Construction and Characterization of Two Recombinant Bacteria That Grow on ortho - and para -Substituted Chlorobiphenyls

Abstract

Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing Comamonas testosteroni VP44 resulted in recombinant pathways allowing growth on ortho-and para-chlorobiphenyls (CBs) as a sole carbon source. The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs). Plasmid pE43 carries the Pseudomonas aeruginosa 142 ohb genes coding for the terminal oxygenase (ISP OHB ) of the ortho-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the Arthrobacter globiformis KZT1 fcb genes, which catalyze the hydrolytic para-dechlorination of 4-CBA. The parental strain, VP44, grew only on low concentrations of 2-and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs. The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively. Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs. Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs.

A novel bacterium that utilizes monochlorobiphenyls and 4-chlorobenzoate as growth substrates

“…If the initial step involved oxygenation of the non-chlorinated ring and if this was the rate limiting step, rapid degradation of the 4-CBA product might explain our lack of 4-CBA detection during 4-CB degradation. Hrywna et al, for example, observed only ephemeral production of 4-CBA during complete degradation of 4-CB by a recombinant strain [8].…”

Artificial selection of microbial ecosystems for 3‐chloroaniline biodegradation

“…A sampling of the recent literature provides examples of the range of current approaches to biodegradation. Researchers have identified strains of bacteria that have the ability to break down or alter environmental pollutants such as petroleum products, polychlorinated biphenyls (PCBs) (Wagner-Do È bler et al, 1998;Hrywna et al, 1999), other halogenated aromatics (Sharak Genthner et al, 1997;Zeyer et al, 1985), toluene (Hubert et al, 1999) and nitrogen-containing heterocyclic aromatic analogues of polycyclic aromatic hydrocarbons (Pfaller et al, 1999). Organisms capable of such activity are often isolated by a screening process in which the target substance is provided as the carbon source or electron acceptor for the growth of a generalized inoculum (Hegeman, 1985;Maymo  -Gatell et al, 1997;Van Agteren et al, 1998).…”

“…Because many of the genes coding for catabolic enzymes can be found on transposons or plasmids, Peters et al (1997) were able to track the horizontal in situ transfer of a plasmid-borne operon coding for phenol metabolism. By introducing specific dehalogenation genes into a bacterial strain capable of co-metabolizing PCBs, Hrywna et al (1999) demonstrated an improvement in the ability of the recipient strain to grow upon and metabolize much higher concentrations of substrate. Novel developments along these lines include the modification of degrading bacteria to fluoresce when actively breaking down a target substance, providing an in situ indicator of degradative progress (Ripp et al, 2000).…”

Cloning, Expression, and Nucleotide Sequence of the Pseudomonas aeruginosa 142 ohb Genes Coding for Oxygenolytic ortho Dehalogenation of Halobenzoates

“…The optimum concentration of 2-CBA for growth was around 2.5 to 3.0 mM for parental strain 142 (61), compared to 1.5 mM for recombinant PB2440(pE43); this could arguably be due to cognate ferredoxin or reductase components in the parent. However, in later experiments we have expressed the ohb genes in biphenyl-degrading C. testosteroni VP44, and the resulting recombinant, VP44(pE43), readily mineralized 10 mM each 2CBA and 2-chlorobiphenyl (36).…”