pathways to dissimilate toxic chemicals released into the environment. .... striata (34) act on a wide range of N-phenylcarbamate herbicides including ..... p m t e r appears to be under positive control, since the 4.2 kb BglII fragment allows slow ..... E. Senior, A.T. Bull, and J.H. Slater, Nature (London), 262, 476-479 (1976).
Biotech. Adv. Vol. 5, pp. 85-99, 1987 Printed in Great Britain. All Rights Reserved.
0734-9750/87 $0.00 + .50 C.pvri~hl ' Pergamon ~ournals Lid
MICROBIAL DEGRADATION OF SYNTHETIC RECALCITRANT COMPOUNDS BETSY FRANTZ, TERI ALDRICH and A. M. CHAKRABARTY Department
of Microbiology and I m m u n o l o g y . University of I l l i n o i s College of M e d i c i n e . Chicago. Illinois 60612. U S A
ABSTRACT Synthetic compounds, particularly highly chlorinated aromatics, comprise the bulk of the environmental pollutants that somehow must be removed from the environment.
Microbial degradation of such compounds is usually very slow, making them
highly persistent in nature. chlorination studies
Some synthetic compounds, with a lower degree of biodegradable;
the evolution of new plasmid-encoded
specifically designed for the chlorinated substrates.
many of the genes encoding
chromosomal genes encoding enzymes catalyzing analogous reactions. In some cases, unique
ments, are present at or near the newly evolved genes. This suggests gene duplication and divergence as well as recombinational events mediated by transposable type elements as key ingredients in the evolution of new degradative functions. An understanding of such evolutionary processes is an essential feature for the development
removing highly chlorinated environmental pollutants from our environment.
KEY WORDS toxic chemicals, gene evolution, repeated sequences, plasmlds
INTRODUCTION Many synthetic compounds are released in the environment for household or industrial applications, resulting in a maze of governmental regulations to prevent the immediate toxic effects on human beings and animals (3,5).
B E T S Y F R A N T Z et(21
and their fate
in the environment.
If a chemical
decidedly toxic, its release is highly regulated and seldom permitted.
ation of the toxicity of a chemical, particularly its slow-acting effect on the human
complexity to the regulatory maze (22) that often allows the chemical industry to continue
environmental contamination is the accidental
release of known hazardous
icals, demonstrated by the infamous Seveso and Bhopal incidences as well as the recent accident in Basle leading to the contamination of the Rhine river
Storage of toxic chemicals, usually as by-products of the chemical industry and the deliberate or accidental dumping of toxic chemicals in the environment have created
there are no particular scientific answers or technologies available. 'Superfund'
program in the United States,
toxic dump sites,
aims only at hauling the to
disposal of these toxic chemicals.
that is purportedly designed to help
toxic chemicals 'safe'
Since natural microorganisms
recycling all kinds of natural wastes but are usually unable
are known for
to attack highly
chlorinated synthetic chemicals, the toxic chemical pollution problem presents a major challenge to the biotechnology industry to develop innovative technologies for the recycling of some of these chemicals. discuss
mostly chlorinated aromatics, how
In this short article, we will
evolve in natural microflora and speculate as to
technologies for the recycling of similar, but more complex toxic chemicals from the environment.
rather rapidly, the highly chlorinated compounds in general are recalcitrant to microbial attack accomplished
Biodegradation of simple chlorinated compounds may be aerobic
cultures (13). It is often possible to isolate pure cultures capable of utilizing known
1,4-dichlorobenzene or 6-aminonaphthalene-2-sulfonic acid from an initial mixed bacterial
chemical as the sole source of carbon and energy (19, 25, 35). bacterial
capable of utilizing
While a number of
been isolated this way, only a few bacterial cultures have been studied intensively with regard to the genetic and molecular basis of such degradation (27).
SYNTHETIC RECALCITRANT C O M P O U N D S
The compounds which are known to be degraded readily by pure cultures comprise the chlorinated benzoic and phenoxyacetic acids, more specifically 3-chlorobenzoate (3Cba) and 2,4-dichlorophenoxyacetate (2,4-D). Most of the genes required for the degradation of these two compounds are known to be borne on plasmids (10,13). There
genes in allowing efficient biodegradation of these compounds. The mode of biodegradation of a naturally-occurring compound such as benzoate and two synthetic CHROMOSOME
O= ~0~ 0 ~ l~............. ~4 ............. ~.~ /
c c, o oo ..
,1 Fig. I.
Pathways for the degradation of benzoate, chlorophenoxyacetates and chlorobenzoates. The benzoate degradative genes are borne on the chromosome while the 2,4-D and 3Cba genes ere borne on plasmids pJP4 and pAC27, respectively. The plasmid-specified pyrocatechase II, cycloisomerase II and hydrolase II have high affinity for chlorinated substrates, while the corresponding chromosomally coded pyrocatechase I, cycloisomerase I and hydrolase I have high affinity for the non-chlorinated substrates with little or no activity towards the chlorinated compounds.
compounds vi__~z. 3-or 4-chlorobenzoate
(4Cba) or 2,4-D is shown in Fig.
plasmid pJP4 is believed to encode the complete pathway for the degradation of 2,4-D (9,38), although plasmid mutations in all the catabolic steps have not been characterized so far (9).
In contrast, the plasmid pAC27 (Fig. i) encodes only a
partial pathway, vi_...~z,that of chlorocatechol. 3-chlorobenzoate
The first two enzymes that convert
to 3-chlorocatechol are specified by the chromosomal genes of
B E T S Y F R A N T Z etal
the host P. putida or A.
Thus both the plasmid and the host
cell chromosomal genes are involved in the total degradation of 3-chlorobenzoate. Other plasmid genes may sometimes be needed for the degradation of other chlorinated compounds.
For example, for the degradation of 4Cba, where the chromosomal
benzoate oxygenase genes are of little value because of the limited substrate specificity
3Cba but not
plasmld derived set of genes is needed for the conversion of 4Cba to 4-chlorocatechol, (7,15).
or other plasmid genes are sometimes
involved, in addition to a naturally-occurring degradative plasmid, for the total dissimilation of a chlorinated compound.
ORGANIZATION AND REGULATION OF THE CHLOROCATECHOL DEGRADATIVE GENES The pathways depicted in Fig.
i show some interesting characteristics regarding
the enzymes and the nature of their substrates.
For example, the pyrocateehase
II, eycloisomerase II, and the hydrolase II, encoded by plasmid pJP4 and involved in the degradation of chlorocatechols derived from 2,4-D, are also involved in the degradation of 3-chlorocatechol derived from 3Cba (9,38).
Thus, the presence
of pJP4 allows the host cells to utilize 3Cba, although the rate of such degradation
constraints, pJP4 will
Analysis of transposon-generated mutations in these genes (termed tfdC, D,
and E), has demonstrated the gene order tfdC, D, and E, similar to the order of the
question in this regard is how much homology the chlorocatechol degradativ~ tfdC, D, and E genes on pJP4 might have with the chlorocatechol genes present in the plasmld
genes are present in a Pl-ineompatlbility 2,4-D degradative plasmid pJP4 to allow degradation of chlorocatechols derived from 2,4-dlchlorophenol,
chol genes borne on an entirely different plasmid pAC27 are expressed specifically to allow degradation of chlorocatechols derived from chlorobenzoates. theless,
Thus, the chlorocateehol genes may have a common ancestry, even
when present on two different plasmids under two different regulatory controls.
The pathways depicted in Fig. 1 also demonstrate the involvement of pyrocatechase I, cycloisomerase I and enol-lactone hydrolase of
(hydrolase I) for the degradation
chromosomal genes, and have rather stringent specificity towards catecbol or its metabolltes.
Thus, Knackmuss, Reineke, and their associates
(32,33) have shown
SYNTHETIC RECALCITRANT C O M P O U N D S
the relative activity of pyrocatechase I towards 3-chlorocatechol at less than i% (relative to catechol) and of cycloisomerase I towards 2-chloromuconate at less than i% (relative to ci___.ss,cis--muconate). Enol-lactone hydrolase has very little activity towards the dienelactone. Such high specificity of the catechol degradarive enzymes readily explains the inability of natural catechol degrading strains to utilize
degradative enzymes pyrocatechase II and cycloisomerase active
for the chlorinated
3- or 4-chlorocatechol
still retain substantial activity
chlorinated catechol or cis,cis-muconate.
II, while being highly
(50% or more)
for the non-
Dienelactone hydrolase, however, has
very little activity towards the enol-lactone
Such enzymatic activity
studies appear to indicate that the plasmid-coded pyrocatechase II and cycloisomerase II might have evolved from the respective analogous chromosomally-coded enzymes, while dienelactone hydrolase may have evolved independently.
on plasmid pAC27,
from plasmid pAC27
cells to utilize 3Cba slowly.
(14) cloned a 4.2
that allows the host P.
This gene cluster was shown to have appreciable
homology with the i0 kb BamHI-EcoRl fragment of plasmid pJP4 that also harbors the chlorocatechol degradative genes.
Rapid growth with 3Cba ensured only when
the gene cluster was amplified to a copy number of 7 or 8 on the plasmid, and the amplification was found to be dependent on the recA + function (13). Amplification was
on this fragment.
A 4.3 kb Bglll
Bglll fragments with a 385 base pair (bp) segment with the promoter sequences and the
sequenced by Frantz and Chakrabarty
Some of the interesting features of
this 4.3 kb BglIl fragment derived from plasmid pAC27 are given in Fig. 2. The steps involved in the conversion of chlorocatechol to maleylacetic acid (which is believed to be converted to 8-ketoadipate, presumably by a chromosomally coded maleylacetate reductase enzyme) require the participation of 3 key chlorocatechol (clc) degradative genes clcA, clcB, and clcD, encoding respectively the pyrocatechese If, the cycloisomerase II and the dienelactone hydrolase Cloning
this 4.3 kb
in broad host
plasmid pMMB22 followed by transfer and subsequent activation of the tac promoter in Escherichia coli resulted in the appearance of all three enzymes in E. coli. The enzymes were subsequently purified in the laboratory of Dr. L.N. Ornston, the N-terminal amino acid sequence determined, and the relative position of the clcA, B, and D genes deduced on the fragment by comparison of the N-terminal amino acid sequences derived
from purified enzymes with those predicted from the DNA se-
BETSY F R A N T Z et al.
**=,,r...* ......, Fig. 2.
The pathway and genes for chlorocatechol degradation. The steps A, B and D are mediated by ehlorocatechol degradative (cl___c_c) genes clcA, clcB, and clcD, respectively, encoding pyrocatechase II, cycloisomerase II and hydrolase II, on the 4.3 kb Bglll fragment of the plasmid pAC27. The location of the promoter upstream of the structural genes is shown by the arrow, and its homology with other promoter sequences of a number of operons known to be under positive control is shown at the bottom. PRCS represents positively regulated conserved sequences at the -I0 and -35 region present upstream of the structural genes of the positively-regulated operons. The ATG of the clcB gene overlaps with the TGA of the clcA gene as shown on the intersection of these two genes. The various restriction sites on the fragment are marked.
quences (11,24). As shown in Fig. 2, the 4.3 kb fragment contains, in addition to the
function is presently unknown.
The clcA and B genes overlap by 4 bp, and this is
believed to result in reduced expression of the clcB gene. The entire clcABD gene cluster appears to be regulated as a single unit, since hyperproduction of both clcA and clcD gene products occurs in E. coli or ~. putida only on activation of the ta___~cpromoter by isopropyl-B-D-thiogalactoside.
S1 nuclease mapping experi-
ments have demonstrated the site of transcription initiation on the adjoining 385 bp Bglll fragment, and the upstream sequences of the clcABD gene cluster show a good deal of homology with the putative promoter sequences of xylCAB and xylDEFG operons, which are under positive control.
A sequence comparison between these
operons, and the E. coli consensus positively-regulated conserved sequences (31) show
Pseudomonas positively-regulated regulatory sequences
(Fig. 2). Since the clcABD
gene cluster has previously been inferred to be under positive control
it would be interesting to find out if all positively-regulated degradative gene clusters sequences.
SYNTHETIC RECALCITRANT C O M P O U N D S
EVOLUTION OF GENES FOR THE DEGRADATION OF SYNTHETIC C0b~0UNDS The determination of complete nucleotlde sequences for the clcABD gene cluster raises the interesting question of how much sequence identity such genes have with chromosomal genes encoding analogous reactions.
It is interesting to note
that the order of the clcABD genes is the same as the order of the degradatlve steps in the pathway, similar to the chlorocatechol genes (tf~CDE) pathway encoded by plasmid pJP4.
in the 2,4-D
of the tfdCDE
cluster have, however, not been determined as yet. It should be reemphasized that while clcA and clcB gene products have activities towards catecho]
muconate, which are normal substrates for catA and catB gene products, the clod gene product appears to catalyze a novel reaction.
How do such genes evolve in
nature? Since oxygenases similar to pyrocatechase II have previously been purified and their amino acid sequences determined (29), it is possible to make comparative studies on sequence homologies between pyrocatechase II and another chromosomally-coded oxygenase such as protocatechuate 3,4-dloxygenase, which is formed by self-assoclatlon of equal amounts of nonidentical a and 8 subunits. tion,
chromosomal catB gene from P. putida which has made a comparative study with the plasmid-borne clcB gene possible. amino
Frantz et al.
(ii) have also determined the site
chromosomally encoded enol-lactene hydrolase and the plasmid-coded dlenelactone hydrolase.
The results of such comparative studies are shown in Fig. 3.
subunlts of protocatechuate
catechase II suggesting a common ancestry among them. the fact
3,4-dioxygenase share extensive homology with pyro-
II retains appreciable
This is also reflected by activity
Similarly, the 52% homology at the nucleotlde level between the chromosomal catB and the plasmid borne clcB gene (I) may indicate the evolutionary origin of clcB gene from an ancestral catB gene, with divergence throughout the gene segment. Such
towards the non-chlorlnated cis,cls-muconate.
In contrast, the hydrolases appear
to have diverged widely, since the N-terminal amino acid sequences of the enzymes are dissimilar. Both the hydrolases are, however, amounts
cysteinyl sequence closely
lying at or near
inactivated by stoichiometric
that each hydrolase
(ii). The enol-lactone Cys-60
(Fig. 3), suggesting that the catalytic region alone might have been
in the enzymatic
catalysis. Various models have been proposed for the evolution of genes involved in the degradation of aromatic compounds and their metabolites in Pseudomonas and other
BETSY FRANTZ el al.
~; ~ ; ,~; ~': ~,~,~~ 7 , ~ h' i ~ : '. ~~~ t :~'[:j :~
Amino acid sequence homology between pyrocatechase II (PYRII) and protocatechuate dioxygenase subunits ~ and (PD= ,PDB), cycloisomerase II (MLE II) and cycloisomerase I (MLE I), and dienelactone hydrolase (DLH) and enol-lactone hydrolase (ELH). The amino acid sequence for PYRII, MLEII, MLEI and DLH has been determined from the DNA nucleotide sequences while that for PD=, PDB and ELH has been determined from the purified proteins.
soil bacteria (8,28,29).
Clear evidence for gene duplication followed by diverg-
ence has been presented for the evolution of nylB gene involved in the degradation of xenobiotic
generally believed to be the result of divergent
and both clcA and
clcB genes might have evolved from the corresponding chromosomal genes by such an evolutionary process.
One of the models proposed for generating gene divergence
involves repeated recombinational events between misaligned DNA segments (28). In this model, double crossovers allow base pair substitutions within genes without altering substantially the translational reading frame or the length of the protein.
They result in nontandem direct repeats and rapid, irreversible divergence
from the ancestral sequence provided subsequent recombination with other genomes does not occur.
This model permits more rapid and drastic gene variation than
could be obtained extent Aldrich
of divergence et al.
two genes have
Such a model may and
ranging from 6 to 13 base pairs throughout their lengths.
The nontandem direct
repeats which have been identified in the first 200 bp of each sequence are shown in Fig. 4.
The repeats usually occur 2 to 4 times within their respective genes.
They are generally unique to the gene in which they are located indicating that they
observations support the model presented above which could account for much of the sequence divergence among B-ketoadipate pathway genes.
SYNTHETIC RECALCITRANT C O M P O U N D S
ROLE OF REPEATED SEQUENCES IN THE EVOLUTION OF DEGRADATIVE GENES Repeated sequences have been discovered
in many prokaryotic
Gram-negative and Gram-positive eubacteria as well as the archaebacteria.
pelling evidence for the role of such sequences in the evolution of degradative functions against synthetic compounds such as nylon oligomers has been provided by Negoro et al. (23) and Okada et al. (26). These authors characterized a plasmid in Flavobacterium sp. K172 which encodes the degradation of 6-aminohexsnoic acid cyclic dimer, a by-product of nylon manufacture, through elaboration of two newly evolved enzymes. and RS-II.
The plasmid contains two kinds of repeated sequence, RS-I
One of the two RS-II sequences, RS-IIA, contains the nylB gene while
the other, RS-II B, contains a homologous gene encoding an enzymatically-nonfunctional protein.
RS-I, which appears 5 times on the plasmid, is thought to be
involved in the rearrangement of the plasmid to translocate the proto-nylB gene, in the same way as insertion sequences mediate gene rearrangements. nylA
is also believed
present on the plasmid, again suggesting gene duplications and further divergence for the evolution of this gene. Circumstantial evidence for the role of repeated sequences in the evolution of genes for the degradation of synthetic compounds comes from the characterization of such an element in the 2,4,5-T degrading strain of ~.
(36). Lessie and Gaffney (21) have recently described the presence of a number of
P. putida catB ATGACAAGTGTGCTGATTGAACGTATCGAGGCAATTATTGTGCATGACCTGCCGACCA TTCGTCCGCCGCACAAGCTGGCGATGCACACCATGCAGACGCAGACCC~GT~-rGATTC GTGTTCGCTGCAGTGATGGCGTGGAAGGCATGGGCGAGGCCACCACCATGGCCGGCCTGG CCTATGGCTACCAAACGCCGGA
P. purida clcB i A~GAACA~CGAA~CATCGAT~GACGCZ~T~ACG~CCCACC~CCCGTCCCArCC
AGATGTCGTTTACCACGGTGCAGAAGCAGAGCTATGCGATCGTGCAGATCCGTGCGGGCG GGCTTGTCGGCATCGGCGAGGGCAGCAGCGTAGGTGGGCCGACTTCGAG~FTCCGAATGCG CTGAAACCATCAAGGTCATCAT
Nontandem direct repeats found in the P. putida catB (encoding cycloisomerase I) and clcB (encoding cycloisomerase II) genes. The first 200 bp of the coding strand of each sequence is shown beginning with the ATG start codons. Arrows above the sequences designate nontandem direct repeats. Letters above the arrows indicate pairs of repeats.
BETSY FRANTZ eta].
insertion sequence elements
in the genome of a strain of P.
well known for its catabolic versatility. They have postulated that some of these insertion sequence elements may be involved not only in recombinational events, but
sequences. The 2,4,5-T degrading strain of [. cepacia ACIlO0 was isolated after prolonged selection in the chemostat in presence of 2,4,5-T as its major source of carbon and energy (19).
It not only is able to utilize 2,4,5-T and 2,4,5-tri-
chlorophenol as its sole source of carbon and energy, but can completely mineralize a number of chlorophenols,
constraints do not allow it to utilize pentachlorophenol as its sole source of carbon and energy (18). In an effort to identify and localize ACII00 genes associated with
insertion mutagenesis with
used to generate mutants blocked in the 2,4,5-T degradative pathway.
PT88, was studied further since it produced a dark brown color in the culture medium
presence of 2,4,5-T.
EcoR1-restricted plasmid and chromosomal DNA from PT88 were
probed with Tn5 DNA and the insertion was localized on the chromosome.
of the kanamycln-resistance marker of Tn5 from this mutant allowed the isolation of a 6 kb Sall fragment that contained half of the Tn5 sequence kanamycin resistance gene and the other half of chromosomal
the insertion site (36). The flanking chromosomal DNA present on this fragment is believed to carry the insertion-lnactivated 2,4,5-T degradatlve gene, presumably coding for an enzyme involved in chlorocatechol metabolism.
When this fragment
was used as a probe against ACII00 chromosomal and plasmid restriction digests to identify the functional gene, a large number of bands lighted up, suggesting the presence of a repeated sequence at or near this gene (36).
This highly repeated
sequence (with at least 20 copies on the chromosome and 9 copies on the plasmlds) was
longer contained the Tn5 sequence. Most interestingly, this repeated sequence did not hybridize with chromosomal DNA isolated from P. number of [. cepacia strains
aeru~inosa, P. putida or a
(36), suggesting that it is unique to the 2,4,5-T
degrading strain of P. cepacia ACt100. Is the presence of such a repeated sequence,
the context of evolution of the 2,4,5-T degradative genes?
Although located near
one of the 2,4,5-T degrading genes, there are at least 20 copies of this element on the chromosome of ACII00 and therefore its location near a 2,4,5-T gene may be just coincidental. It is nonetheless important to note that the 2,4,5-T degrading ability
repeated sequence has been undetectible in a large number of Pseudomonas species isolated from soil (37). and
It is therefore not unlikely that both the 2,4,5-T gene
SYNTHETIC RECALCITRANT COMPOUNDS
rearrangements may have involved transfer of genes from nonpseudomonal bacteria through transposable elements. sequence
In order to define the nature of the repeated al.
chromosomal fragments harboring the repeat, and determined the sequence of the common element
The structure of this element is shown in Fig. 5.
There is an 8 bp direct repeat, which is due to duplication of the host chromosomal target site. This is followed by a 39 bp terminal inverted repeat (38 bp in the other orientation) with a few mismatches.
There is an open reading frame of
1314 bp immediately bounded by the terminal inverted repeats interposed sequences).
(with 9 and 5 bp
The element has therefore all the structural character-
istics of a transposable element (20) which may also explain the large number of copies
on the chromosome
The 8 bp host
chromosome duplication at the site of insertion is reminiscent of similar target duplications (17).
That RSII00-I may indeed be a transposable element is seen from the amino
acid sequences predicted from the 1314 bp open reading frame.
A part of the 48
kilodalton polypeptide contains the typical DNA-blndlng domain as demonstrated by the presence
of a helix-turn-helix motif,
and the similarity of the critical
amino acid sequence with those encoded by the transposase gene (MuA) of phage Mu (16) and the lacR gene (30) is shown in Fig. 5. reading frame encodes element,
It is thus likely that the open
a transposase enzyme for efficient
translocatlon of the
although the origin of such an element in AC1100 and its role in the
recruitment of 2,4,5-T degradative genes remains undefined.
Structure of a repeated sequence element present in multiple copies on the chromosome of [. cepacia ACII00. The open arrows represent the duplication of the host chromosome target site while the solid arrows represent the terminal inverted repeats with the number of bases designated on top. The thin lines represent sequences which are mismatches with the number of bases shown. The bottom part shows the amino acid sequence homology of the DNA-blndlng polypeptldes containing the helix-turn-hellx motif.
BETSY FRANTZ e t a l .
CONCLUDING REMARKS The
industry and the government regulatory agencies simply help recognize the extent of the problem and seeking ACII00,
but do not address the problem of
have been shown to be capable of removing large concentrations of the
toxic chemical (2,4,5-T) from the contaminated soil, and such cells appear to die off quickly once the target chemical is gone (13). Microbial degradation thus represents the major route to solving this problem, if only the problem of microbial recalcitrance to synthetic compounds is understood and overcome.
review emphasizes the mode of evolution of new degradative functions in bacteria with the understanding that such studies will lead t2 the development of newer strains against newer synthetic chemicals through enhanced evolution of degradative genes under highly selective conditions in the chemostat. emphasized
(6) the roles played by microbial products
biopolymers in pollution control.
We have previously
such as surfactants
Selective evolution not only allows evolution
of the degradative genes, but also the ancilliary genes for the production of emulsifiers and surfactants that might prove to be important in enabling the host microorganisms to take up and utilize highly hydrophobic synthetic compounds (4). An understanding of the mode of evolution of new genetic functions in bacteria is an
ACKNOWLEDGEMENT This work was supported by a Public Health Service grant ES 04050 from the National Institute of Environmental Health Science and in part by a grant from the National Science Foundation (DMB-8514671).
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