NahR

Overview

The nahR gene originated from the 83 kb naphthalene degradation plasmid NAH7 of Pseudomonas putida encodes a 34 kDa protein which binds to nah and sal promoters to activate transcription of the degradation genes in response to the inducer salicylate. This plasmid encodes enzymes for the metabolism of naphthalene or salicylate as the sole carbon 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 metabolism for naphthalene to salicylate and pyruvate, and sal (nahG-M), encoding eight enzymes which metabolize salicylate to intermediates of TCA cycle (Fig. 1b) ^[2].































Previous work have confirmed that the cloned nah, sal, and nahR genes can be expressed and normally regulated in heterologous host Escherichia coli resembling the situation in Pseudomonas putida [3]. NahR is a member of LysR-type transcriptional factors, which have a conserved N-terminal segment that contains the helix-turn-helix DNA-binding motif. It is sigma70 dependent and functions via contacting the α-unit of RNAP [4]. Mutagenesis experiments also largely facilitated the localization of functional domains in the NahR protein [5,6]. N terminal portion (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 multimerization through a different protein domain [6]. Gel filtration analysis done by Mark A. Schell showed that the active NahR transcription factor may be a tetramer [5]. Additionally, mutations among residues 140-200 and 207-266 largely affected specificity of inducers, indicating that those residues might serve as a ligand-binding crevice (Fig. 2) [6].





As for promoters it regulates, the -82 to -47 region nal and sal promoters is highly homologous, which suggests a consensus NahR-binding site (Fig. 3) [7].








Several experiments all 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 engineered E. coli and its native host Pseudomonas putida [7]. This fact, along with the evidence from methylation protection experiments, suggested a conformational change in the NahR•DNA complex which results in transcriptional activation (Fig. 4)[8].
















Wide Type of NahR responds to its authentic inducer salicylate with the induction ratio over 20 [7]. In the attempt of building different whole-cell biosensors, NahR has been artificially evolved or somehow reshaped by mutagenesis to respond to new signals such as substituted salicylates and substituted benzoates [6,9]. New inducers obtained from mutagenesis are summarized in Table 1.





















We ligated BBa_J61051 which contains the constitutively expressed NahR and sal promoter with 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 sensor strain NahR following test protocol 1 (hyperlink is needed). NahR strain with no inducer showed low basal expression of sfGPF and 18 compounds showed apparent activation effect with the induction ratios over 20 (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, our sensor strain 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 strain was subjected to induction 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.

Fig. 1. Degradation pathway of naphthalene in Pseudomonas putida and the gene cluster encoding this function. (a) Gene cluster on the NAH7 plasmid that degrades naphthalene: Naphthalene is transformed into salicylate under the 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 metabolic intermediate in the pathway. (b) Metabolism of naphthalene encoded by the NAH7 plasmid: Naphthalene is degraded by a series of 13 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.