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

Abstract

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.

Metabolic Resource Allocation in Individual Microbes Determines Ecosystem Interactions and Spatial Dynamics

“…In media with lactose and methylamine, the Δ hprA M. extorquens strain relies on acetate from E. coli , while providing the other two species with a source of nitrogen due to dissimilation of methylamine (Fig 4A). To our knowledge this is the first metabolically-engineered obligate mutualism between three species (but see (Miller et al, 2010) and (Kim et al, 2008) for systems that were not metabolically engineered, and (Hernández-Sánchez et al, 2013) for a non-obligate system).…”

Metagenomic Analysis of a Biphenyl-Degrading Soil Bacterial Consortium Reveals the Metabolic Roles of Specific Populations

“…Thus, the biphenyl skeleton is not degraded through this pathway. Aerobic biodegradation on the contrary is better suited for low chlorinated congeners ( Pieper, 2005 ; Furukawa and Fujihara, 2008 ; Pieper and Seeger, 2008 ) and biphenyl can be aerobically mineralized either by a single microorganism or by a consortium ( Hernandez-Sanchez et al, 2013 ). Aerobic bioremediation of PCBs has been one of the main approaches to alleviate their persistence ( Harkness et al, 1993 ; Pieper, 2005 ; Sharma et al, 2017 ) and usually occurs through its cometabolism by enzymes of the biphenyl upper degradation pathway, encoded by the bphABCDEFG gene cluster ( Furukawa and Fujihara, 2008 ), although gene clusters for ethylbenzene ( etb ) and naphthalene ( nar ) degradation have also been shown to contribute to biphenyl and aerobic degradation of PCBs ( Kimura et al, 2006 ; Iwasaki et al, 2007 ), resulting in the formation of (chloro)benzoic acid using biphenyl as carbon and energy source ( Pieper, 2005 ; Pieper and Seeger, 2008 ).…”

“…On the other hand, plant–microorganism interaction also plays a major role in degradation of PCBs ( Leigh et al, 2006 ; Gerhardt et al, 2009 ; Vergani et al, 2017b ). The use of PCB-degrading strains together with others that are capable of degrading their metabolic products (i.e., chlorinated benzoic acids) has also shown to extend the degradation rate of PCBs and results in complete mineralization of certain chlorobiphenyls ( Fava et al, 1994 ; Hernandez-Sanchez et al, 2013 ).…”

Phytoremediation: State-of-the-art and a key role for the plant microbiome in future trends and research prospects

“…The very few bacteria capable of mineralising di-and polychlorinated congeners that have been isolated and cultured (Pieper 2005, Pieper and Seeger 2008), or were obtained upon genetic manipulation and/or plasmid transfer, was considered a main impediment to highly chlorinated congener biodegradation (Villacieros et al 2005, Rein et al 2007, Hernandez-Sanchez, Lang and Wittich 2013. The value of cultivation-independent analysis to detect PCB-degrading bacteria and functional genes was demonstrated by Leigh et al in 2007.…”

“…Solutions come from the use of natural plasmids and natural gene transfer, microbes that can be "domesticated" and conditioned to e.g. higher chlorinated PCB"s (Taghavi, Barac, Greenberg et al 2005, Hernandez-Sanchez, Lang andWittich 2013).…”