The Ferredoxin ThnA3 Negatively Regulates Tetralin Biodegradation Gene Expression via ThnY, a Ferredoxin Reductase That Functions as a Regulator of the Catabolic Pathway

Laura Ledesma-García,Eduardo Santero,Francisca Reyes-Ramírez,Dongsheng Zhou

doi:10.1371/journal.pone.0073910

Abstract

The genes for tetralin (thn) utilization in Sphingomonas macrogolitabida strain TFA are regulated at the transcriptional level by ThnR, ThnY and ThnA3. ThnR, a LysR-type transcriptional activator activates transcription specifically in response to tetralin, and ThnY is an iron-sulfur flavoprotein that may activate ThnR by protein-protein interaction. ThnA3, a Rieske-type ferredoxin that transfers electrons to the tetralin dioxygenase, prevents transcription of thn genes when the inducer molecule of the pathway is a poor substrate for the dioxygenase. The mechanism by which ThnA3 transduces this signal to the regulatory system is a major question concerning thn gene regulation. Here, we have confirmed the discriminatory function of ThnA3 and the negative role of its reduced form. We have generated ThnY variants with amino acid exchanges in the [2Fe-2S], FAD and NAD(P) H binding domains and their regulatory properties have been analyzed. Two variants, ThnY-C40S and ThnY-N201G,S206P have completely lost the discriminatory function of the regulatory system because they induced thn gene expression with different molecules such us cis-decalin, cyclohexane, trans-decalin, or benzene, which are not real inducers of the pathway. These results support a model in which ThnA3 exerts its negative modulation via the regulator ThnY.

Highlights

Tetralin (1,2,3,4-tetrahydronaphthalene) is a bicyclic molecule composed of an aromatic and an alicyclic moiety, which is found at low concentrations in different crude oils, and it is industrially produced for its use as an organic solvent
Based on the phenotype of mutants affected in each component of the initial tetralin dioxygenase enzymatic complex, which catalyzes the first reaction, we proposed a model in which ThnA3, an intermediate ferredoxin that transfers electrons to the tetralin dioxygenase, plays a role in this signal transduction [3]
Involvement of ThnA3 in thn regulation in vivo is confirmed in this study (i) by reproducing tetralin regulation in a genetic background deleted for all thn genes, to which different factors involved were sequentially added (Figure 2), and (ii) by complementing thnY thnA1 and thnY thnA3 double mutants with wild type or mutant thnY alleles (Figure 6B)

Summary

Introduction

Tetralin (1,2,3,4-tetrahydronaphthalene) is a bicyclic molecule composed of an aromatic and an alicyclic moiety, which is found at low concentrations in different crude oils, and it is industrially produced for its use as an organic solvent. The genes coding for the enzymes involved in the tetralin catabolic pathway (thn genes) are arranged into four closely linked operons, two of which contain internal promoters (Figure 1). With the exception of the weak constitutive thnR promoter, transcription from the thn promoters is induced in response to tetralin but is repressed in the presence of preferred carbon sources [1,2]. ThnR activates thn gene transcription in response to tetralin by binding to palindromic sites present at each of the four thn promoter regions (PB, PC, PH, and PM). A mechanism of transcriptional co-regulation of the divergent thnB and thnC promoters has been reported, which involves the formation of a DNA loop and a higher order structure maintained by interaction between ThnR molecules bound to each promoter region, generating an octameric protein structure [5]

Methods

Results

Conclusion