The Naphthalene Catabolic ( nag ) Genes of Ralstonia sp. Strain U2 Are an Operon That Is Regulated by NagR, a LysR-Type Transcriptional Regulator

Abstract

In Ralstonia sp. strain U2, the nag catabolic genes, which encode the enzymes for the pathway that catabolizes naphthalene via the alternative ring cleavage gentisate pathway, are transcribed as an operon under the same promoter. nagR, which encodes a LysR-type transcriptional regulator, is divergently transcribed compared to the nag catabolic genes. A 4-bp frameshift deletion in nagR demonstrated that NagR is required for expression of the nag operon. The transcriptional start of the nag operon was mapped, and a putative ؊10, ؊3570 -type promoter binding site was identified. Further upstream, a site proximal to the promoter was identified as a site that has bases which have been found to be conserved in the activator-binding motif of other naphthalene pathways. Transcriptional fusion studies demonstrated that NagR regulates the expression of the nag operon positively in the presence of salicylate and to a lesser extent in the presence of 2-nitrobenzoate. Mutation of the LysR-type activator-binding motif in the nag promoter-proximal region resulted in a loss of inducibility of a lacZ reporter gene transcriptionally fused to nagAa, the first gene of the operon. However, other mutations in the region increased the effectiveness of salicylate as an inducer.The nag genes of Ralstonia sp. strain U2 code for the enzymes of the alternative pathway for the catabolism of naphthalene, which converts naphthalene to fumarate and pyruvate via salicylate (2-hydroxybenzoate) and gentisate (2,5-dihydroxybenzoate), and they are organized in a continuous sequence of adjacent genes (8,28,29). This is in contrast to the classical naphthalene pathway enzymes, which are encoded on two separate operons, the upper-pathway operon (genes nahAa to nahF) for the conversion of naphthalene to salicylate and the lower-pathway (or meta-pathway) operon (genes nahG to nahM) for the conversion of salicylate to acetyl coenzyme A and pyruvate via catechol (5, 27).

A New Classification System for Bacterial Rieske Non-Heme Iron Aromatic Ring-Hydroxylating Oxygenases

“…In many cases, RHO oxygenases have not been identified together with ETC components, thus it has been a challenging task to find a cognate ETC partner that can function in cooperation with these "incomplete" oxygenase components [9,[35][36][37][38][39]. Since some ETCs are interchangeable with each other and can be often replaced by outside ETC sources and it is the terminal oxygenase component that has the catalytic active site, less emphasis was given to ETC components in Nam's classification.…”

Nitroaromatic Compounds, from Synthesis to Biodegradation

“…Sequence comparisons revealed that the regulators in strains JS42 and JS765 (NtdR and NbzR, which are identical in sequence) differ from the regulator in strain U2 (NagR) by only five amino acids. The LysR binding sites and promoters are identical in all three strains, and both regulators activate gene expression in the presence of salicylate, which is an intermediate of the naphthalene degradation pathway and the natural inducer of the naphthalene degradation genes in strain U2 (87,112). However, neither naphthalene nor salicylate serves as a growth substrate for Comamonas sp.…”

“…strain U2, the product of a divergently transcribed lysR-type regulatory gene located upstream of each dioxygenase operon (Fig. 19) activates gene expression in response to recognized inducer compounds (87,89,112). Sequence comparisons revealed that the regulators in strains JS42 and JS765 (NtdR and NbzR, which are identical in sequence) differ from the regulator in strain U2 (NagR) by only five amino acids.…”

“…strains R34 and DNT that their pathways have evolutionary origins in a naphthalene degradation pathway like that present in Ralstonia sp. strain U2 (87,111,112,186,221). In all of these strains, the genes for the dioxygenase system are organized in very similar operons, and the deduced protein sequences share Ͼ85% identity (Fig.…”

Gentisate 1,2-dioxygenase, in the third naphthalene catabolic gene cluster of Polaromonas naphthalenivorans CJ2, has a role in naphthalene degradation

“…Because LysR-type regulatory genes associated with naphthalene catabolism are known to be induced by salicylate (Jones et al, 2003) and the promoter of the third catabolic gene cluster was identical to that of the first gene cluster (see above and Supplementary Fig. S2), real-time RT-PCR analysis was conducted to investigate whether the transcription of the third gene cluster was controlled by the nagR regulator of the first gene cluster.…”