Enhancing PCB bioremediation - Bioremediation of Recalcitrant Compounds - page 179

respectively, as we previously reported (Tsoi et al., 1999). Plasmid pOCC1,
in addition, contains the clc genes for the chlorocatechol pathway from
D-plasmid pAC27 (Ghosal and You, 1989) under control of the lac promoter.
The plasmids may conveniently be used for transformation of laboratory or
indigenous biphenyl-degrading organisms, which customarily belong to
either the Rhodococcus genus or a variety of gram-negative bacteria. Addition
of the clc genes presumably would allow recombinant PCB degraders to
grow on congeners that contain di- or trichlorinated rings, yielding respec-
tive di- or trichlorobenzoates that would be ortho-dechlorinated by the ohb
genes to the respective mono- or dichlorocatechols.
Plasmids pRO41 and pOCC1 were introduced in four different biphe-
nyl-degrading strains: VP44 and LB400 (gram negative), and Rhodococcus
NY05 and RHA1 (gram positive). Recipient VP44 served as a control because
we reported previously expression of the ohb genes in this host (Hrywna et
al., 1999). Controls were the same recipients transformed with vector pRT1.
After 8 (VP44) to 12 (LB400 and NY05) weeks of incubation, fully grown
single colonies (a few per each plating) grew on 2-CBA in the case of plasmids
pRO41 and pOCC1. No growth was detected for pRT1, as expected. No
growth was detected for pOCC1 on dichlorobenzoates (dCBA). No growth
has been detected in the case of Rhodococcus RHA1. From each original
transformant, a few randomly chosen colonies grown on 2-CBA were serially
transferred in 2-CBA and BP batches and shown to fully grow on 2 m M
2-CBA in 2 to 5 days, depending on the recipient (fastest, VP44; slowest,
NY05). The best-growing GEM was LB400(pRO41), which was used in fur-
ther studies. In our previous experiments (see above sections), resting cells
of LB400 partially oxidized 2-CBA; this correlated with a less than expected
yield of 2-CBA from oxidation of 2-CBP and 2,2′-CBP. However, control
variant LB400(pRT1) did not grow on 2-CBA, thus implicating the cometa-
bolic nature of the 2-CBA oxidation by LB400. Recombinant LB400(pRO41)
(Figure 6.15) efficiently grew on 2-CBA with stoichiometric release of Cl - .
It appears that many biphenyl degraders can normally grow on 4-CB
and 2-CBP via oxidation of the nonchlorinated ring (pentadiene) (Hrywna
et al., 1999). Control strain LB400(pRT1) was grown on BP and inoculated
in K1 medium with 1 m M 2-CBP. LB400(pRT1) grew on 1 m M 2-CBP, accu-
mulating 2-CBA and Cl - in culture liquid. The residual 2-CBA and Cl - total
to approximately 1.2 m M , as measured, indicating complete consumption
of 2-CB. Again, less than stoichiometric production of 2-CBA indicated its
partial oxidation. The recombinant LB400(pRO41) was grown on BP and
inoculated in 1 mM 2-CBP (Figure 6.16). The GEM completely oxidized both
2-CBP and 2-CBA, yielding more biomass than the control LB400(pRT1),
with an estimated 1.2 m M Cl - released.
Haddock et al. (1995) purified biphenyl dioxygenase from LB400 and
showed that 97% of 2-CBP is dihydroxylated at the 2,3 positions of the
nonchlorinated ring and only 3% is dihydroxylated/dehalogenated in the 2,3
positions of the chlorinated ring. Therefore, most of the 0.7 m M Cl - released
by control strain LB400(pRT1) supposedly came from dechlorination of