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recruitment of genes that encode degradative enzymes from other pathways. Thus, pathway
prediction using bioinformatics is complicated by degradation genes that are not in a single
operon or are surrounded by genes that are not involved in degradation.
An integrated “omics” approach including proteomics and transcriptomics provided some
insight about
cis
-DCE degradation pathways in JS666. The approach was based on the premise
that proteins or messenger RNA (mRNA) transcripts that are upregulated by growth on
cis
-DCE compared to growth on a reference substrate are more likely to be involved in
cis
-DCE
degradation pathways. Proteomics using two-dimensional (2-D) gel electrophoresis revealed
that genes annotated as a cyclohexanone monooxygenase (CMO), glutathione S-transferase
(GST), and haloacid dehalogenase (HAD) were upregulated during growth on
cis
-DCE
(Jennings et al.,
2009
). Transcriptomics experiments using complementary deoxyribonucleic
acid (cDNA) microarrays confirmed that the above genes were among the most highly
upregulated genes, while identifying many others that could play an important role in the
initial attack on
cis
-DCE (e.g., cytochrome P450) (Jennings et al.,
2009
).
Comparative genomics also can aid in predicting degradation pathways. Genes that are
present in JS666 but not in the closely related
P. napthalenivorans
are more likely to be
involved with
cis
-DCE degradation in JS666. Selected genes upregulated by
cis
-DCE in
JS666 were compared to similar genes in
P. napthalenivorans
using the BLASTP algorithm.
The CMO, GST, and HAD seem not to be present in
P. napthalenivorans
- supporting the
hypothesis that the enzymes are important in
cis
-DCE degradation (Jennings et al.,
2009
).
Monooxygenases can catalyze the addition of oxygen to a double bond and form an
epoxide. The oxidation of halogenated alkenes in bacteria is thought to occur primarily by
monooxygenase-catalyzed epoxidation (Figure
7.3
) (Ensign,
2001
; Van Hylckama Vlieg and
Janssen,
2001
). The upregulation of a monooxygenase and the ability to convert ethene and
propene to the corresponding epoxides supports the hypothesis that a monooxygenase is
involved in
cis
-DCE degradation in JS666 (Coleman et al.,
2002a
). However, compound
specific isotope analysis (CSIA) indicates that the first step in the primary
cis
-DCE degradation
pathway in JS666 does not involve a monooxygenase (Jennings et al.,
2009
). The results suggest
that there may be two
cis
-DCE degradation pathways in JS666.
CSIA can be used to discriminate between biodegradation and abiotic losses of contami-
nants. It also can reveal the initial mechanism of degradation since the degree of fractionation
depends on the type of bond being broken in the first step (Hirschorn et al.,
2007
). The degree of
fractionation can be described by the isotopic enrichment factor,
. A large fractionation (larger
negative number) can be expected from the cleavage of bonds between heavy atoms (e.g.,
carbon-chloride bond, C-Cl). A small fractionation (smaller negative number) can be expected
from the cleavage of bonds between small atoms (e.g., carbon-carbon double bond, C
e
C).
In JS666, the carbon isotopic fractionation associated with aerobic
cis
-DCE degradation is
¼
22.4
‰
(Jennings et al.,
2009
), which is consistent with theoretical predictions for
C-Cl cleavage (Glod et al.,
1997
) and experimental fractionation values for C-Cl cleavage in
chloroethenes (Bloom et al.,
2000
; Hunkeler et al.,
2002
; Lee et al.,
2007
; Slater et al.,
2001
) and
17.4 to
DCE epoxide
cis
-DCE
O
2
NAD(P)H
H
H
H
O
H
C=C
C C
Cl
Cl
Cl
Cl
Figure 7.3. Monooxygenase-catalyzed formation of cis-DCE epoxide from cis-DCE in Rhodococcus
sp. strain AD45 (adapted from Van Hylckama Vlieg and Janssen,
2001
).
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