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

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.