Enhancing PCB bioremediation - Bioremediation of Recalcitrant Compounds - page 170

from the degradation of PCBs, based on analogy to the biphenyl pathway
(Omori et al., 1986), are uncharacterized, and therefore their fate remains
unknown, although it was suggested that chlorinated pentadiene can be
metabolized through formation of chloroacetate followed by dehalogenation
to acetate (Brenner et al., 1994). Strain VP44 did not grow on chloroacetate.
In summary, the ohb and fcb operons for ortho- and para-dechlorination
of chlorobenzoates, respectively, were successfully expressed in C. testosteroni
strain VP44. Although the parent VP44 was incapable of either growth on
or dechlorination of chlorobenzoates, the introduced genes encoding deha-
logenation of these compounds permitted both. The resulting transgenic
strains were capable of growth on and dechlorination of the respective chlo-
robenzoates and chlorobiphenyls. Degradation of ortho-chlorinated PCB
congeners is especially significant given their predominance among the
products of anaerobic PCB dechlorination. Up to 80% molar of the PCBs
present following anaerobic dechlorination of Aroclor 1242 consists strictly
of ortho-chlorinated congeners; 2-CB alone may constitute as much as 40%
molar of the total PCBs in anaerobically dechlorinated sediments (Bedard
and Quensen, 1995). These results were summarized in a recent paper by
Hrywna et al. (1999) and demonstrated an alternative approach for the
construction of PCB-degrading bacteria, i.e., using genes encoding periph-
eral enzymatic activities for modification of xenobiotics into substrates for
the central metabolic pathway for degradation of aromatic compounds. The
introduction of specific dechlorination genes and their expression in the
biphenyl-degrading bacterium C. testosteroni strain VP44 demonstrated the
efficacy of this method for extending the substrate range for PCB degradation
by biphenyl-degrading bacteria.
6.4.1.3 Developing gene transfer system for G+/G- PCB-degrading
bacteria
Because of inefficient rates of degradation of important PCB congeners such
as 2,2′-, 2,4′-, and 2,4,2′-CBs by strain VP44, other BP degraders, particularly
gram-positive RHA1 and NY05, as well as the most active strain LB400, were
chosen for subsequent design of PCB-growing GEMs.
Gram-positive bacteria, especially Rhodococcus strains, offer a number of
advantages for environmental use, including higher growth yields on biphe-
nyl, the presence of multiple PCB metabolic systems allowing co-oxidation
of a wider range of PCB congeners (Masai et al., 1997; Seto et al., 1995), and
more tolerance to environmental stresses such as drought or exposure to
toxic compounds (Warhust and Fewson, 1994; Tsoi et al., 1991). Genetic
engineering of catabolic pathways in Rhodococcus , however, is not well devel-
oped. We constructed a broad-host-range shuttle vector pRT1 suitable for
transferring (dehalogenase) gene cassettes into Rhodococcus as well as
gram-negative strains, based on RP4/RK2 derivative pSP329 and Rhodococ-
cus -specific replicon pRC1 (Rodrigues et al., 2001). We then cloned the gene
cassette carrying 4-CBA degradation operon fcbABC into pRT1, yielding