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
are,
however,
demonstrate
Some synthetic compounds, with a lower degree of biodegradable;
biochemical,
genetic,
the evolution of new plasmid-encoded
and molecular
enzymatic
activities
specifically designed for the chlorinated substrates.
Nucleotide
sequences of
many of the genes encoding
activities
demonstrate
considerable
throughout
the molecules
homology
either
near
the
such enzymatic
active
sites
or
with
the
chromosomal genes encoding enzymes catalyzing analogous reactions. In some cases, unique
repeated
sequences,
reminiscent
of prokaryotlc
insertion sequence
ele-
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
of genetically-lmproved
bacteria
capable
of utilizing
and
thereby
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).
85
The regulations
86
B E T S Y F R A N T Z et(21
are
usually
based
on
two major
toxicological properties
characteristics
and their fate
of
the
chemicals,
in the environment.
viz.
their
If a chemical
decidedly toxic, its release is highly regulated and seldom permitted.
is
Determin-
ation of the toxicity of a chemical, particularly its slow-acting effect on the human
immune
system
is,
however,
tricky,
variable
and
controversial,
adding
complexity to the regulatory maze (22) that often allows the chemical industry to continue
to dump
suspected
chemicals
into
the
environmental contamination is the accidental
environment.
Another
route
release of known hazardous
of
chem-
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
(34).
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
massive
pollution
problems
in
the
industrialized
countries
for which
there are no particular scientific answers or technologies available. 'Superfund'
program in the United States,
decontaminate from
the
toxic dump sites,
site
storage.
No
of
contamination
technology
is
aims only at hauling the to
some
presently
disposal of these toxic chemicals.
Even the
that is purportedly designed to help
EPA-designated
available
for
toxic chemicals 'safe'
the
site
safe
and
Since natural microorganisms
recycling all kinds of natural wastes but are usually unable
for
away their
economical
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
how
new
degradative
mostly chlorinated aromatics, how
an understanding
of
functions
In this short article, we will
against
simple
chlorinated
compounds,
evolve in natural microflora and speculate as to
such
evolutionary
processes
may
help
us
develop
new
technologies for the recycling of similar, but more complex toxic chemicals from the environment.
It
is
known
that
while
simple
chlorinated
compounds
are
usually
biodegraded
rather rapidly, the highly chlorinated compounds in general are recalcitrant to microbial attack accomplished
(2,8).
either
by
Biodegradation of simple chlorinated compounds may be aerobic
or
anaerobic
microbial
consortia,
or
by
pure
cultures (13). It is often possible to isolate pure cultures capable of utilizing known
synthetic
compounds
such
as
2,4,5-trichlorophenoxyacetic
acid
(2,4,5-T),
1,4-dichlorobenzene or 6-aminonaphthalene-2-sulfonic acid from an initial mixed bacterial
community
through
prolonged
chemostatic
selection
chemical as the sole source of carbon and energy (19, 25, 35). bacterial
strains
capable of utilizing
a variety
with
the
target
While a number of
of xenobiotic
compounds have
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
87
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
are some
interesting
interrelationships
between
chromosomal
and plasmid
genes in allowing efficient biodegradation of these compounds. The mode of biodegradation of a naturally-occurring compound such as benzoate and two synthetic CHROMOSOME
i......... o~
pat27
DJP4
i coo.
cl
i............ i
.
............... i
O= ~0~ 0 ~ l~............. ~4 ............. ~.~ /
"]i"" .....
l ...........
@
O
c c, o oo ..
s.,.,..,.
,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.
i.
The
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
88
B E T S Y F R A N T Z etal
the host P. putida or A.
eutrophus cells.
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
of
their
gene
products
(only
towards
3Cba but not
4Cba),
a TOL-
plasmld derived set of genes is needed for the conversion of 4Cba to 4-chlorocatechol, (7,15).
the
latter being
Consequently,
a
substrate
for
either chromosomal
the pAC27
plasmid encoded
enzymes
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
is very
genetic (14).
slow due
rearrangements
to
regulatory
before
plasmid
constraints, pJP4 will
which
allow
must
rapid
be
overcome
growth with
by
3Cba
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
enzymes
involved
in
the
2,4-D
degradative
pathway
(9).
An
interesting
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
pAC27.
It
should
be noted
that while
the
chlorocatechol
degradative
genes are present in a Pl-ineompatlbility 2,4-D degradative plasmid pJP4 to allow degradation of chlorocatechols derived from 2,4-dlchlorophenol,
the chlorocate-
chol genes borne on an entirely different plasmid pAC27 are expressed specifically to allow degradation of chlorocatechols derived from chlorobenzoates. theless,
homology
demonstrated
studies
homology
genes (13,14).
only
between
these
between
the
two
plasmids
fragments
(pJP4
harboring
and
the
Never-
pAC27)
have
chlorocatechol
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
non-chlorlnated
catechol.
Such
enzymes
(hydrolase I) for the degradation
are
known
to
be
specified
by
the
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
89
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
chlorocatechols.
In contrast,
the plasmid-specffied
degradative enzymes pyrocatechase II and cycloisomerase active
for the chlorinated
3-chloromuconate
substrates,
viz.
3- or 4-chlorocatechol
still retain substantial activity
chlorinated catechol or cis,cis-muconate.
chlorocatechol
II, while being highly
(50% or more)
and
2- or
for the non-
Dienelactone hydrolase, however, has
very little activity towards the enol-lactone
(32,33).
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.
To
obtain
an
degradative
insight
regarding
genes present
kilobase pair
the mode
of
on plasmid pAC27,
(kb) segment
evolution Ghosal
from plasmid pAC27
cells to utilize 3Cba slowly.
of
et al.
the
chlorocatechol
(14) cloned a 4.2
that allows the host P.
putida
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
postulated
sequences
to
be
necessary
on this fragment.
due
to
the
A 4.3 kb Bglll
absence
of
the
regulatory
fragment containing
gene
two adjoining
Bglll fragments with a 385 base pair (bp) segment with the promoter sequences and the
larger
fragment
harboring
the
sequenced by Frantz and Chakrabarty
chlorocatechol (12).
genes
have
been
completely
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
of
this 4.3 kb
fragment
under
the
ta_..~cpromoter
(hydrolase II).
in broad host
range
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-
90
BETSY F R A N T Z et al.
cl
~>
~
o
(OnF3)
/\
**=,,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
clcA,
B,
and D
genes,
another
function is presently unknown.
putative
open
reading
frame
(ORF3)
whose
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
interesting
conservation
of
upstream
sequences,
Pseudomonas positively-regulated regulatory sequences
characteristic
of
(Fig. 2). Since the clcABD
gene cluster has previously been inferred to be under positive control
(13,14),
it would be interesting to find out if all positively-regulated degradative gene clusters sequences.
in
Pseudomonas
will
demonstrate
the
presence
of
these
conserved
SYNTHETIC RECALCITRANT C O M P O U N D S
91
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.
The nucleotide
sequences
in the 2,4-D
of the tfdCDE
gene
cluster have, however, not been determined as yet. It should be reemphasized that while clcA and clcB gene products have activities towards catecho]
and cis,cis-
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,
Aldrich
et
al.
(i) have
recently
cloned
and
completely
In addi-
sequenced
the
chromosomal catB gene from P. putida which has made a comparative study with the plasmid-borne clcB gene possible. amino
acid
sequence
homology
Frantz et al.
near
a catalytic
(ii) have also determined the site
Cys
residue
between
the
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
The two
3,4-dioxygenase share extensive homology with pyro-
that pyrocatechase
II retains appreciable
This is also reflected by activity
towards
catechol.
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
divergence
may
still
allow
the
evolved
towards the non-chlorlnated cis,cls-muconate.
enzyme
to
retain
some
activity
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
of ~-chloromercuribenzoate,
cysteinyl sequence closely
side
chain
lying at or near
surrounding resembles
hydrolase
suggesting
one
the
of
amino
the acid
the
cysteine sequence
inactivated by stoichiometric
that each hydrolase
active residues
site in
neighboring
(ii). The enol-lactone Cys-60
contains amino
a
acid
hydrolase
in dienelactone
(Fig. 3), suggesting that the catalytic region alone might have been
constrained
against
divergence because
of
its critical
role
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.
92
~; ~ ; ,~; ~': ~,~,~~ 7 , ~ h' i ~ : '. ~~~ t :~'[:j :~
Fig. 3.
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
compounds
such
as nylon
(23,26).
generally believed to be the result of divergent
Dissimilatory
evolution,
genes
are
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
by multiple
of divergence et al.
(i).
seen These
point mutations.
throughout
the
catB
two genes have
Such a model may and
numerous
clcB
genes
as
explain
the
reported
by
short duplicated
ranging from 6 to 13 base pairs throughout their lengths.
segments
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
were
acquired
by
intra-
rather
than
inter-gene
recombinations.
These
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
93
ROLE OF REPEATED SEQUENCES IN THE EVOLUTION OF DEGRADATIVE GENES Repeated sequences have been discovered
in many prokaryotic
systems
including
Gram-negative and Gram-positive eubacteria as well as the archaebacteria.
Com-
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
gene,
encoding
extremely
low enzymatic
activity
A duplicate
is also believed
to be
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 ~.
cepacia ACII00
(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
Fig. 4.
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].
94
insertion sequence elements
in the genome of a strain of P.
cepacia, which
is
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
also
in
facilitating
gene
expression
by
providing
appropriate
promoter
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,
including pentachlorophenol,
although regulatory
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
2,4,5-T degradation,
transposon
insertion mutagenesis with
used to generate mutants blocked in the 2,4,5-T degradative pathway.
Tn5 was
One mutant,
PT88, was studied further since it produced a dark brown color in the culture medium
characteristic of
presence of 2,4,5-T.
chlorocatechol
or ortho-chloroquinone
accumulation
in
EcoR1-restricted plasmid and chromosomal DNA from PT88 were
probed with Tn5 DNA and the insertion was localized on the chromosome.
Cloning
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
including the
sequences flanking
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
further
localized
to
a
1.27
kb
Sall-Pvull
restriction
fragment
which
no
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,
termed RSII00-1,
the context of evolution of the 2,4,5-T degradative genes?
relevant in
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
evolved
after months
of
selective
pressure
in the
chemostat,
and
the
repeated sequence has been undetectible in a large number of Pseudomonas species isolated from soil (37). and
the
repeated
It is therefore not unlikely that both the 2,4,5-T gene
sequence
element
may
have
nonpseudomonal
origin
and
genetic
SYNTHETIC RECALCITRANT COMPOUNDS
95
rearrangements may have involved transfer of genes from nonpseudomonal bacteria through transposable elements. sequence
element,
Tomasek
et
In order to define the nature of the repeated al.
(37) have
completely
sequenced
a number
of
chromosomal fragments harboring the repeat, and determined the sequence of the common element
(RSII00-I).
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
of RSII00-I
on the chromosome
and plasmids
of ACII00.
The 8 bp host
chromosome duplication at the site of insertion is reminiscent of similar target duplications (17).
generated
upon
integration
of
prokaryotic
transposable
elements
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.
ebp
Fig. 5.
Im
19
2
,
I
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 .
96
CONCLUDING REMARKS The
manufacturing,
essential
in
environmental remedied.
a
modern
distribution industrial
pollution,
Toxicological
that
not
studies,
and
use
of
synthetic
society,
have
created
only must
be
recognized,
which
have
been
the
chemicals,
severe but
focus
while
problems also
of
must
the
of be
chemical
industry and the government regulatory agencies simply help recognize the extent of the problem and seeking ACII00,
a
its causative
solution.
Laboratory
agent(s),
but do not address the problem of
selected
microorganisms
such
as
[.
cepacia
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.
This short
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
and
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
essential
strains
prerequisite
capable
for
of enhanced
the
construction
degradation of
and
toxic,
development
of
microbial
synthetic pollutants
in the
environment.
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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