Insights in the regulation of the degradation of PAHs in Novosphingobium sp. HR1a and utilization of this regulatory system as a tool for the detection of PAHs.

Novosphingobium sp. HR1a is able to grow using diverse polycyclic aromatic hydrocarbons (PAHs) as the sole carbon sources. We have identified two transposons that contain genes encoding several ring-hydroxylating dioxygenases and we have demonstrated the crucial role of one of these dioxygenases in the PAH metabolism in this strain; a mutant in the large subunit of this dioxygenase was unable to growth with 2-, 3-, or 4-rings aromatic hydrocarbons. Using a construction of lacZ gene fused with the pathway promoter, we determined that the expression of the dioxygenase gene was specifically induced in the presence of some PAHs and intermediates of their metabolic pathway. In silico analysis of the ORFs within the transposons and construction of the corresponding knock-out mutants allowed us to identify the main regulatory protein involved in PAH degradation in Novosphingobium sp. HR1a. To our knowledge this is the first time that a regulatory protein controlling the degradation pathway of high-molecular weight PAHs has been investigated. A deeper knowledge of the regulatory circuits that control the expression of PAH degradation has allowed us to design two biosensors for monitoring environments contaminated with oil-derived mixtures. Novosphingobium sp. HR1a (pKSR-1), the biosensor based on the promoter of the regulatory protein PahR, was more sensitive and faster in the detection of aromatic contaminants in environmental samples than Novosphingobium sp. HR1a (pKSA-1), the biosensor that is based on the PAHs-dioxygenase promoter (PpahA). Novosphingobium sp. HR1a (pKSR-1) was able to detect PAHs in the range of μgl-1 (ppb).