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).

New family of biosensors for monitoring BTX in aquatic and edaphic environments.

Benzene, toluene, ethylbenzene and xylenes (BTEX) contamination is a serious threat to public health and the environment, and therefore, there is an urgent need to detect its presence in nature. The use of whole-cell reporters is an efficient, easy-to-use and low-cost approach to detect and follow contaminants outside specialized laboratories; this is especially important in oil spills that are frequent in marine environments. The aim of this study is the construction of a bioreporter system and its comparison and validation for the specific detection of monocyclic aromatic hydrocarbons in different host bacteria and environmental samples. Our bioreporter system is based on the two component regulatory system TodS-TodT of P. putida DOT-T1E, and the PtodX promoter fused to the GFP protein as the reporter protein. For the construction of different biosensors, this bioreporter was transferred into three different bacterial strains isolated from three different environments, and their performance was measured. Validation of the biosensors on water samples spiked with petrol, diesel and crude oil on contaminated waters from oil spills and on contaminated soils demonstrated that they can be used in mapping and monitoring some BTEX compounds (specifically benzene, toluene and two xylene isomers). Validation of biosensors is an important issue for the integration of these devices into pollution-control programmes.

The Three-Species Consortium of Genetically Improved Strains Cupriavidus necator RW112, Burkholderia xenovorans RW118, and Pseudomonas pseudoalcaligenes RW120 Grows with Technical Polychlorobiphenyl, Aroclor 1242.

Burkholderia xenovorans LB400, Cupriavidus necator H850, and Pseudomonas pseudoalcaligenes KF707 are bacterial strains able to mineralize biphenyl and to co-oxidize many of its halogenated derivatives (PCBs). Only strain LB400 also mineralizes a few mono- and dichlorobiphenyls, due to the presence of a functioning chlorocatechol pathway. Here, we used a Tn5-based minitransposon shuttle system to chromosomically introduce genes tcbRCDEF, encoding the chlorocatechol pathway into KF707, and genes cbdABC encoding a 2-chlorobenzoate 1,2-dioxygenase into KF707 and LB400, as well as transposon Tn4653 from the TOL plasmid, providing genes xylXYZL, encoding a broad-range toluate (methylbenzoate) dioxygenase and its dihydrodiol dehydrogenase, to extend the range for the mineralization of halogenated benzoates in LB400 and in KF707 through co-oxidation of halobenzoates into chlorocatechols. The engineered derivatives of LB400 and KF707 thus gained the ability for the mineralization of all isomeric monochloro- and bromobenzoates of the so-called lower pathway which, consequently, also allowed the mineralization of all monochlorobiphenyls and a number of di- and trichlorobiphenyls, thus preventing the accumulation of halobenzoates and of catabolites thereof. LB400 and KF707 also grow with the two commercial PCB formulations, Aroclor 1221 and Aroclor 1232, as the sole carbon and energy sources, but not with higher halogenated PCB mixtures, similar to the already published strain RW112. Repeated exposition of the modified LB400 to short pulses of UV light, over a prolonged period of time, allowed the isolation of a derivative of LB400, termed RW118, capable of growth with Aroclor 1016 still containing only traces of biphenyl, and in co-culture with modified KF707 termed RW120, and modified H850 (RW112) with Aroclor 1242, the commercial mixture already void of biphenyl and monochlorobiphenyls.