5. Schematic diagram for the NahR biosensor circuit. The Standard Biologicla Part BBa_J61051 was placed preceding reporter sfGFP in the backbone pSB1C3. Promoters are presented in orange, RBS in light green, coding sequence in dark blue and terminators in red.

Fig. 6. Response of sensor NahR biosensor to various aromatic species. (For the full name of the compounds, CLICK HERE). (a) The induction ratio of various aromatic species in the ON-OFF test. NahR could respond to 18 out of 78 aromatics with the induction ratio over 20. (b) The aromatics-sensing profile of NahR biosensor.The aromatic species that can elicit strong responses of NahR biosensor are highlighted in green in the aromatics spectrum. The structure formula of typical inducer is also listed around the spectrum. Induction ratio is calculated by dividing the fluorescence intensity of biosensor exposed to inducers by the basal fluorescence intensity of the biosensor.

Fig. 7. Dose response curves of NahR biosensor. (a) Dose response curves for salicylate, its homologs and derivatives; (b) Dose response curves for benzoate, its derivatives and special inducers like 5-ClSaD and 2,4,6-TClPhl. Induction ratio is calculated by dividing the fluorescence intensity of biosensor exposed to inducers by the basal fluorescence intensity of the biosensor.For the full name of the compounds, CLICK HERE).

References:
[1] Dunn, N. W., and I. C. Gunsalus.(1973) Transmissible plasmid encoding early enzymes of naphthalene oxidation in Pseudomonas putida. J. Bacteriol. 114:974-979
[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

Table 1. Various salicylate analogs that activate NahR mutants

Amino acid substitution	Inducers	Reference
Arg248→Cys	SaA, salicylamide, BzO, 2-ClBzO, 3-ClBzO	Lorenzo et al [6]
Asn169→Asp	SaA, BzO, 2-ClBzO, 3-ClBzO	Lorenzo et al [6]
Arg132→Cys	SaA, salicylamide, BzO, 3-ClBzO	Lorenzo et al [6]
Asn169→Asp / Arg248→Cys	SaA, BzO	Hae Ja Shin et al [9]
Asn169→Asp / Arg248→Lys	SaA, BzO	Hae Ja Shin et al [9]