Difference between revisions of "Part:BBa J61051"

Revision as of 16:14, 23 September 2013

[Psal1] J61051 pSB1A2 Bca9015 Salicylate promoter

(Edited by Zheng Pu, Peking iGEM 2013) (Please visit[http://2013.igem.org/Team:Peking/Project/BioSensors/NahR]for more details)

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 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 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].

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 degraded 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 metabolite intermediate in the pathway. (b) Metabolism of naphthalene encoded by the NAH7 plasmid: Naphthalene is degraded by a series of enzymatic reactions in 13 steps, 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.

Fig. 2. The organization of NahR protein domains. 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 in the transcription activation.

Fig. 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 marked in color. NahR binding sequence and RNAP binding sequence are boxed in green and yellow, respectively. The -82 to -47 region nal and sal promoters is highly homologous, which suggests a consensus NahR-binding site.

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 transcription activation (Fig. 4)[8].

Sequence and Features