United States
Environmental Protection
Agency
Hazardous Waste Engineering
Research Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/M-86/025 Jan. 1987
ENVIRONMENTAL
RESEARCH BRIEF
Microbial Degradation of 2,4,5-T and Chlorinated Dioxins
A. M. Chakrabarty
This report from the University of Illinois College of
Medicine at Chicago covers three years of research under
a Cooperative Agreement.
A pure culture of Pseudomonas cepacia strain AC1100,
isolated from a chemostat enrichment culture experiment,
is capable of growing on 2,4,5-T as its sole source of carbon
and energy. Metabolic pathway studies indicate the activity
of both constitutive and inducible enzymes. In laboratory
experiments, soil contaminated with 2,4,5-T could be
detoxified by AC1100 treatment, with the liter of AC11 00
rapidly falling to nearly undetectable levels after the 2,4,5-
T was substantially degraded.
Extensive homology observed between plasmids points to
the role of plasmid genes in the evolution and spread of
degradative characters against toxic chemical compounds.
A 1.3 kilobase pair DNA repeated sequence was found
in strain AC1100. The specificity of this sequence to
AC1100 suggests that this unique sequence may be a
useful genetic probe of strain AC1100 and other novel
Pseudomonas strains which are under consideration for
deliberate release to aquatic and terrestrial environments.
Introduction
This research brief describes the effort conducted under
a cooperative agreement between the U.S. Environmental
Protection Agency and the Department of Microbiology
of the University of Illinois College of Medicine at Chicago.
The research relates to the development and continued
biochemical and genetic studies of a specific bacterial
strain, Pseudomonas cepacia AC1100. These microorga-
nisms can utilize a recalcitrant compound such as 2,4,5-
trichlorophenoxyacetic acid (2,4,5-T) as a sole source of
carbon and energy, and can dechlorinate a variety of
chlorophenols. Additionally, such a strain is shown to be
highly effective in removing large quantities of 2,4,5-T from
contaminated soil, thereby allowing the growth of plants
that are normally sensitive to the presence of small
quantities of 2,4,5-T. This work attempted to elucidate the
organisms' metabolic pathways of degradation and gene
recombination involved in the evolution of the strain.
The Role of Plasmids
Plasmids are known to play a major role in the biodeg-
radation of a variety of complex organic compounds.
However, only a few plasmids, such as pAC25, pWR1,
and pAC31 are known to allow complete dechlorination
of the compounds. The evolution of a chlorobenzoate
degradative plasmid such as pAC25 specifying a number
of new enzymes having maximal activity towards the
chlorinated substrates, raises the interesting question as
to how new degradative functions evolve in nature.
Several lines of evidence suggest that plasmids interact
with one another to greatly extend the substrate range
of plasmid-specified enzymes. Growth in a chemostat of
3-chlorobenzoate-degrading Pseudomonas species B13
with 4-chloro- or 3,5-dichlorobenzoate allows emergence
of strains capable of degrading these compounds only
when other Pseudomonas strains harboring the TOL
plasmid are introduced into the chemostat. This project
demonstrated that under such conditions, a part of the
TOL plasmid undergoes transpostion on the chromosome
to provide the broad substrate specific benzoate oxygenase
function necessary for the conversion of chlorinated
benzoates to the corresponding chlorocatechols.
Since growth with 3,5-dichlorobenzoate needs the
participation of additional enzymes capable of dechlori-
nating the chlorolactone and chloromaleyl acetic acid, a
segment of TOL containing the replication/incompatibility
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genes and a segment of the chlorobenzoate plasmid pAC27
are recombined. This recombination gives rise to a
separate plasmid, which then undergoes mutational
divergence to generate new enzymatic activities for the
dechlorination of the chlorinated intermediates. Thus,
plasmids appear to play a vital role in nature as carriers
of duplicated genes for their ultimate mutational
divergence to generate new degradative functions.
Gene Regulation
Other useful information emerging from the studies of
degradative plasmids relates to the extent of clustering
of degradative genes on such plasmids. A physical map
of the chlorobenzoate degradative plasmid pAC27 was
constructed to demonstrate that the degradative genes are
clustered within a single EcoFM fragment of the plasmid.
The cloned chlorobenzoate degradative genes, when
transferred to Escherichia coif, are not expressed. The
specificity of plasmid promoter sequences in Pseudomonas
may help explain why, in spite of the transmissible nature
of such plasmids, pseudomonads are the predominant
scavengers in nature.
Based on the premise that plasmids evolve by recruitment
of genes from other plasmids or from the chromosome,
and that new genetic functions are acquired through
mutational divergence of genes specifying analogous
biological functions, laboratory culture conditions were
developed which have yielded a strain of Pseudomonas
cepacia (strain AC1100) that can utilize 2,4,5/T as a sole
source of carbon and energy. Also, the AC1100 resting
cell suspensions were found to be capable of oxidizing
and dachlorinating a number of chlorophenols. Dehaloge-
nation studies revealed that different halogen atoms on
analogous molecules can lead to different efficiencies of
dehalogenation. These results suggest that halogen atoms
are removed in decreasing order FI>CI>Br>l, consistent
with the general literature information on dehalogenation
of halogenated aromatic molecules. The implication is that
other halogenated compounds can be considered similarly
biodegradable.
The regulation of the dehalogenating functions of AC1100
has been partially elucidated. The enzyme(s) for the
conversion of 2,4,5-T to 2,4,5-TCP are constitutive, but
the enzymes needed for 2,4,5-TCP degradation are
inducible. Also, 2,4,5-TCP (or some metabolite), but not
pentachlorophenol (PCP), can serve as an inducer.
Development of Strain AC 1100
Pseudomonas cepacia AC1100 was developed in the
laboratory using a chemostat. When the chemostat was
switched over to 2,4,5-T as the sole source of carbon and
energy, it was acting as a continuous enrichment culture
and breeding ground for bacteria with the ability to rapidly
metabolize 2,4,5-T and derive carbon and energy from it.
Thus, as the genetic information necessary for the
complete degradation of 2,4,5-T was evolving, growth on
2,4,5-T alone was the only selective pressure present.
There was no selective pressure on this organism to evolve
an effective means of regulating the 2,4,5-T degradative
pathway in the presence of alternate carbon sources.
Therefore, the manner by which the 2,4,5-T degradative
pathway is regulated is thought to reflect the "evolutionary
youth" of this organism.
Homologies
Plasmid pJP4, like other 2,4-D-degradative plasmids such
as pJP1, pJP2, etc., was originally isolated from
Alcaligenes sp., whereas both pAC25 and pWR1 were
originally isolated from Pseudomonas sp. The question that
arises is whether the difference in expression is due to
(1) an inherent variation in the structural and regulatory
genes on the 2 plasmids or (2) to a mismatch of the signal
sequences present on plasmid pJP4 with the transcription-
translation machinery of the new host, i.e., Pseudomonas
sp. The second explanation seems plausible, since
Pseudomonas plasmids are known to be expressed poorly
in other hosts, particularly the enteric bacteria. However,
the presence of unique structural features present in the
plasmid pJP4.aD.pea;;?.Jojrjdica.tejhajt a^variatipnjnjhe
continuity of the structural and regulatory genes pertinent
to 3-chlorobenzoic acid degradation is the major difference
between pAC25 and pJP4.
Physical maps of pJP4 and pAC27 were constructed with
extensive homology observed between the chlorobenzoate
degradative plasmid pAC27 and the 2,4-D degradative
plasmid pJP4 concerning the chlorocatechol-degradative
genes and that between the same regions of pJP4 and
the resident plasmids of the 2,4,5-T-degrading strain of
P. cepacia AC1100. This points to the role of plasmid genes
in the evolution and spread of degradative characters
against toxic chemical compounds. Although this pheno-
menon is well-known in the evolution and dissimilation
of antibiotic-resistance genes, no clear evidence for the
occurrence of a similar trait in soil bacteria was available
until now.
The structural studies performed in this work point to the
important role of direct and inverted repeats in the genetic
rearrangements necessary for rapid transition from growth
on one substrate to that of a different substrate. The
amplification of the structural genes in absence of a
putative regulatory element is analogous to that observed
in case of antibiotic resistance to allow the host
microorganisms to withstand high and toxic concentra-
tions of such antibiotics. Many toxic chemicals, resemble
antibiotics in terms of their inherent toxicity, both towards
animals as well as microorganisms, and it is little wonder
that microorganisms employ essentially identical tools, i.e.,
deletion, fusion, insertion, and other modes of genetic
rearrangements, to help evolve new degradative functions.
Continued studies of such processes are expected to
provide unique insights regarding the mode of evolution
of new biological functions in bacteria.
A DNA Repeated Sequence
In an effort to identify and localize AC1100 genes
associated with 2,4,5-T degradation, transposon insertion
mutagenesis was used to generate mutants blocked in
the 2,4,5-T degradative pathway. A 1.3 kilobase pair DNA
repeated sequence was found in the 2,4,5-T-degrading
Pseudomonas cepacia strain AC1100. One copy of this
sequence RSnoo-l was located near the chromosomal
transposon 5 insertion site of a 2,4,5-T-negative mutant
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designated PT-88. Thus, RSnoo-l appears to be closely
associated with at least one 2,4,5-T gene and may have
played a role in gene rearrangement. As judged by
Southern hybridization analysis, the repeated sequence
was not homologous to DNAs from several other P. cepacia
DNAs and was absent in strains of P. aeruginosa, P. putida,
P. mendocina, and other pesudomonads isolated from soil
samples. The specificity of RSnoo-l to AC1100 suggests
that this unique sequence may be a useful genetic probe
of strain AC1100 and other novel Pseudomonas strains
which are under consideration for deliberate release to
aquatic and terrestrial environments.
7. Tomasek, P., B. Frantz, D. K. Chatterjee, and A. M.
Chakrabarty. 1986. "Genetic and Molecular Basis
of the Microbial Degradation of Herbicides and
Pesticides," In: Biotechnology for Solving Agricul-
tural Problems, Augustine, P. C., H. D. Danforth, and
M. R. Bakst, eds., Marinus Nijhoff Publishers,
Dordrecht, pp. 355-368.
Detox/cation of Contaminated Soil
In laboratory experiments, soil previously contaminated
with as much as 5,OOO /ug of 2,4,5-T/g :of soil could be
detoxified by AC1100 treatment, allowing the growth of
plants sensitive to less than 10 /ug 2,4,5-T/g of soil. Soil
contaminated with as much as 20,000 /ug of 2,4,5-T/g
of soil showed greater than 90% degradation after six
weekly AC1100 treatments. After 2,4,5-T has been
substantially degraded in contaminated soil, the titer of
AC1100 rapidly falls to nearly undetectable levels.
Publications
The following publications describe research supported by
Cooperative Agreement, CR809666, between the U.S.
Environmental Protection Agency and the University of
Illinois College of Medicine at Chicago:
1. Chakrabarty, A. M., 1985. Genetically manipulated
microorganisms and their products in the oil service
industries. Trends in Biotechnology, 3:32-38.
2. Frantz, B., and A. M. Chakrabarty. 1986. "Degrad-
ative Plasmids in Pseudomonas." In: The Bacteria,
Sokatch, J. R., and L. N. Ornston, eds.. Vol. 10, Ch.
9, Academic Press, New York, pp. 295-323.
.^i. „ ^?.;'i* s-:*n' i? ir-rW-."! :?«siV} Vi **%!--; -£~%i ?r- •=;.?, i •- ^ -"-*>•.; *.^. -5*:^;;.
3. Ghosal, D., I.-S. You, D. K. Chatterjee, and A. M.
Chakrabarty. 1985. Genes specifying degradation of
3-chlorobenzoic acid in plasmids pAC27 and pJP4,
Proc. Natl. Acad. Sci. USA. 82:1638-1642.
4. Ghosal, D., I.-S. You, D. K. Chatterjee, and A. M.
Chakrabarty. 1985. Microbial degradation of halo-
genated compounds. Science, 222:135-142.
5. Ghosal, D., I.-S. You, D. K. Chatterjee, and A. M.
Chakrabarty. 1985. "Plasmids in the Degradation of
Chlorinated Aromatic Compounds." In: Plasmids in
Bacteria, Helinski, D. R. et al, eds. Plenum Press,
New York. pp. 667-686.
6. Tomasek, P., B. Frantz, and A. M. Chakrabarty: a
forthcoming paper on repeated sequences of DNA
discovered in Pseudomonas cepacia strain AC1100.
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