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.
Highlights
PCBs represent poorly water-soluble, highly persistent, and toxic halo-organic compounds in the biosphere (Harris et al, 1993; Carpenter, 1998)
We report the construction of two recombinant bacterial strain, based on Burkholderia xenovorans LB400, termed RW118, and on Pseudomonas pseudoalcaligenes KF707, termed RW120, upon genetic manipulations and exposure to UV as a potential mutagenic agent, and capable of good growth together with the earlier constructed strain RW112 based on Cupriavidus necator H850, on Aroclor 1242, containing 44% of chlorine per molecule, as the sole source of carbon and energy
The initial pH was raised to 7.5 when growing on chlorobenzoates or chlorobiphenyls (5 mM), and to initially pH 8.0 when growing it on Aroclor 1232, 1016, or 1242, respectively, to compensate for acidification of the culture medium due to the release of protons, and to avoid phosphate buffer concentrations above 27 mM which inhibited growth. 20 ml cultures with derivatives of LB400 and individual chlorobiphenyls or PCB mixtures were grown in 100 ml, PTFE-sealed glass Erlenmeyer flasks on an overhead spinner between 10 and 50 rpm
Summary
PCBs represent poorly water-soluble, highly persistent, and toxic halo-organic compounds in the biosphere (Harris et al, 1993; Carpenter, 1998). The technical mixtures of mid and highly halogenated PCBs are rather recalcitrant toward bacterial degradation and have accumulated in the ecosphere. Their congeners are subject of anaerobic bacterial dehalogenation (Tiedje et al, 1993) which proceeds slowly under strictly anoxic environmental conditions due to the overall low energy yield of these reactions they are thermodynamically favorable, whilst aerobic bacteria can use the entire carbon skeleton of (halo-) biphenyl for efficient growth. Halogenated PCBs yield less chlorinated congeners in anoxic environments which, upon migration in soil or sediments to more oxic zones can be attacked by enzyme systems of aerobic bacteria. The literature published on the biodegradation of PCBs, including general, biochemical, genetic, and applied aspects, has been reviewed only a few years ago (Field and Sierra-Alvarez, 2008; Furukawa and Fujihara, 2008; Pieper and Seeger, 2008)
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