U.S. patent application number 09/898238 was filed with the patent office on 2002-04-04 for isolated and purified dna molecule and protein for the degradation of triazine compounds.
This patent application is currently assigned to Regents of the University of Minnesota. Invention is credited to de Souza, Mervyn L., Sadowsky, Michael J., Wackett, Lawrence P..
Application Number | 20020039778 09/898238 |
Document ID | / |
Family ID | 24182036 |
Filed Date | 2002-04-04 |
United States Patent
Application |
20020039778 |
Kind Code |
A1 |
Wackett, Lawrence P. ; et
al. |
April 4, 2002 |
Isolated and purified DNA molecule and protein for the degradation
of triazine compounds
Abstract
Abstract of the Disclosure An isolated and purified DNA
molecule, and an isolated and purified protein, that are involved
in the degradation of s-triazine compounds (e.g., atrazine) are
provided. A method for the purification of this protein is also
provided.
Inventors: |
Wackett, Lawrence P.; (St.
Paul, MN) ; Sadowsky, Michael J.; (Roseville, MN)
; de Souza, Mervyn L.; (St. Paul, MN) |
Correspondence
Address: |
MUETING, RAASCH & GEBHARDT, P.A.
P.O. BOX 581415
MINNEAPOLIS
MN
55458
US
|
Assignee: |
Regents of the University of
Minnesota
|
Family ID: |
24182036 |
Appl. No.: |
09/898238 |
Filed: |
July 3, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09898238 |
Jul 3, 2001 |
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08546793 |
Oct 23, 1995 |
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6284522 |
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Current U.S.
Class: |
435/196 ;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/14 20130101; A62D
2101/26 20130101; A62D 3/02 20130101; C07K 16/40 20130101; B09C
1/10 20130101 |
Class at
Publication: |
435/196 ;
435/325; 435/320.1; 435/69.1; 536/23.2 |
International
Class: |
C12N 009/16; C07H
021/04; C12P 021/02; C12N 005/06 |
Goverment Interests
[0001] This invention was made with government support from the
United States Department of Agriculture-BARD program, Grant No.
94-34339-112. The government may have certain rights in this
invention.
Claims
What is claimed is:
1. An isolated and purified DNA molecule encoding atrazine
chlorohydrolase; the DNA molecule hybridizes to DNA complementary
to DNA having the sequence shown in FIG. 6 (SEQ ID NO:1), beginning
at position 236 and ending at position 1655, under the stringency
conditions of hybridization in buffer containing 0.25 M
Na.sub.2HPO.sub.4, 7% SDS, 1% BSA, 1.0 mM EDTA at 65.degree. C.,
followed by washing with 0.1% SDS and 0.1.times.SSC at 65.degree.
C.
2. The isolated and purified DNA molecule of claim 1 encoding the
atrazine chlorohydrolase having an amino acid sequence shown in
FIG. 7 (SEQ ID NO:2).
3. The isolated and purified DNA molecule of claim 1 having the
nucleotide sequence shown in FIG. 6 (SEQ ID NO:1) beginning at
position 236 and ending at position 1655.
4. The isolated and purified DNA molecule of claim 1 having the
nucleotide sequence shown in FIG. 6 (SEQ. ID NO:1).
5. An isolated and purified protein having a molecular weight of
about 245 kilodaltons that converts atrazine to
hydroxyatrazine.
6. The isolated and purified protein of claim 5 which is a
homotetramer.
7. The isolated and purified protein of claim 5 which has the amino
acid sequence shown in FIG. 7 (SEQ. ID NO:2).
8. The isolated and purified protein of claim 7 bound to an
immobilization support.
9. An isolated and purified protein encoded by the DNA molecule of
claim 1.
10. An isolated and purified protein encoded by the DNA molecule of
claim 3.
11. A polyclonal antibody preparation produced from the isolated
and purified protein of claim 5.
12. A polyclonal antibody preparation produced from the isolated
and purified protein of claim 7.
13. A vector comprising the DNA molecule of claim 1.
14. The vector of claim 13 wherein the DNA molecule of claim 1 is
derived from a Pseudomonas strain.
15. A non-Pseudomonas bacterial cell comprising the vector of claim
14.
16. An isolated and purified oligonucleotide of about 7-300
nucleotides which hybridizes to DNA having the sequence shown in
FIG. 6 (SEQ ID NO:1), beginning at position 236 and ending at
position 1655, under the stringency conditions of hybridization in
buffer containing 0.25 M Na.sub.2HPO.sub.4, 7% SDS, 1% BSA, 1.0 mM
EDTA at 65.degree. C., followed by washing with 0.1% SDS and
0.1.times.SSC at 65.degree. C.
17. A method for the purification of atrazine chlorohydrolase in at
least about 90% yield consisting of a step of adding ammonium
sulfate to an aqueous cell-free extract of an atrazine
chlorohydrolase-containing bacterium.
18. The method of claim 17 wherein ammonium sulfate is added in an
amount of no greater than about 20% of saturation.
19. A method for the degradation of compounds have the following
general formula: 2wherein R.sup.1.dbd.Cl,
R.sup.2.dbd.NR.sup.4R.sup.5 (wherein R.sup.4 and R.sup.5 are each
independently H or a C.sub.1-3 alkyl group), and R.sup.3
.dbd.NR.sup.6R.sup.7 (wherein R.sup.6 and R.sup.7 are each
independently H or a C.sub.1-3 alkyl group), with the proviso that
at least one of R.sup.2 or R.sup.3 is an alkylamino group; said
method comprising adding atrazine chlorohydrolase to a sample
containing said compound.
20. The method of claim 19 wherein the sample is a soil sample.
21. The method of claim 20 wherein the soil sample is contaminated
with a nitrogen-containing fertilizer.
22. The method of claim 19 wherein the step of adding atrazine
chlorohydrolase comprises adding a recombinant bacterium that
expresses atrazine chlorohydrolase.
23. The method of claim 19 wherein the step of adding atrazine
chlorohydrolase comprises adding the bacterial cell of claim
15.
24. An isolated and purified protein that converts atrazine to
hydroxyatrazine, wherein the protein comprises an amino acid
sequence encoded by a DNA molecule having a compliment that
hybridizes to a DNA having the sequence shown in FIG. 6 (SEQ ID
NO:1), beginning at position 236 and ending at position 1655, under
the stringency conditions of hybridization in buffer containing
0.25 M Na.sub.2HPO.sub.4, 7% SDS, 1% BSA, 1.0 mM EDTA at 65.degree.
C., followed by washing with 0.1% SDS and 0.1.times.SSC at
65.degree. C.
25. An isolated and purified protein and biologically active
derivatives thereof that convert atrazine to hydroxyatrazine,
wherein the protein comprises an amino acid sequence encoded by a
DNA molecule having a compliment that hybridizes to a DNA having
the sequence shown in FIG. 6 (SEQ ID NO:1), beginning at position
236 and ending at position 1655, under the stringency conditions of
hybridization in buffer containing 0.25 M Na.sub.2HPO.sub.4, 7%
SDS, 1% BSA, 1.0 mM EDTA at 65.degree. C., followed by washing with
0.1% SDS and 0.1 .times.SSC at 65.degree. C.
26. An isolated and purified protein that converts atrazine to
hydroxyatrazine, wherein the protein comprises an amino acid
sequence having greater than about 80% sequence identity to the
amino acid sequence depicted at SEQ ID NO:2.
27. An isolated and purified protein and biologically active
derivatives thereof that convert atrazine to hydroxyatrazine,
wherein the protein comprises an amino acid sequence having greater
than about 80% sequence identity to the amino acid sequence
depicted at SEQ ID NO:2.
Description
BACKGROUND OF THE INVENTION
[0002] Atrazine
[2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine- )] is a
widely used s-triazine (i.e., symmetric triazine) herbicide for the
control of broad-leaf weeds. Approximately 800 million pounds were
used in the United States between 1980 and 1990. As a result of
this widespread use, for both selective and nonselective weed
control, atrazine and other s-triazine derivatives have been
detected in ground and surface water in several countries.
[0003] Numerous studies on the environmental fate of atrazine have
shown that atrazine is a recalcitrant compound that is transformed
to CO.sub.2 very slowly, if at all, under aerobic or anaerobic
conditions. It has a water solubility of 33 mg/l at 27.degree. C.
Its half-life (i.e., time required for half of the original
concentration to dissipate) can vary from about 4 weeks to 57 weeks
if in soils at low concentration (i.e., less than about 2 parts per
million (ppm)). High concentration of atrazine, such as those
occurring in spill sites have been reported to dissipate even more
slowly.
[0004] As a result of its widespread use, atrazine is often
detected in ground water and soils in concentrations exceeding the
maximum contaminant level (MCL) of 3 .mu.g/l (i.e., 3 parts per
billion (ppb)), a regulatory level that took effect in 1992. Point
source spills of atrazine have resulted in levels as high as 25 ppb
in some wells. Levels of up to 40,000 mg/l (i.e., 40,000 parts per
million (ppm)) atrazine have been found in the soil of spill sites
more than ten years after the spill incident. Such point source
spills and subsequent runoff can cause crop damage and ground water
contamination.
[0005] There have been numerous reports on the isolation of
s-triazine-degrading microorganisms (see, e.g., Behki et al., J.
Agric. Food Chem., 34, 746-749 (1986); Behki et al., Appl. Environ.
Microbiol. 59, 1955-1959 (1993); Cook, FEMS Microbiol. Rev., 46,
93-116 (1987); Cook et al., J. Agric. Food Chem., 29 1135-1143
(1981); Erickson et al., Critical Rev. Environ. Cont., 19, 1-13
(1989); Giardina et al., Agric. Biol. Chem. 44, 2067-2072 (1980);
Jessee et al., Appl. Environ. Microbiol., 45, 97-102 (1983);
Mandelbaum et al., Appl. Environ. Microbiol., 61, 1451-1457 (1995);
Mandelbaum et al., Appl. Environ. Microbiol., 59, 1695-1701 (1993);
Mandelbaum et al., Environ. Sci. Technol., 27, 1943-1946 (1993);
Radosevich et al., Appl. Environ. Microbiol., 61, 297-302 (1995);
and Yanze-Kontchou et al., Appl. Environ. Microbiol., 60, 4297-4302
(1994)). Many of the organisms described, however, failed to
mineralize atrazine (see, e.g., Cook, FEMS Microbiol. Rev., 46,
93-116 (1987); and Cook et al., J. Agric. Food Chem., 29, 1135-1143
(1981)). While earlier studies have reported atrazine degradation
only by mixed microbial consortia, more recent reports have
indicated that several isolated bacterial strains can degrade
atrazine. For example, we previously reported the isolation of a
pure bacterial culture, identified as Pseudomonas sp. strain ADP
(Mandelbaum et al., Appl. Environ. Microbiol., 61, 1451-1457
(1995); Mandelbaum et al., Appl. Environ. Microbiol., 59, 1695-1701
(1993); and Mandelbaum et al., Environ. Sci. Technol., 27,
1943-1946 (1993)), which degraded a high concentration of atrazine
(>1,000 .mu.g/ml) under growth and non-growth conditions. See
also, Radosevich et al., Appl. Environ. Microbiol., 61, 297-302
(1995) and Yanze-Kontchou et al., Appl. Environ. Microbiol., 60,
4297-4302 (1994). Pseudomonas sp. strain ADP (Atrazine Degrading
Pseudomonas) uses atrazine as a sole source of nitrogen for growth.
The organism completely mineralizes the s-triazine ring of atrazine
under aerobic growth conditions. That is, this bacteria is capable
of degrading the s-triazine ring and mineralizing organic
intermediates to inorganic compounds and ions (e.g., CO.sub.2).
[0006] Little information is available concerning the genes and
enzymes involved in the metabolism of s-triazine compounds.
Although genes that encode the enzymes for melamine
(2,4,6-triamino-s-triazine) metabolism have been isolated from a
Pseudomonas sp. strain, and that encode atrazine degradation
activity from Rhodococcus sp. strains, to date there have been no
reports identifying the genes encoding atrazine dechlorination.
SUMMARY OF THE INVENTION
[0007] The present invention provides an isolated and purified DNA
molecule that encodes atrazine chlorohydrolase. The DNA molecule
hybridizes to DNA complementary to DNA having the sequence shown in
FIG. 6 (SEQ ID NO: 1), beginning at position 236 and ending at
position 1655, under the stringency conditions of hybridization in
buffer containing 0.25 M Na.sub.2HPO.sub.4, 7% SDS, 1% BSA, 1.0 mM
EDTA at 65.degree. C., followed by washing with 0.1% SDS and
0.1.times.SSC at 65.degree. C. Preferably, the present invention
provides an isolated and purified DNA molecule encoding the
atrazine chlorohydrolase having an amino acid sequence shown in
FIG. 7 (SEQ ID NO:2). Preferably, the DNA molecule has the
nucleotide sequence shown in FIG. 6 (SEQ ID NO: 1) beginning at
position 236 and ending at position 1655. The present invention
also provides a vector comprising the DNA molecule described
herein, a transformed cell line, and isolated and purified
oligonucleotides of about 7-300 nucleotides.
[0008] The present invention also provides an isolated and purified
protein having a molecular weight of about 245 kilodaltons that
converts atrazine to hydroxyatrazine. Preferably, this protein has
the amino acid sequence shown in FIG. 7 (SEQ. ID NO:2). Also
provided is an isolated and purified preparation of polyclonal
antibodies produced from this isolated and purified protein.
[0009] The present invention also provides a method for the
purification of atrazine chlorohydrolase in at least about 90%
yield consisting of a step of adding ammonium sulfate to an aqueous
cell-free extract of an atrazine chlorohydrolase-containing
bacterium. This ammonium sulfate is present in an amount of no more
than about 20% of saturation. Finally, the present invention
provides a method for degrading s-triazine compounds.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1. Physical relationship of clones expressing atrazine
degradation ability. Cosmid pMD1 is a 21.5 kb EcoRI fragment in
pLAFR3. Plasmid pMD2 is a 8.7 kb EcoRI fragment from pMD1 cloned
into pACYC184. Plasmid pMD3 is a 3.3 kb ClaI/Bam HI fragment from
pMD2 cloned into pACYC184. Plasmid pMD4 is a 1.9 kb AvaI fragment
from pMD3 cloned into pACYC 184. All four clones express atrazine
degrading activity in Escherichia coli DH5.alpha..
[0011] FIG. 2. Southern hybridization analysis of genomic DNA from
Pseudomonas sp. strain ADP and other atrazine degrading
microorganisms. A .sup.32P-labelled 0.6 kb ApaI/PstI fragment from
pMD4 was used as probe. Lanes: 1, EcoRI-digested genomic DNA from a
consortium degrading atrazine (Stucki et al., Water Res., 1,
291-296 (1995)); 2, EcoRI-digested genomic DNA from a newly
isolated atrazine-degrading bacterium; 3, AvaI-digested genomic DNA
from Pseudomonas sp. strain ADP; 4, EcoRI-digested genomic DNA from
Pseudomonas sp. strain ADP. Values in margin are in kilobase
pairs.
[0012] FIG. 3. Physical and genetic map of the 21.5 kb EcoRI
genomic DNA fragment from Pseudomonas sp. strain ADP cloned in
cosmid pMD1. Forty-six independent transposon Tn5 insertions within
the cloned DNA fragment are indicated. The open and solid circles
represent clearing and nonclearing phenotypes, respectively.
[0013] FIG. 4. Pathway for atrazine degradation in Pseudomonas sp.
strain ADP. The first step is encoded by a gene region located on
pMD4 and generates hydroxyatrazine, which is subsequently
metabolized to carbon dioxide and ammonia.
[0014] FIG. 5. Sequencing strategy for DNA fragment cloned in pMD4.
Arrows indicate direction of primer sequencing reactions.
[0015] FIG. 6. Nucleotide sequence of atzA (SEQ ID NO: 1),
beginning at position 236 and ending at position 1655. The complete
nucleotide sequence of the approximately 1.9-kb AvaI DNA fragment,
cloned in pMD4, was determined on both strands using subcloning and
the primer walking method and PCR. The ORF designated atzA is
indicated by the arrow and a potential Pseudomonas ribosome binding
site is underlined. The double underlined sequence is the stop
codon.
[0016] FIG. 7. Amino acid sequence of the AtzA enzyme (SEQ ID NO:2)
determined by translating the atzA ORF.
[0017] FIG. 8. Enzyme kinetics. Michaelis Menton (A) and Lineweaver
Burke (B) plots for purified AtzA. The estimated Km is 125
.mu.M.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention provides an isolated and purified DNA
molecule, and an isolated and purified protein, involved in the
degradation of s-triazine compounds. More specifically, the
isolated and purified DNA molecule and the protein it encodes are
involved in the dechlorination of s-triazine compounds containing a
chlorine atom and at least one alkylamino side chain. Such
compounds have the following general formula: 1
[0019] wherein R.sup.1.dbd.Cl, R.sup.2 .dbd.NR.sup.4R5 (wherein
R.sup.4 and R.sup.5 are each independently H or a C-.sub.1-3 alkyl
group), and R.sup.3 .dbd.NR.sup.6R.sup.7 (wherein R.sup.6 and
R.sup.7 are each independently H or a C .sub.1-3 alkyl group), with
the proviso that at least one of R.sup.2 or R.sup.3 is an
alkylamino group. As used herein, an "alkylamino" group refers to
an amine side chain with one or two alkyl groups attached to the
nitrogen atom. Examples of such compounds include atrazine
(2-chloro-4-ethylamino-6-isopropylamino-1,3,5-s-triazine),
desethylatrazine (2-chloro-4-amino-6-isopropylamino-s-triazine),
desisopropylatrazine (2-chloro-4-ethylamino-6-amino-s-triazine),
and simazine (2-chloro-4,6-diethylamino-s-triazine).
[0020] Triazine degradation activity is localized to a 21.5-kb
EcoRI fragment, and more specifically to a 1.9-kb AvaI fragment of
the genome of Pseudomonas sp. ADP bacterium. Specifically, these
genomic fragments are involved in s-triazine dechlorination. In
fact, the rate of degradation of atrazine that results from the
expression of these fragments in E. coli is comparable to that seen
for native Pseudomonas sp. strain ADP; however, in contrast to what
is seen with native Pseudomonas sp. strain ADP, this degradation is
unaffected by the presence of inorganic nitrogen sources like
ammonium chloride. This is particularly advantageous for regions
contaminated with nitrogen-containing fertilizers, for example. The
expression of atrazine degradation activity in the presence of
inorganic nitrogen compounds broadens the potential use of
recombinant organisms for biodegradation of atrazine in soil and
water. Thus, the background in which these fragments are expressed
can provide advantageous results.
[0021] The 1.9-kb AvaI genomic fragment includes the gene that
encodes an enzyme that transforms atrazine to hydroxyatrazine,
atrazine chlorohydrolase. As used herein, this gene is referred to
as "atzA", whereas the protein that it encodes is referred to as
"AtzA". Hydroxyatrazine formation in the environment was previously
thought to result solely from the chemical hydrolysis of atrazine
(Armstrong et al., Environ. Sci. Technol. 2, 683-689 (1968);
deBruijn et al., Gene, 27, 131-149 (1984); and Nair et al.,
Environ. Sci. Technol., 26, 1627-1634 (1992)). In contrast to
reports that the first step in atrazine degradation by
environmental bacteria is dealkylation, this suggests that
biological transformation of atrazine to hydroxyatrazine may be
widespread in natural systems.
[0022] The AtzA protein can be purified to homogeneity (i.e., about
95% purity) in two steps involving precipitation in an aqueous
NH.sub.4SO.sub.4 solution and anion exchange chromatography.
Preferably, the aqueous NH.sub.4SO.sub.4 solution contains no more
than about 20% NH.sub.4SO.sub.4, based on its saturation level in
water, typically at about 4.degree. C. Advantageously, the initial
NH.sub.4SO.sub.4 precipitation step alone provides the protein in a
level of purity of at least about 90%. It may be further purified
using anion exchange chromatography, typically performed with
DEAE-cellulose or DEAE Sepharose CL-6B, to separate, at least
partially, different activities. Other chromatographic techniques
that may be used in the purification of such enzymes include
hydroxylapatite and gel filtration, preferably in combination with
one or more of a variety of affinity chromatographic columns with
varying degrees of specificity. Affinity columns that may be used
include Affi-Gel Blue, ATP-agarose chromatography,
heparin-Sepharose, ADP-agarose, PAP-agarose,
Estradiol-17.beta.-Sepharose- , and p-hydroxyphenylacetic
acid-agarose.
[0023] The availability of purified AtzA enzyme makes it possible
to characterize the enzyme and to develop antibodies that can be
used, for example, to screen DNA expression libraries. The fact
that AtzA is precipitated from cell-free supernatants in such a low
concentration of NH.sub.4SO.sub.4 is surprising and fortuitous and
will facilitate the large scale production of AtzA for remediation
technologies. For example, a 250 liter culture of recombinant E.
coli could yield 10 kilograms wet cell paste that would give 50
liters of crude protein extract, which could be processed by adding
ammonium sulfate to 20% saturation, followed by filtration to give
50 grams of purified protein. Even higher yields are possible with
more optimized expression cassette components.
[0024] In addition, the ability of the AtzA enzyme to dechlorinate
substrates such as atrazine and simazine, for example, make this
protein unique and potentially very useful for environmental
remediation of xenobiotic triazine compounds, particularly because
it is very efficient. For example, a protein concentration of 50
mg/liter could degrade about 100 .mu.M of atrazine to about 1 .mu.M
in about 1.35 hours, and about 30 ppm of atrazine to about 3 ppb in
about 2.6 hours. Various environmental remediation techniques are
known that utilize high levels of proteins. For example, proteins
can be bound to immobilization supports, such as beads, particles,
films, etc., made from latex polymers, alginate, polyurethane,
plastic, glass, polystyrene, and other natural and man-made support
materials. Such immobilized protein can be used in packed-bed
columns for treating water effluents. Other environmental samples
could also be treated with the protein of the present invention
(e.g., soil samples).
[0025] Specifically, the present invention is directed to the
isolation and expression of atrazine chlorohydrolase DNA as well as
the characterization and production of an atrazine chlorohydrolase
protein. To that end, the invention provides an isolated and
purified DNA molecule encoding an atrazine chlorohydrolase protein
(i.e., an enzyme) or biologically active derivative thereof. More
preferably, the DNA molecule encodes the protein represented by the
amino acid sequence shown in FIG. 7 (SEQ ID NO:2). Most preferably,
the DNA molecule is represented by the complete nucleotide sequence
shown in FIG. 6 (SEQ ID NO:1), beginning at position 236 and ending
at position 1655. Isolated and purified proteins encoded by this
DNA molecule that convert atrazine to hydroxyatrazine are also
within the scope of the invention.
[0026] As used herein, the terms "isolated and purified" refer to
in vitro isolation of a DNA molecule or protein from its natural
cellular environment, and from association with other coding
regions of the bacterial genome, so that it can be sequenced,
replicated, and/or expressed. Preferably, the isolated and purified
DNA molecules of the invention comprise a single coding region.
Thus, the present DNA molecules are preferably those consisting of
a DNA segment encoding an atrazine chlorohydrolase protein or
biologically active derivative thereof. Although the DNA molecule
includes a single coding region, it can contain additional
nucleotides that do not detrimentally affect the function of the
DNA molecule, i.e., the expression of the atrazine chlorohydrolase
protein or biologically active derivative thereof. For example, the
5' and 3' untranslated regions may contain variable numbers of
nucleotides. Preferably, additional nucleotides are outside the
single coding region.
[0027] The present invention provides an isolated and purified DNA
molecule that encodes atrazine chlorohydrolase protein and that
hybridizes to a DNA molecule complementary to the DNA molecule
shown in FIG. 7 (SEQ ID NO:1), beginning at position 236 and ending
at position 1655, under high stringency hybridization conditions.
As used herein, "high stringency hybridization conditions" refers
to hybridization in buffer containing 0.25 M Na.sub.2HPO.sub.4 (pH
7.4), 7% sodium dedecyl sulfate (SDS), 1% bovine serum albumin
(BSA), 1.0 mM ethylene diamine tetraacetic acid (EDTA, pH 8) at
65.degree. C., followed by washing 3.times. with 0.1% SDS and
0.1.times.SSC (0.1.times.SSC contains 0.015 M sodium chloride and
0.0015 M trisodium citrate, pH 7.0) at 65.degree. C.
[0028] The present invention also provides an isolated and purified
(preferably chemically synthesized) oligonucleotide of at least
about seven nucleotides (i.e., a primer or a probe preferably
containing no more than about 300 nucleotides) which hybridizes to
the DNA molecules of the present invention, preferably the DNA
molecule shown in FIG. 7, beginning at position 236 and ending at
position 1655, under the high stringency hybridization conditions
described above. Oligonucleotide probes and primers are segments of
labeled, single-stranded DNA which will hybridize, or noncovalently
bind, with complementary single-stranded DNA to be identified. If
desired, the probe and primer can be labeled with any suitable
label known to those skilled in the art, including radioactive and
nonradioactive labels. Typical radioactive labels include .sup.32p,
.sup.125I, .sup.35S, and the like. Nonradioactive labels include,
for example, ligands such as biotin or digoxigenin as well as
enzymes such as phosphatase or peroxidases, or the various
chemiluminescers such as luciferin, or fluorescent compounds like
fluorescein and its derivatives. The probe or primer may also be
labeled at both ends with different types of labels for ease of
separation, as, for example, by using an isotopic label at one end
and a biotin label at the other end.
[0029] As used herein, the terms atrazine chlorohydrolase (AtzA)
protein, atrazine chlorohydrolase (AtzA) enzyme, or simply atrazine
chlorohydrolase (AtzA), are used interchangeably, and refer to an
atrazine chlorohydrolase enzyme involved in the degradation of
atrazine and similar molecules as discussed above. A "biologically
active derivative thereof" is an atrazine chlorohydrolase that is
modified by amino acid deletion, addition, substitution, or
truncation, or that has been chemically derivatized, but that
nonetheless converts atrazine to hydroxyatrazine. For example, it
is known in the art that substitutions of aliphatic amino acids
such as alanine, valine, and isoleucine with other aliphatic amino
acids can often be made without altering the structure or function
of a protein. Similarly, substitution of aspartic acid for glutamic
acid, in regions other than the active site of an enzyme, are
likely to have no appreciable affect on protein structure or
function. The term "biologically active derivative" is intended to
include AtzA's as thus modified. The term also includes fragments,
variants, analogs or chemical derivatives of AtzA enzyme. The term
"fragment" is meant to refer to any polypeptide subset of AtzA
enzyme. Fragments can be prepared by subjecting AtzA to the action
of any one of a number of commonly available proteases, such as
trypsin, chymotrypsin or pepsin, or to chemical cleavage agents,
such as cyanogen bromide. The term "variant" is meant to refer to a
molecule substantially similar in structure and function to either
the entire AtzA molecule or to a fragment thereof. A molecule is
said to be "substantially similar" to AtzA or a fragment thereof if
both molecules have substantially similar amino acid sequences,
preferably greater than about 80% sequence identity, or if the
three-dimensional backbone structures of the molecules are
superimposable, regardless of the level of identity between the
amino acid sequences. Thus, provided that two molecules possess
atrazine chlorohydrolase activity, they are considered variants as
that term is used herein even if the structure of one of the
molecules is not found in the other, or if the sequences of amino
acid residues are not identical. The term "analog" is meant to
refer to a protein that differs structurally from the wild type
enzyme AtzA, but converts atrazine to hydroxyatrazine.
[0030] The present invention also provides a vector comprising an
isolated and purified DNA molecule encoding atrazine
chlorohydrolase, preferably the atrazine chlorohydrolase having the
amino acid sequence shown in FIG. 7 (SEQ ID NO:2) beginning at
position 236 and ending at position 1655. That is, preferably, the
vector includes a single atrazine chlorohydrolase coding region. It
can also include other DNA segments operably linked to the coding
sequence in an expression cassette as required for expression of
atrazine chloprohydrolase, such as a promoter region operably
linked to the 5' end of the coding DNA sequence, a selectable
marker gene, a reporter gene, and the like.
[0031] The present invention also provides a recombinant cell line,
preferably a bacterial cell line, the genome of which has been
augmented by chromosomally integrated non-native DNA encoding
atrazine chlorohydrolase as herein described. For example, DNA that
expresses atrazine chlorohydrolase and is isolated from a
Pseudomonas sp. bacterial strain, can be transferred to a
non-Pseudomonas sp. strain, such as other Pseudomonas bacterial
strains as well as bacterial genera Escherichia, Rhizobium,
Bacillus, Bradyrhizobium, Arthrobacter, Alcaligenes, and other
rhizosphere and nonrhizosphere soil microbe strains. Such strains
may possess advantageous properties not present in the native
Pseudomonas sp. strain. For example, inorganic nitrogen-containing
fertilizers in soils can shut off activity in the native
Pseudomonas sp. strain, but not in other strains such as E.
coli.
[0032] The present invention also provides a preparation of
polyclonal antibodies produced in response to the AtzA protein of
the present invention. Preferably, the polyclonal antibodies are of
the IgG class, although other classes are possible. The polyclonal
antibody preparation can be used, for example, to screen bacteria
for the presence of the AtzA protein. It can also be used in the
isolation of the atzA gene and detection of the AtzA protein
expressed in another host organism. Furthermore, the antibody can
also be bound to immobilization supports, such as commercially
available matrices like Activated Affinity Supports Affi-Gel 15+10
by Biorad Laboratories (Hercules, Calif.) and used in affinity
chromatography columns for purifying the AtzA protein.
[0033] Several different methods are available for isolating atzA
DNA. This includes, for example, purifying enzyme protein, and then
subjecting it to amino acid microsequencing, either directly or
after limited cleavage. The partial amino acid sequence that is
obtained can be used to design degenerate oligonucleotide probes or
primers for use in the generation of unique, nondegenerate
nucleotide sequences by polymerase chain reaction (PCR), sequences
that can in turn be used as probes for screening DNA libraries.
Antibodies raised against purified protein may also be used to
isolate DNA clones from DNA expression libraries. Alternatively,
the sequences of DNA molecules for related enzymes may be used as
starting points in a cloning strategy. This method is often
referred to as "cloning by homology." Another way of utilizing
sequence information from different species is to take advantage of
shorter areas of high sequence homology among related DNA molecules
from different species and to perform the polymerase chain reaction
sequencing amplification method (PCR) to obtain "species-specific"
nondegenerate nucleotide sequences. Such a sequence can then be
used for DNA library screening or even for direct PCR-based DNA
cloning.
[0034] Using standard biochemical procedures well-known in the art,
oligonucleotide probes can be used to detect and amplify an atzA
DNA molecule in a wide variety of samples. For example, Southern or
Northern blotting hybridization techniques using labeled probes can
be used. Alternatively, PCR techniques can be used, and nucleic
acid sequencing of amplified PCR products can be used to detect
mutations in the DNA.
[0035] Detection of the DNA can involve the use of PCR using novel
primers. The method involves treating extracted DNA to form
single-stranded complementary strands, treating the separate
complementary strands of DNA with two oligonucleotide primers,
extending the primers to form complementary extension products that
act as templates for synthesizing the desired nucleic acid
molecule, and detecting the amplified molecule.
[0036] DNA primer pairs of known sequence positioned 10-300 base
pairs apart that are complementary to the plus and minus strands of
the DNA to be amplified can be prepared by well known techniques
for the synthesis of oligonucleotides. Conveniently, one end of
each primer can be extended and modified to create restriction
endonuclease sites when the primer is annealed to the target DNA.
These restriction sites facilitate the use of the amplified product
for cloning at a later date. The PCR reaction mixture can contain
the target DNA, the DNA primer pairs, four deoxyribonucleoside
triphosphates, MgCl.sub.2, DNA polymerase, and conventional
buffers. The DNA can be amplified for a number of cycles. It is
generally possible to increase the sensitivity of detection by
using a multiplicity of cycles, each cycle consisting of a short
period of denaturation of the target DNA at an elevated
temperature, cooling of the reaction mixture, and polymerization
with the DNA polymerase.
[0037] Cloning of the open reading frame encoding atzA into the
appropriate replicable vectors allows expression of the gene
product, AtzA enzyme, and makes the coding region available for
further genetic engineering. Expression of AtzA enzyme or portions
thereof, is useful because these gene products can be used to
degrade atrazine and similar compounds, as discussed above.
[0038] 1. Isolation of DNA
[0039] DNA containing the region encoding AtzA may be obtained from
a DNA library, containing either genomic or complementary DNA,
prepared from bacteria believed to possess the atzA DNA and to
express it at a detectable level. Libraries are screened with
appropriate probes designed to identify the DNA, either genomic or
complementary DNA, of interest. Preferably, for DNA libraries,
suitable probes include oligonucleotides that consist of known or
suspected portions of the atzA DNA from the same or different
species; and/or complementary or homologous DNA molecules or
fragments thereof that consist of the same or a similar DNA. For
DNA expression libraries (which express the protein), suitable
probes include monoclonal or polyclonal antibodies that recognize
and specifically bind to the AtzA protein. Appropriate probes for
screening DNA libraries include, but are not limited to,
oligonucleotides, cDNA molecules, or fragments thereof that consist
of the same or a similar gene, and/or homologous genomic DNA
molecules or fragments thereof. Screening the DNA library with the
selected probe may be accomplished using standard procedures.
[0040] Screening DNA libraries using synthetic oligonucleotides as
probes is a preferred method of practicing this invention. The
oligonucleotide sequences selected as probes should be of
sufficient length and sufficiently unambiguous to minimize false
positives. The actual nucleotide sequence(s) of the probe(s) is
usually designed based on regions of the atzA DNA that have the
least codon redundancy. The oligonucleotides may be degenerate at
one or more positions, i.e., two or more different nucleotides may
be incorporated into an oligonucleotide at a given position,
resulting in multiple synthetic oligonucleotides. The use of
degenerate oligonucleotides is of particular importance where a
library is screened from a species in which preferential codon
usage is not known.
[0041] The oligonucleotide can be labeled such that it can be
detected upon hybridization to DNA in the library being screened. A
preferred method of labeling is to use ATP and polynucleotide
kinase to radiolabel the 5' end of the oligonucleotide. However,
other methods may be used to label the oligonucleotide, including,
but not limited to, biotinylation or enzyme labeling.
[0042] Of particular interest is the atzA nucleic acid that encodes
a full-length mRNA transcript, including the complete coding region
for the gene product, AtzA enzyme. Nucleic acid containing the
complete coding region can be obtained by screening selected DNA
libraries using the deduced amino acid sequence. An alternative
means to isolate the DNA encoding AtzA enzyme is to use PCR
methodology. This method requires the use of oligonucleotide primer
probes that will hybridize to the DNA encoding AtzA.
[0043] 2. Insertion of DNA into a Vector
[0044] The nucleic acid containing the atzA coding region is
preferably inserted into a replicable vector for further cloning
(amplification of the DNA) or for expression of the gene product,
AtzA enzyme. Many vectors are available, and selection of the
appropriate vector will depend on: 1) whether it is to be used for
DNA amplification or for DNA expression; 2) the size of the nucleic
acid to be inserted into the vector; and 3) the host cell to be
transformed with the vector. Most expression vectors are "shuttle"
vectors, i.e., they are capable of replication in at least one
class of organism but can be transfected into another organism for
expression. Each replicable vector contains various structural
components depending on its function (amplification of DNA or
expression of DNA) and the host cell with which it is compatible.
These components are described in detail below.
[0045] Construction of suitable vectors employs standard ligation
techniques known in the art. Isolated plasmids or DNA fragments are
cleaved, tailored, and relegated in the form desired to generate
the plasmids required. Typically, the ligation mixtures are used to
transform E. coli DH5.alpha. and successful transformants are
selected by ampicillin or tetracycline resistance where
appropriate. Plasmids from the transformants are prepared, analyzed
by restriction endonuclease digestion, and/or sequenced by methods
known in the art. See, e.g., Messing et al., Nucl. Acids Res., 9,
309 (1981) and Maxam et al., Methods in Enzymology, 65, 499
(1980).
[0046] Replicatable cloning and expression vector components
generally include, but are not limited to, one or more of the
following: a signal sequence, an origin of replication, one or more
marker genes, and a promoter. Such vector components are well known
to one of skill in the art. For example, a signal sequence may be
used to facilitate extracellular transport of a cloned protein. To
this end, the atzA gene product, AtzA enzyme, may be expressed not
only directly, but also as a fusion product with a heterologous
polypeptide, preferably a signal sequence or other polypeptide
having a specific cleavage site at the N-terminus of the cloned
protein. The signal sequence may be a component of the vector, or
it may be a part of the atzA DNA that is inserted into the vector.
The heterologous signal sequence selected should be one that is
recognized and processed (i.e., cleaved by a signal peptidase) by
the host cell. For prokaryotic host cells, a prokaryotic signal
sequence may be selected, for example, from the group of the
alkaline phosphatase, penicillinase, lpp or heat-stable intertoxin
II leaders.
[0047] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, phages, and viral systems. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, for example.
[0048] Expression and cloning vectors may contain a marker gene,
also termed a selection gene or selectable marker. This gene
encodes a protein necessary for the survival or growth of
transformed host cells grown in a selective culture medium. Host
cells not transformed with the vector containing the selection gene
will not survive in the culture medium. Typical selection genes
encode proteins that: (a) confer resistance to antibiotics or other
toxins, e.g., ampicillin, neomycin, methotrexate, streptomycin or
tetracycline; (b) complement auxotrophic deficiencies; or (c)
supply critical nutrients not available from complex media, e.g.,
the gene encoding D-alanine racemase for Bacillus. One example of a
selection scheme utilizes a drug to arrest growth of a host cell.
Those cells that are successfully transformed with a heterologous
gene express a protein conferring drug resistance and thus survive
the selection regimen.
[0049] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the atzA nucleic acid. Promoters are untranslated sequences located
upstream (5') to the start codon of a structural gene (generally
within about 100 to 1000 bp) that control the transcription and
translation of a particular nucleic acid sequence, such as the atzA
nucleic acid sequence, to which they are operably linked. Such
promoters typically fall into two classes, inducible and
constitutive. Inducible promoters are promoters that initiate
increased levels of transcription from DNA under their control in
response to some change in culture conditions, e.g., the presence
or absence of a nutrient or a change in temperature. In contrast,
constitutive promoters produce a constant level of transcription of
the cloned DNA segment.
[0050] At this time, a large number of promoters recognized by a
variety of potential host cells are well known in the art.
Promoters are removed from their source DNA using a restriction
enzyme digestion and inserted into the cloning vector using
standard molecular biology techniques. Native or heterologous
promoters can be used to direct amplification and/or expression of
atzA DNA. Heterologous promoters are preferred, as they generally
permit greater transcription and higher yields of expressed protein
as compared to the native promoter. Well-known promoters suitable
for use with prokaryotic hosts include the beta-lactamase and
lactose promoter systems, alkaline phosphatase, a tryptophan (trp)
promoter system, and hybrid promoters such as the tac promoter.
Such promoters can be ligated to atzA DNA using linkers or adapters
to supply required restriction sites. Promoters for use in
bacterial systems may contain a Shine-Dalgarno sequence for RNA
polymerase binding.
[0051] The genetically engineered plasmid of the invention can be
used to transform a host cell. Typically, prokaryotic host cells
are used in the expression system according to the invention,
although eukaryotic cells may also be used. Suitable prokaryotes
include eubacteria, such as Gram-negative or Gram-positive
organisms, for example, E. coli, Bacilli such as B. subtilis,
Pseudomonas species such as P. aeruginosa, Salmonella typhimurium,
or Serratia marcsecans. Preferably the host cell should secrete
minimal amounts of proteolytic enzymes. Alternatively, in vitro
methods of cloning, e.g., PCR or other nucleic acid polymerase
reactions, are suitable.
[0052] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable hosts for atzA-encoding
vectors. Saccaromyces cerevisiae, or common baker's yeast, is the
most commonly used among lower eukaryotic host microorganisms.
However, a number of other genera, species, and strains are
commonly available and useful herein, such as Schizosaccaromyces
pombe, Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis,
K. bulgaricus, K. thermotolerans, and K. marxianus, yarrowia,
Pichia pastoris, Candida, Trichoderma reesia, Neurospora crassa,
and filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium, and Aspergillus hosts such as A. nidulans.
[0053] 4. Transfection and Transformation
[0054] Host cells are transfected and preferably transformed with
the above-described expression or cloning vectors of this invention
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences. Transfection refers to the
taking up of an expression vector by a host cell whether or not any
coding sequence are in fact expressed. Numerous methods of
transfection are known to the ordinarily skilled artisan, for
example, the calcium phosphate precipitation method and
electroporation are commonly used. Successful transfection is
generally recognized when any indication of the operation of the
vector occurs within the host cell.
[0055] Transformation means introducing DNA into an organism so
that the DNA is replicable, either as an extrachromosomal element
or by chromosomal integrant. Depending on the host cell used,
transformation is done using standard techniques appropriate to
such cells. Calcium chloride is generally used for prokaryotes or
other cells that contain substantial cell-wall barriers.
Transformations into yeast are typically carried out according to
the method of Van Solingen et al., J. Bact., 130, 946 (1977) and
Hsiao et al., Proc. Natl. Acad. Sci. (USA), 78 3829 (1979).
However, other methods for introducing DNA into cells such as by
nuclear injection, electroporation, or protoplast fusion may also
be used.
[0056] 5. Cell Culture
[0057] Prokaryotic cells used to produce the atzA gene product,
atzA protein, are cultured in suitable media, as described
generally in Maniatis et al., Molecular Cloning: A Laboratory
Manual; Cold Spring Harbor Press: Cold Spring Harbor, N.Y. (1989).
Any necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to those skilled in the art. Induction of
cells, to cause expression of the AtzA protein, is accomplished
using the procedures required by the particular expression system
selected. The host cells referred to in this disclosure encompass
in in vitro culture as well as cells that are within a host animal.
Cells are harvested, and cell extracts are prepared, using standard
laboratory protocols. The AtzA protein can be isolated from cell
extracts. Optionally, cell extracts may be assayed directly for
atrazine degradation activity.
[0058] AtzA variants in which residues have been deleted, inserted,
or substituted are recovered in the same fashion as native AtzA
enzyme, taking account of any substantial changes in properties
occasioned by the variation. For example, preparation of an AtzA
fusion with another protein or polypeptide, e.g., a bacterial or
viral antigen, facilitates purification; an immunoaffinity column
containing antibody to the antigen can be used to adsorb the fusion
polypeptide. Immunoaffinity columns such as a rabbit polyclonal
anti-AtzA column can be employed to absorb the AtzA variant by
binding it to at least one remaining immune epitope. Alternatively,
the AtzA enzyme may be purified by affinity chromatography using a
purified AtzA-IgG coupled to a (preferably) immobilized resin such
as Affi-Gel 10 (Bio-Rad, Richmond, Calif.) or the like, by means
well-known in the art. A protease inhibitor such as phenyl methyl
sulfonyl fluoride (PMSF) also may be useful to inhibit proteolytic
degradation during purification, and antibiotics may be included to
prevent the growth of adventitious contaminants.
[0059] General atrazine chlorohydrolase activity may be assayed by:
monitoring the degradation of substrates like atrazine and simazine
using HPLC; monitoring the clearing of atrazine on plates;
monitoring the amount of chlorine released, as described by Bergman
et al., Anal. Chem., 29 241-243 (1957); evaluating the derivitized
product using gas chromatography and/or mass spectroscopy.
[0060] The invention will be further described by reference to the
following detailed examples.
EXAMPLES
Materials and Methods
[0061] Bacterial strains and growth conditions. Pseuidomonas sp.
strain ADP (Mandelbaum et al., Appl. Environ. Microbiol., 59,
1695-1701 (1993)) was grown at 37.degree. C. on modified minimal
salt buffer medium, containing 0.5% (wt/vol) sodium citrate
dihydrate. The atrazine stock solution was prepared as described in
Mandelbaum et al.,Appl. Environ. Microbiol., 61, 1451-1457 (1995)).
Escherichia coli DH5.alpha. was grown in Luria-Bertani (LB) or M63
minimal medium, which are described in Maniatis et al., Molecular
Cloning: A Laboratory Manual; Cold Spring Harbor Press: Cold Spring
Harbor, N.Y. (1989). Tetracycline (15 .mu.g/ml), kanamycin (20
.mu.g/ml), and chloramphenicol 30 (.mu.g/ml) were added as
required.
[0062] Genomic library construction. Genomic DNA from Pseudomonas
sp. strain ADP was isolated as follows. Briefly, cells grown as
described above were centrifuged at 10,000.times.g for 10 minutes
at 4.degree. C., washed once in TEN buffer (50 mM Tris, 10 mM
disodium EDTA, 50 mM NaCl, pH 8.0), and suspended in TEN buffer.
Lysozyme (Sigma Chemical Co., St. Louis, Mo.) was added to a final
concentration of 0.5 mg/ml, and cell suspensions were incubated at
37.degree. C. for 30 minutes. Predigested protease solution (2 ml;
5 mg of protease [Type X; Sigma] per ml in TEN buffer heated at
37.degree. C. for 1 hour) was added, and the suspensions were
incubated at 37.degree. C. for 30 minutes. A 2-ml fraction of 20%
(wt/vol) Sarkosyl (N-lauroylsarcosine; Sigma) was added, and the
mixtures were incubated at 37.degree. C. for 1 hour. CsCl (31 g),
7.5 ml of TEN buffer, and 1.6 ml of ethidium bromide solution (10
mg/ml) were added to the cell lysates; and the mixtures were
centrifuged at 40,000 rpm for 48 hours at 20.degree. C. in a
fixed-angle rotor (60 Ti; Beckman Instruments, Inc., Fullerton,
Calif.). The high molecular weight DNA band was removed, and the
DNA was repurified by ethidium bromide equilibrium density
centrifugation, as described above. Genomic DNA was partially
digested with EcoRI and size-selected by using sucrose density
gradient centrifugation as described in Maniatis et al., Molecular
Cloning: A Laboratory Manual; Cold Spring Harbor Press: Cold Spring
Harbor, N.Y. (1989). DNA fragments, 18-22 kb in size, were ligated
into EcoRI-digested cosmid vector pLAFR3, which is described in
Staskawicz et al., J. Bacteriol., 169, 5789-5794 (1987). Ligated
DNA was packaged in vitro using the Packagene DNA packaging system
(Promega, Madison, Wis.). E. coli DH5.alpha. was transfected with
the packaging mix and colonies were selected on LB medium
containing 15 .mu.g/ml tetracycline and 50 .mu.g/ml
5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (X-Gal). The
final library contained 2000 clones.
[0063] Library screening. All colonies from the genomic DNA library
were replica-plated onto LB medium containing 15 .mu.g/ml
tetracycline and 500 .mu.g/ml crystalline atrazine. Plates were
incubated at 37.degree. C. for two weeks. Colonies expressing
atrazine degradation activity had clearing zones surrounding them
due to atrazine metabolism in the vicinity of the colony.
[0064] DNA manipulations. Subcloning, plasmid and cosmid DNA
isolation procedures, Southern blotting, and hybridizations were
performed as described in Maniatis et al., Molecular Cloning: A
Laboratory Manual; Cold Spring Harbor Press: Cold Spring Harbor,
N.Y. (1989). Transformation of E. coli DH5.alpha. was done
according to the method of Hanahan, DNA Cloning Vol. 11; D. M.
Glover; Ed.; IRL Press Limited: Oxford, England; p. 120 (1985).
Specifically, plasmid pACYC184, which is described in Chang et al.,
J. Bacteriol., 134, 1141-1156 (1978) was used as the vector for all
subcloning steps.
[0065] Tn5 mutagenesis. Random Tn5 mutagenesis, using .lambda.::Tn5
(.lambda.467, b221 rex::Tn5 c1857, Oam29, Pam80) was done as
described by de Bruijn et al., Gene, 27, 131-149 (1984). E. coli
strain SE5000 was used as the host for cosmid pMD1 and plasmid MD2
during mutagenesis. Tn5 insertions in cloned, insert DNA were
identified and mapped by restriction enzyme analysis and by
Southern hybridization.
[0066] DNA Sequencing. The nucleotide sequence of the approximately
1.9-kb AvaI DNA fragment in vector pACYC 184, designated pMD4, was
determined on both strands. DNA was sequenced by using a PRISM
Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit
(Perkin-Elmer Corp., Norwalk, Conn.) and a ABI Model 373A DNA
Sequencer (Applied Biosystems, Foster City, Calif.). Nucleotide
sequence was determined initially by subcloning and subsequently by
using primers designed based on sequence information obtained from
subcloned DNA fragments. The GCG sequence analysis software package
(Genetics Computer Group, Inc., Madison, Wis.) was used for all DNA
and protein sequence comparisons. DNA and protein sequences were
compared to entries in Genbank and PIR, SwissProt sequence
databases.
[0067] Analytical methods: plate assays, high performance liquid
chromatography (HPLC), thin layer chromatography (TLC), and
liquid-liquid partitioning analyses. Atrazine or hydroxyatrazine
were incorporated in solid LB or minimal medium, as described in
Mandelbaum et al., Appl. Environ. Microbiol., 61, 1451-1457 (1995),
at a final concentration of 500 .mu.g/ml to produce an opaque
suspension of small particles in the clear agar. The degradation of
atrazine or hydroxyatrazine by wild-type and recombinant bacteria
was indicated by a zone of clearing surrounding colonies.
[0068] High performance liquid chromatography (HPLC) analysis was
performed using a Hewlett-Packard HP 1090 Liquid Chromatograph
system equipped with a photodiode array detector and interfaced to
an HP 79994A Chemstation. Atrazine metabolites were resolved by
using an analytical C.sub.18 reverse-phase HPLC column (Waters,
Nova-Pak, 4 .mu.m spherical packing, 150.times.3.9 mm) and an
acetonitrile gradient, in water, at a flow rate of 1.0 ml/minute.
Linear gradients of 0-6 minutes, 10-25% acetonitrile (ACN); 6-21
minutes, 25-65% ACN; 21-23 minutes, 65-100% ACN; and 23-25 minutes
100% ACN, were used. Spectral data of the column eluent was
acquired between 200-400 nm (12 nm bandwidth per channel) at a
sampling frequency of 640 milliseconds. Spectra were referenced
against a signal at 550 nm.
[0069] Thin layer chromatography analysis was done using precoated
silica gel 60 F254 TLC plates (Alltech Associates, Chicago, Ill.)
and developed using a chloroform:methanol:formic acid:water
(75:20:4:2 v/v) solvent system. E. coli strains containing pMD 1,
pMD2, or pMD3 and Pseudomonas sp. strain ADP were grown in LB
medium supplemented with appropriate antibiotics as required. After
24 hours of growth, cells were harvested by centrifugation at
10,000.times.g for 10 minutes, washed twice in 0.1 M phosphate
buffer (pH 7.5) and resuspended in the same buffer to an absorbance
of 25 at 600 nm. Reaction mixtures consisted of 100 .mu.l of cell
suspension, 390 .mu.l 0.1 M phosphate buffer (pH 7.5), 5 .mu.l of
unlabeled atrazine stock solution (10.59 mg/ml), and 5 .mu.l of
uniformly-labelled [.sup.14C]-atrazine (51,524 cpm/.mu.l). After
incubation for 30 minutes at 37.degree. C., a 40 .mu.l aliquot of
each reaction mixture was spotted onto a TLC plate. A radiolabelled
hydroxyatrazine standard was prepared by mixing 8 .mu.l of
uniformly ring-labelled [.sup.14C]-hydroxyatrazine (30,531
cpm/.mu.l) in 492 .mu.l phosphate buffer, pH 7.5, and a 40 .mu.l
aliquot of the standard (containing 19,500 cpm) was spotted on the
TLC plate. After developing, plates were scanned using a model BAS
1000 Bio-Imaging Analyzer System (Fugix Co., Japan).
[0070] Liquid-liquid partitioning analysis was done as described in
Mandelbaum et al., Appl. Environ. Microbiol., 61, 1451-1457 (1995),
except that a 50:50 (vol/vol) mixture of ethyl acetate:n-hexane was
used as the organic extractant.
[0071] Chemicals. Authentic samples of atrazine
(2-chloro-4-ethlyamino-6-i- sopropylamino-1,3,5-s-triazine),
desethylatrazine (2-chloro-4-amino-6-isop- ropylamino-s-triazine),
deisopropylatrazine (2-chloro-4-ethylamino-6-amino- -s-triazine),
hydroxyatrazine (2-hydroxy-4-ethylamino-6-isopropylamino-s-t-
riazine), desethylhydroxyatrazine
(2-hydroxy-4-amino-6-isopropylamino-s-tr- iazine),
desisopropylhydroxyatrazine (2-hydroxy-4-amino-6-isopropylamino-s-
-triazine), desethyldesisopropylatrazine
(2-chloro-4,6-diamino-s-triazine)- , simazine
(2-chloro-4,6-diethylamino-s-triazine), terbutylazine
(2-chloro-4-ethylamino-6-terbutylamino-s-triazine, and melamine
(2,4,6-triamino-s-triazine) were obtained from Ciba Geigy Corp.,
Greensboro, N.C. Ammelide (2,4-dihydroxy-6-amino-s-triazine),
ammeline (2-hydroxy-4,6,-diamino-s-triazine), and cyanuric acid
(1,3,5-triazine-2,4,6-triol) were obtained from Aldrich Chemical
Co., Milwaukee, Wis. Radiolabelled chemicals (Table 1) were
obtained from Ciba Geigy Corp., Greensboro, N.C.
1TABLE 1 Chemical and physical properties of
[.sup.14C]-ring-labelled compounds used in this study. Specific
Activity Chemical Radiolabel Rf Compound (.mu.Ci/mg) Purity (%)
Purity (%) value.sup.a Atrazine 14.6 97.3 98.6 0.91 Hydroxyatrazine
44.2 96.7 98.6 0.52 Desisopropyl- 22.6 95.8 92.7 0.23
hydroxyatrazine Desethyl- 20.9 96.3 96.2 0.30 hydroxyatrazine
Ammelide 8.6 99.4 86.1 0.27 Ammeline 11.1 99.0 99.9 0.07 Cyanuric
acid 12.2 99.7 98.5 0.27 .sup.aDetermined by TLC analysis according
to procedures described in the materials and methods. Unlabelled
desisopropylatrazine and desethylatrazine had R.sub.f vales of 0.79
and 0.83, respectively.
[0072] Protein Purification. E. coli transformed with pMD4 was
grown over night at 37.degree. C. in eight liters of LB medium
containing 25 .mu.g/ml chloramphenicol. The culture medium was
centrifuged at 10,000 .times.g for 10 minutes at 4.degree. C.,
washed in 0.85% NaCl, and the cell pellet was resuspended in 50 ml
of 25 mM MOPS buffer (3-[N-morpholino]propane-sulfonic acid, pH
6.9), containing phenylmethylsulfonylfluoride (100 .mu.g/ml). The
cells were broken by three passages through a Amicon French
Pressure Cell at 20,000 pounds per square inch (psi) at 4.degree.
C. Cell-free extract was obtained by centrifugation at
10,000.times.g for 15 minutes. The supernatant was clarified by
centrifugation at 18,000.times.g for 60 minutes and solid
NH.sub.4SO.sub.4 was added, with stirring, to a final concentration
of 20% (wt/vol) at 4.degree. C. The solution was stirred for 30
minutes at 4.degree. C. and centrifuged at 12,000 .times.g for 20
minutes. The precipitated material was resuspended in 50 ml of 25
mM MOPS buffer (pH 6.9), and dialyzed overnight at 4.degree. C.
against 1 liter of 25 mM MOPS buffer (pH 6.9).
[0073] The solution was loaded onto a Mono Q HR 16/10 Column
(Pharmacia LKB Biotechnology, Uppsala, Sweden). The column was
washed with 25 mM MOPS buffer (pH 6.9), and the protein was eluted
with a 0-0.5 M KCl gradient. Protein eluting from the column was
monitored at 280 nm by using a Pharmacia U.V. protein detector.
Pooled fractions containing the major peak were dialyzed overnight
against 1 liter 25 mM MOPS buffer (pH 6.9). The dialyzed material
was assayed for atrazine degradation ability by using HPLC analysis
(see above) and analyzed for purity by sodium dodecyl sulfate (SDS)
polyacrylamide gel electrophoreses (Laemlli).
[0074] Protein Characterization. Protein subunit sizes were
determined by SDS polyacrylamide gel electrophoresis by comparison
to known standard proteins, using a Mini-Protean II gel apparatus
(Biorad, Hercules, Calif.). The size of the holoenzyme was
determined by gel filtration chromatography on a Superose 6 HR
(1.0.times.30.0 cm) column, using an FPLC System (Pharmacia,
Uppsala, Sweden). The protein was eluted with 25 mM MOPS buffer (pH
6.9) containing 0.1 M NaCl. Proteins with known molecular weights
were used as chromatography standards. Isoelectric point
determinations were done using a Pharamacia Phast-Gel System and
Pharamacia IEF 3-9 media. A Pharamacia broad-range pI calibration
kit was used for standards.
[0075] Amino acid analysis. The amino acid composition and
N-terminal amino acid sequence of purified atzA protein was
determined using a Beckman 6300 Amino Acid Analyzer.
[0076] Metal analysis. The metal content of atzA protein was
determined by inductively coupled plasma emission spectroscopy.
[0077] Enzyme Kinetics. Purified AtzA protein, 50 .mu.g/ml, was
added to 500 .mu.l of different concentrations of atrazine (23.3
.mu.M, 43.0 .mu.M, 93 .mu.M. 233 .mu.M, and 435 .mu.M in 25 mM MOPS
buffer, pH 6.9) and reactions were allowed to proceed at room
temperature for 2, 5, 7, and 10 minutes. The reactions were stopped
by boiling the reaction tubes at specific times, the addition of
500 .mu.l acetonitrile and rapid freezing at -80.degree. C. Thawed
samples were centrifuged at 14,000 rpm for 10 minutes, the
supernatants were filtered through a 0.2 .mu.M filter, and placed
into crimp-seal HPLC vials. HPLC analysis was done as described
above. Based on HPLC data, initial rates of atrazine degradation
and hydroxyatrazine formation were calculated and Michaelis Menton
and Lineweaver Burke plots were constructed.
[0078] Effect of simple nitrogen sources on atrazine degradation.
From experiments done with Pseudomonas species strain ADP on solid
media with 500 ppm atrazine and varying concentrations of ammonium
chloride, ammonium chloride concentrations as low as 0.6-1.2 mM
were sufficient to inhibit visible clearing on the plates, even
after 2 weeks of incubation either at 28.degree. C. or 37.degree.
C. With similar experiments using E. coli DH5.alpha. (pMD1 or pMD2)
clearing, atrazine degradation was observed in the presence of
ammonium chloride concentrations as high as 48 mM. This value is
almost 40-80 fold higher than the wild-type tolerance for ammonium
chloride with concomitant atrazine degradation.
[0079] Protocol for polyclonal antibody production. On Day 1
rabbits were pre-bled and immunized by a series of subcutaneous
injections (approximately 10 injection sites, each with
approximately 100 .mu.l of antigen plus adjuvant). Up to 1
milligram of antigen per rabbit was used either in Complete Freund
Adjuvent or polyacrylamide as adjuvent. On Days 14 and 21 the first
booster was given with up to 500 micrograms of antigen per rabbit
in Incomplete Freund Adjuvent or polyacrylamide. On Days 25 and 28
the first bleed was accomplished withdrawing a small amount (5 ml)
of blood from an artery or vein using a 23 6A, 1 inch butterfly
needle. On Days 26-34 testing was done for the presence of
antibodies. On Day 42 the second booster was given with up to 500
micrograms of antigen per rabbit in Incomplete Freunds adjuvent or
polyacrylamide. On Day 49 the second bleed was completed in the
same manner as the first bleed. On Days 50-55 testing was again
done for the presence of antibodies. The third booster was given on
Day 63 with up to 200 micrograms of antigen per rabbit in
Incomplete Freunds Adjuvant or polyacrylamide. The third bleed was
completed on Day 70 in the same manner as the first bleed. On Day
71 testing was done for the presence of antibodies. If the antibody
titre was sufficient, on Day 72 the rabbits were anesthetized with
22-44 mg of ketamine and a cardiac puncture was performed to drain
the blood. The rabbits were then euthanized with an IV injection of
B euthanasia. The chests were opened to be sure the euthanasia was
complete. For the blood samples mentioned above, 0.1 cc of
acepromazine or topical xylene was used. The acepromazine was
injected (intramuscularly) IM.
[0080] Atrazine degradation gene expressed in Bradyrhizobium
japonicum. A cosmid clone, pMD 1, which contains the 22 kb DNA
region from atrazine degrading Pseudomonas strain (ADP), was
successfully transferred to Bradyrhizobium japonicum strain USDA
123. The pMD1 was transferred from E. coli DH5.alpha. (pMD1) to B.
japonicum strains USDA 123 by conjugation. This was done by using
helper plasmid pRK2073 and the triparental mating procedure of
Leong et al., J. Bio. Chem, 257, 8724-8730 (1982). This was done
using a modified patch mating technique. Equal quantities of E.
coli DH5.alpha. (pMD1), B. japonicum strain USDA plate and spread
to the size of a nickel. The patch was incubated for 4 days at
28.degree. C. and the resulting bacterial growth was removed from
the pate, serially diluted in 0.85% NaCl plus 0.01% tween 80, and
spread onto the surface of minimal AG plates (Ag without yeast
extract) containing 60 .mu.g/ml tetracycline. Plates were incubated
for 2 weeks at 28.degree. C. Colonies arising in the plates were
checked for atrazine degradation activity by using the plate
clearing assay. Atrazine degradation was verified by HPLC analysis.
The identity of transconjugants was verified by using strain
specific fluorescent antibodies prepared according to Schmidt et
al., J. Bacteriol., 95, 1987-1992 (1968). B. japonicum strain can
express the atrazine degradation genes located in the cosmid clone
pMD 1. B. japonicum strain that carries pMD1 can clear atrazine in
10 days on AG media plates containing up to 500 ppm atrazine. HPLC
analysis the overnight culture broth shows that all the supplied
atrazine (33 ppm) is degraded and there is no atrazine detectable
in the culture broth. The control strains of B. japonicum strain
does not carry the cosmid clone, pMD1 failed to degrade atrazine
both in plates and in culture broth.
Results
[0081] Cloning of genes involved in atrazine degradation. Atrazine
degradation genes from Pseudomonas sp. strain ADP were cloned and
expressed in E. coli DH5.alpha.. The cloning strategy was based on
the ability of wild-type and recombinant bacteria to form clearing
zones surrounding colonies on atrazine-amended solid medium.
Clearing of atrazine on solid nutrient media by both Pseudomonas
sp. strain ADP and E. coli DH5 .alpha. (PMD1, pMD2, pMD3 or pMD4)
provided a convenient visual assay for atrazine degradation during
the cloning and subcloning procedures. Atrazine degradation was
verified by HPLC, TLC, and liquid-liquid partitioning analyses (see
below).
[0082] To construct the Pseudomonas sp. strain ADP genomic library,
total genomic DNA was partially digested with EcoRI, ligated to the
EcoRI-digested cosmid vector pLAFR3 DNA, and packaged in vitro. The
completed genomic DNA library contained 2000 colonies.
[0083] To identify the atrazine degrading clones, the entire gene
library was replica-plated onto LB medium containing 500 .mu.g/ml
atrazine and 15 .mu.g/ml tetracycline. Fourteen colonies having
clearing zones were identified. All fourteen clones degraded
atrazine, as determined by HPLC analysis. Cosmid DNA isolated from
the fourteen colonies contained cloned DNA fragments which were
approximately 22 kb in length. The fourteen clones could be
subdivided into six groups on the basis of restriction enzyme
digestion analysis using EcoRI. All fourteen clones, however,
contained the same 8.7 kb EcoRI fragment. Thirteen of the colonies,
in addition to degrading atrazine, also produced an opaque material
that surrounded colonies growing on agar medium. Subsequent
experiments indicated that the opaque material only was observed in
E. coli clones which accumulated hydroxyatrazine. Thus, the cloudy
material surrounding E. coli pMD2-pMD4 colonies was due to the
deposition of hydroxyatrazine in the growth medium. The one colony
that degraded atrazine without the deposition of the opaque
material was selected for further analysis. The cosmid from this
colony was designated pMD1 (FIG. 1).
[0084] Subcloning of pMD1. To more precisely localize the DNA
region involved in the initial steps in atrazine degradation,
cosmid pMD1 was digested with EcoRI and the mixture was ligated
into EcoRI-digested pACYC184, as described in Mandelbaum et al.,
Appl. Environ. Microbiol., 61, 1451-1457 (1995). An atrazine
degrading subclone was identified by using the plate clearing
assay. This subclone, pMD2, contained the 8.7 kb EcoRI fragment
identified in pMD1 (FIG. 1). Plasmid pMD2 was further subcloned by
digestion with BamHI and ClaI, followed by ligation into BamHI and
ClaI-digested pACYC 184. An atrazine degrading subclone, pMD3,
containing a 3.3 kb BamHI/ClaI fragment, was identified by using
the plate-clearing assay (FIG. 1). Plasmid pMD3 was further
subcloned by digestion with AvaI, and ligated into AvaI-digested
pACYC 184. This strategy led to the isolation of pMD4 (FIG. 1),
which contained a 1.9 kb AvaI fragment encoding atrazine
degradation activity.
[0085] All the clones and subclones had clearing zones surrounding
single colonies in about one week, although clearing appeared
sooner in the more heavily-inoculated area of streak plates. E.
coli DH5.alpha. cells containing pMD2, pMD3, and pMD4 produced a
clearing phenotype on LB or minimal medium containing 500 .mu.g/ml
atrazine, but they also produced an opaque secreted product, or
precipitate, in the medium surrounding colonies. No secreted
material was seen with E. coli DH5.alpha.(pMD1). Of the four
plasmids examined, only E. coli (pMD1) produced a clearing zone on
medium containing hydroxyatrazine, suggesting that a gene or genes
encoding for hydroxyatrazine metabolism were located on this large
cosmid.
[0086] Hybridization analyses. To determine whether the 1.9 kb AvaI
fragment, which encodes atrazine degradation activity in
Pseudomonas sp. strain ADP, was also present in other
atrazine-degrading microorganisms, the internal 0.6 kb ApaI/IPstI
fragment from pMD4 was hybridized to EcoRI-digested genomic DNA
from SG1, a recently-isolated, atrazine-degrading pure culture
isolate from St. Gabriel, La., and an atrazine-degrading microbial
consortium described in Stucki et al., Water Res., 1, 291-296
(1995). Results shown in FIG. 2 indicate that the internal 0.6 kb
ApaI/PstI fragment from pMD4 hybridized to a 8.7 kb EcoRI fragment
and a 1.9 kb AvaI genomic DNA fragment from Pseudomonas sp. strain
ADP and that the gene probe hybridized to a 8.7 kb genomic DNA
fragment in strain SG1 and to a 9.3 kb fragment in DNA from the
consortium. DNA from P. cepacia G4, which does not metabolize
atrazine, did not hybridize to the probe. DNA from a P. cepacia G4,
an organism that does not degrade atrazine, did not hybridize to
the 0.6-kb probe. These results indicated that the isolated gene
region, which encodes atrazine-degradation activity, was not
restricted to Pseudomonas sp. strain ADP, but was present in at
least two independently obtained atrazine-degrading bacteria
obtained from geographically diverse locations.
[0087] To determine if the cloned gene region encoding atrazine
degradation activity was located on an indigenous plasmid, a
.sup.32P-labelled 0.6 kb ApaI/PstI fragment from pMD4 was
hybridized to EcoRI- and AvaI-digested plasmid DNA from Pseudomonas
sp. strain ADP. While the Pseudomonas strain harbored at least one
large plasmid of approximately 60 kb, there was no hybridization
between the probe and the plasmid DNA, suggesting that the isolated
gene region is located on the chromosome or on a plasmid that could
not be isolated by the method used here.
[0088] Tn5 mutagenesis analyses. To more precisely localize the
gene region(s) involved in atrazine dechlorination and to determine
if other regions of pMD1 were involved in the transformation of
atrazine, random Tn5 mutagenesis in E. coli was used to generate
mutations in the cloned genomic DNA fragments from Pseudomonas sp.
strain ADP. Forty-six unique Tn5 insertions in the cloned DNA were
mapped using restriction enzyme digestions and Southern
hybridization analysis (FIG. 3). Cosmids containing single Tn5
insertions were transformed into E. coli DH5.alpha. and the Tn5
mutants were screened for their ability to clear atrazine on solid
media. All of the transposon-containing mutants that had lost the
ability to clear atrazine from the growth medium mapped within the
1.9 kb AvaI fragment (i.e., the genomic DNA fragment cloned in
pMD4). The Tn5 insertions in all other regions of cosmid pMD 1 did
not affect their ability to clear atrazine from the growth medium.
Results of this mutagenesis study delimited the region essential
for atrazine dechlorination to 1.3 kb and indicated that other
regions of pMD1 were not required for atrazine dechlorination in E.
coli.
[0089] Analysis of atrazine metabolism by E. coli clones. The
extent and rate of atrazine degradation was determined in liquid
culture. E. coli clones containing plasmids pMD1, pMD2, or pMD3
were compared to Pseudomonas sp. strain ADP for their ability to
transform ring-labelled [.sup.14C]-atrazine to water-soluble
metabolites. This method, which measures [.sup.14C]-label
partitioning between organic and aqueous phases, had previously
been used with Pseudomonas sp. ADP to show the transformation of
atrazine to metabolites that partition into the aqueous phase, in
Mandelbaum et al., Appl. Environ. Microbiol., 61, 1451-1457 (1995).
When Pseudomonas sp. strain ADP, E. coli (pMD1), E. coli (pMD2), or
E. coli (pMD3), was incubated for 2 hours with [.sup.14C]-atrazine,
98%, 97%, 88%, and 92%, respectively, of the total recoverable
radioactivity was found in the aqueous phase (Table 2). Greater
than 90% of the initial radioactivity was accounted for as atrazine
plus water soluble metabolites, indicating that little or no
[.sup.14]CO.sub.2 was formed. In contrast, forty-four percent of
the radioactivity was lost from the Pseudomonas ADP culture after
18.5 hours. In previous studies done with Pseudomonas sp. strain
ADP and ring-labelled .sup.14C-atrazine, radiolabel was lost from
culture filtrates as .sup.14CO.sub.2 (see, e.g., Mandelbaum et al.,
Appl. Environ. Microbiol., 61, 1451-1457 (1995). With E. coli
(pMD1), E. coli (pMD2) and E. coli (pMD3) cultures, essentially all
the radioactivity was retained. It was found in the culture
filtrate as one or more metabolites.
2TABLE 2 [.sup.14C] Atrazine transformation to water soluble
metabolites by E. coli clones and Pseudomonas sp. strain ADP.
[.sup.14C] Atrazine Transformation Percent Water Soluble
Metabolites.sup.a Percent Recovered.sup.b Organism 2 hr 18.5 hr 2
hr 18.5 hr Pseudomonas strain 98 100 92 56 ADP E. coli (pMD1) 97
100 94 91 E. coli (pMD2) 88 97 96 92 E. coli (pMD3) 92 98 98 93
.sup.aValues equal (cpm in aqueous phase) .div. (cpm in aqueous
phase + cpm in organic phase) .times. 100. .sup.bValues equal (cpm
in aqueous phase + cpm in organic phase) .div. (cpm of starting
atrazine) .times. 100.
[0090] These results show that pMD1 contains genes that encode for
one or more enzymes that catalyze the conversion of hydroxyatrazine
to more water soluble metabolites. This data suggests that
hydroxyatrazine is the first intermediate in the atrazine
degradation pathway by Pseudomonas sp. strain ADP. This result is
consistent with earlier studies (Mandelbaum et al., Appl. Environ.
Microbiol., 61, 1451-1457 (1995)) which showed that hydroxyatrazine
was transiently produced during transformation of atrazine by a
bacterial consortium, from which Pseudomonas sp. strain ADP was
isolated (Mandelbaum et al., Appl. Environ. Microbiol., 59,
1695-1701 (1993)).
[0091] To ascertain the nature of the accumulating metabolite(s),
thin layer chromatography on silica gel plates was conducted. The
R.sub.f values of [.sup.14C] metabolites, obtained from culture
filtrates, were compared to authentic triazine compounds that could
be possible metabolites (Table 1). With E. coli clones containing
pMD2 or pMD3, a metabolite accumulated with an R.sub.f value
identical to standard hydroxyatrazine (R.sub.f=0.52). The amount of
radioactivity in the metabolite fraction was equivalent to the
starting radioactivity of [.sup.14C]-atrazine. A radioactive spot
corresponding to the R.sub.f value of hydroxyatrazine was observed
with E. coli (pMD1) after a few minutes of incubation. Over time,
however, this spot decreased in intensity and another somewhat more
polar compound (R.sub.f=0.43) was observed to increase
concomitantly. The Rf value of the unknown metabolite was not
equivalent to any of the tested standard compounds (Table 1).
[0092] Further evidence for the identity of the metabolite obtained
from the E. coli clones was obtained by using HPLC analyses.
Culture filtrates from E. coli containing pMD2, pMD3, or pMD4
contained a compound with a retention time of 6.5 minutes. This
compound was not observed with the E. coli DH5.alpha. wild-type
control. The hydroxyatrazine standard had a retention time of 6.5
minutes. Coinjection of hydroxyatrazine and culture filtrates from
the recombinant strains yielded a single uniform peak. Moreover,
the absorption spectrum of authentic hydroxyatrazine was identical
to that obtained from the 6.5 minute peak eluting from culture
filtrates. E. coli (pMD1) cleared atrazine from culture filtrates,
but the compound eluting at 6.5 minutes was not observed even after
18 hours of incubation. HPLC analysis did not reveal another
metabolite, but there was a significant amount of polar material
from the growth medium that eluted between 1-4 minutes and could
have obscured accumulating polar metabolites. Taken together,
results of this study indicate that hydroxyatrazine is the first
metabolite in the degradation of atrazine by Pseudomonas sp. strain
ADP (FIG. 4).
[0093] DNA and protein sequence of the atzA gene. The nucleotide
sequence of the approximately 1.9-kb AvaI DNA fragment in pMD4 was
determined on both strands. Nucleotide sequence was determined
initially by subcloning and subsequently using primers based on
sequence information obtained from subcloned DNA fragments. The
sequencing strategy used is shown in FIG. 5 and the nucleotide
sequence is shown in FIG. 6. DNA sequence analysis revealed several
possible open reading frames (ORFs) beginning with ATG. One large
ORF, beginning at base number 236 gave a translation product of 473
amino acids, was designated as the atzA gene. The atzA gene
consists of 1419 nucleotides that encodes a polypeptide of 473
amino acids with an estimated M, of 52,421 and a pl of 6.6. A
typical Pseudomonas ribosome binding site, beginning with GGAGA, is
located 11 bp upstream from the proposed start codon. A potential
stop codon is located at position 1655.
[0094] Several lines of evidence support the conclusion that the
designated ORF constitutes the atrazine chlorohydrolase gene: 1) E.
coli transformed with pMD4, gained the ability to degrade atrazine
as demonstrated by clearing zones surrounding colonies on solid
media containing crystalline atrazine, 2) the dechlorination
activity was abolished by transposon Tn5 insertions specifically
within the 1.9-kb AvaI fragment and the Tn5 insertion was located
within the ORF, 3) there is also significant homology between the
atzA ORF (40.987% identity over 484 amino acid residues) and a
protein from Rhodococcus corallinus NRRL B-15444R which possesses
an analogous catalytic activity, a triazine hydrolase which is
responsible for the deamination of melamine
(2,4,6-triamino-1,3-5-triazine) and dechlorination of
deethylsimazine. While no typical E. coli-10 sequence was seen
preceding the predicted start of AtzA, a potential Pseudomonas
ribosome binding site was found 11 base pairs upstream of the ATG
(V. Shigler et al., J. Bacteriol., 174, 711-724 (1992)). This is
interesting given the fact that atzA was expressed in E. coli.
[0095] The protein sequence derived from a translation of the ORF
is show in FIG. 7. N-terminal sequence analysis of AtzA indicated
that the 10 amino acids detect were identical to those predicted by
translating the ORF.
[0096] Homology of AtzA to other proteins. The AtzA amino acid
sequence was compared to other proteins in the Swiss Prot and
translated genes in Genbank/EMBL databases. The AtzA protein has
the highest sequence identity, at the amino acid level, with TrzA,
40.9% (Table 3). A comparison of the sequence shows that there is a
much higher degree of amino acid conservation towards the
C-terminus of the proteins. Other proteins showing amino acid
similarities with AtzA include: urease-alpha subunit (urea
amidohydrolase), cytosine deaminase, and immidazolone-5-propionate
hydrolase (IPH). The homologous proteins do not belong to any one
particular group of bacteria. The AtzA protein (atrazine
chlorohydrolase) is more related to TrzA and
imidazolone-5-propionate hydrolase than it is to the other proteins
having some amino acid similarity with atrazine chlorohydrolase.
The urease proteins were tightly clustered to one another and as a
group were less related to AtzA.
3TABLE 3 Relationship of AtzA to other proteins at the amino acid
level. % Amino Accession Acid Identity Designator Enzyme Name
Organism to AtzA Swiss Prot Database P18314 Urea Amidohydrolase
Klebsiella 20.3 aerogenes P16122 Urease Alpha Subunit Proteus
vulgaris 17.3 P17086 Urease Alpha Subunit Proteus mirabilis 17.1
P25524 Cytosine Deaminase E. coli 22.2 P41020 Urease Alpha Subunit
Bacillus pasteuri 17.7 GenBank/EMBL Database RERTRZA
N-ethylammeline Rhodococcus 41.0 chlorohydrolase corallinus S69145
Urease Alpha Subunit Rhizobium meliloti 22.8 X63656 Cytosine
Deaminase E. coli 21.8 D31856 Imidazolone-5- Bacillus subtilus 21.7
proprionate hydrolase
[0097] A comparison of the molecular and biochemical properties of
AtzA and TrzA (Table 4) indicate that while both enzymes have a
significant amount of amino acid similarity, there are major
differences between these two triazine hydrolases. First, AtzA
appears to only catalyze dechlorination reactions while TrzA is
capable of both dechlorination and deamination reactions. Second,
both enzymes have different substrate ranges and TrzA does not
degrade either atrazine or simazine, both of which are
environmentally important substrates for AtzA. It appears from
limited substrate analysis that the substrates degraded by AtzA
require a chlorine atom and an alkyamino side chain. In addition,
AtzA does not degrade melamine, the primary substrate for TrzA.
However, both enzymes have the ability to dechlorinate
deisopropylatrazine (desethylsimazine). Taken together, these
results indicate that despite amino acid similarities, both enzymes
are biochemically different and catalyze significantly different
reactions.
4TABLE 4 Properties of triazine hydrolases from Pseudomonas sp.
strain ADP and Rhodococcus corallinus NRRL B-1544R Enzyme
N-ethylammeline Atrazine chlorohydrolase chlorodydrolase (TrzA)
(AtzA) Substrate Melamine Atrazine Deethylsimazine Products
Ammeline Hydroxyatrazine N-ethylammeline Reaction Deamination and
Dechlorination Dechlorination Holoenzyme 200,000 Daltons 240,000
Daltons Subunit MW 54 KD .about.53 KD Number of 4 4 subunits
[0098] Purification of AtzA. The atrazine chlorohydrolase was
purified from cell-free extracts of E. coli (pMD4) by precipitation
with 20% (wt/vol) NH.sub.4SO.sub.4. That is, solid NH.sub.4SO.sub.4
was added to a buffered solution of the extract up to 20% of its
saturation point at 4.degree. C. The 0-20% NH.sub.4SO.sub.4
fraction was isolated and further purified by anion exchange
chromatography on a Mono Q HR16/10 column. The resultant material
was eluted with 0-0.5 M KCl gradient and one of the peaks was found
to yield a single major band of approximately 60 kDa when subjected
to SDS-PAGE.
[0099] Enzyme characterization. The molecular size of the native
protein was estimated by gel filtration chromatography on a
Superose 6 column to be approximately 240,000 daltons. These
results, combined with SDS-PAGE analysis suggest that the enzyme is
a homotetramer. No metals were detected in the native enzyme and
the isoelectric point of the protein was 5.25 (Table 5).
5TABLE 5 Molecular properties of purified AtzA. Property AtzA
Subunit structure (.alpha.).sub.4 Subunit molecular mass (kDa)
SDS-Page 60 Calculated (aa quantification) 52.42 Native molecular
mass (kDa) Gel filtration 240-250 Calculated (aa quantification)
210 Metal content None detected pI Observed 5.25 Calculated (aa
quantification) 6.6
[0100] The AtzA protein was examined for its ability to degrade
various triazine compounds in vitro. Results in Table 6 show that
only substrates containing a chlorine atom and an alkyamino side
chain were degraded. Melamine and tertbutylazine were not
substrates for AtzA.
6TABLE 6 Substrate range of Atrazine chlorohydrolase (AtzA) from
Pseudomonas sp. strain ADP.sup.a. Substrate Degraded Not Degraded
Atrazine Desethyldesisopropylatrazine Desethylatrazine Melamine
Desisopropylatrazine Tertbutylazine Simazine .sup.aDegradation of
substrates determined by using purified enzyme in vitro.
[0101] Enzyme Kinetics. Using several concentrations of atrazine,
the K.sub.m of AtzA for atrazine was estimated to be approximately
125 .mu.M (FIG. 8). This value is slightly higher than those
reported for the related triazine hydrolase TrzA which had a
K.sub.m value of 82 .mu.M for desethylsimazine and 61 .mu.M for
desethyl-s-triazine.
[0102] All publications, patents and patent documents are
incorporated by reference herein, as though individually
incorporated by reference. The invention has been described with
reference to various specific and preferred embodiments and
techniques. However, it should be understood that many variations
and modifications may be made while remaining within the spirit and
scope of the invention.
Sequence CWU 1
1
* * * * *