U.S. patent application number 10/658564 was filed with the patent office on 2004-02-26 for materials and methods for detection of oxalobacter formigenes.
Invention is credited to Peck, Ammon B., Sidhu, Harmeet.
Application Number | 20040038286 10/658564 |
Document ID | / |
Family ID | 27500741 |
Filed Date | 2004-02-26 |
United States Patent
Application |
20040038286 |
Kind Code |
A1 |
Peck, Ammon B. ; et
al. |
February 26, 2004 |
Materials and methods for detection of Oxalobacter formigenes
Abstract
The subject invention concerns the novel use of formyl-CoA
transferase enzyme together with oxalyl-CoA decarboxylase enzyme
for the detection and measurement of oxalate in biological samples.
The use of the enzyme system according to the subject invention
results in the conversion of oxalate into carbon dioxide and
formate. Because the production of formate is directly correlated
to the concentration of oxalate present in a sample, the
determination of the resulting formate concentration provides an
accurate, sensitive and rapid means for detecting even low levels
of oxalate. The subject invention further concerns the cloning,
sequencing and expression of the genes that encode the formyl-CoA
transferase enzyme and the oxalyl-CoA decarboxylase enzyme of
Oxalobacter formigenes. The subject invention also concerns methods
for detecting the presence of Oxalobacter formigenes organisms in a
sample, and the polynucleotide probes and primers used in the
detection method.
Inventors: |
Peck, Ammon B.;
(Gainesville, FL) ; Sidhu, Harmeet; (Gainesville,
FL) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK
A PROFESSIONAL ASSOCIATION
2421 N.W. 41ST STREET
SUITE A-1
GAINESVILLE
FL
326066669
|
Family ID: |
27500741 |
Appl. No.: |
10/658564 |
Filed: |
September 8, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10658564 |
Sep 8, 2003 |
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09829094 |
Apr 9, 2001 |
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09829094 |
Apr 9, 2001 |
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08936094 |
Sep 23, 1997 |
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6214980 |
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08936094 |
Sep 23, 1997 |
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08883610 |
Jun 26, 1997 |
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6090628 |
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08883610 |
Jun 26, 1997 |
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08717587 |
Sep 27, 1996 |
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5912125 |
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08717587 |
Sep 27, 1996 |
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08493197 |
Jun 20, 1995 |
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5837833 |
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08493197 |
Jun 20, 1995 |
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08262424 |
Jun 20, 1994 |
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5604111 |
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Current U.S.
Class: |
435/6.12 ;
435/232; 435/252.3; 435/320.1; 435/6.14; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/10 20130101; C12Q
1/527 20130101; G01N 2333/9104 20130101; G01N 2333/988 20130101;
C12Q 1/48 20130101; C12N 9/1014 20130101; C12Q 1/04 20130101; C12Q
1/689 20130101; C12N 9/88 20130101; C12N 9/1022 20130101 |
Class at
Publication: |
435/6 ; 435/69.1;
435/232; 435/252.3; 435/320.1; 536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/88; C12N 001/21; C12N 015/74 |
Goverment Interests
[0003] This invention was made with government support under
National Institutes of Health Grant No. DK 20586. The government
has certain rights in this invention.
Claims
We claim:
1. A purified polynucleotide molecule, comprising a nucleotide
sequence that encodes an oxalyl-CoA decarboxylase polypeptide, or a
fragment of said oxalyl-CoA decarboxylase that retains functional
enzymatic activity.
2. The polynucleotide molecule according to claim 1, wherein said
nucleotide sequence is derived from Oxalobacter formigenes.
3. The polynucleotide molecule according to claim 1, wherein said
nucleotide sequence encodes a polypeptide comprising the amino acid
sequence shown in SEQ ID NO. 4, or a fragment of said oxalyl-CoA
decarboxylase that retains functional enzymatic activity.
4. The polynucleotide molecule according to claim 1, comprising the
nucleotide sequence shown in SEQ ID NO. 3.
5. The polynucleotide molecule according to claim 1, wherein said
polynucleotide molecule hybridizes under standard high-stringency
conditions with a polynucleotide molecule comprising the nucleotide
sequence shown in SEQ ID NO. 3, or the complementary sequence
thereof.
6. The polynucleotide molecule according to claim 1, wherein said
polynucleotide consists of a nucleotide sequence that encodes
oxalyl-CoA decarboxylase comprising the amino acid sequence shown
in SEQ ID NO. 4, or a fragment of said oxalyl-CoA decarboxylase
that retains functional enzymatic activity.
7. The polynucleotide molecule according to claim 6, wherein said
polynucleotide hybridizes under high stringency conditions with a
nucleotide sequence comprising nucleotides 181 through 1884 of the
nucleotide sequence shown in SEQ ID NO. 3 or the complementary
sequence thereof.
8. The polynucleotide according to claim 6, wherein said nucleotide
sequence consists of nucleotides 181 through 1884 of the nucleotide
sequence shown in SEQ ID NO. 3 or the complementary sequence
thereof.
9. A polynucleotide probe, comprising a nucleotide sequence that is
substantially complementary with a polynucleotide sequence present
in an Oxalobacter formigenes genome, wherein the polynucleotide
sequence present in the Oxalobacter formigenes genome comprises a
gene selected from the group consisting of the formyl-CoA
transferase gene and the oxalyl-CoA decarboxylase gene.
10. A polynucleotide PCR primer, comprising a nucleotide sequence
that is substantially complementary with a polynucleotide sequence
present in an Oxalobacter formigenes genome, wherein said
polynucleotide sequence present in said Oxalobacter formigenes
genome comprises a gene selected from the group consisting of the
formyl-CoA transferase gene and the oxalyl-CoA decarboxylase gene,
and wherein said PCR primer is capable of priming PCR amplification
of said polynucleotide sequence present in said Oxalobacter
formigenes genome.
11. A method for detecting Oxalobacter formigenes in a sample,
comprising the steps of: (a) contacting said sample with a
polynucleotide probe according to claim 9 under conditions
sufficient for selective hybridization of said polynucleotide probe
with a DNA fragment specific for Oxalobacter formigenes; and (b)
detecting said probe hybridized to said DNA fragment.
12. A polynucleotide vector comprising a polynucleotide molecule
according to claim 1.
13. The polynucleotide vector according to claim 12, wherein said
polynucleotide molecule consists of a nucleotide sequence that
encodes an oxalyl-CoA decarboxylase polypeptide comprising the
amino acid sequence shown in SEQ ID NO. 4, or a fragment of said
oxalyl-CoA decarboxylase that retains functional enzymatic
activity.
14. The polynucleotide vector according to claim 12, wherein said
polynucleotide vector hybridizes under high stringency conditions
with a nucleotide sequence consisting of nucleotides 181 through
1884 of the nucleotide sequence shown in SEQ ID NO. 3 or the
complementary sequence thereof.
15. The polynucleotide vector according to claim 12, wherein said
nucleotide sequence of said polynucleotide molecule consists of
nucleotides 181 through 1884 of the nucleotide sequence shown in
SEQ ID NO. 3 or the complementary sequence thereof.
16. A recombinant host cell which comprises the polynucleotide
vector of claim 12, and wherein said cell expresses said oxalyl-CoA
decarboxylase or a fragment of said oxalyl-CoA decarboxylase that
retains enzymatic activity.
17. The cell according to claim 16, wherein said polynucleotide
vector hybridizes under high stringency conditions with a
nucleotide sequence consisting of nucleotides 181 through 1884 of
the nucleotide sequence shown in SEQ ID NO. 3 or the complementary
sequence thereof.
18. The cell according to claim 16, wherein said nucleotide
sequence of said polynucleotide molecule consists of nucleotides
181 through 1884 of the nucleotide sequence shown in SEQ ID NO. 3
or the complementary sequence thereof.
Description
[0001] Cross-Reference to a Related Applications
[0002] This application is a continuation of U.S. application Ser.
No. 09/829,094, filed Apr. 9, 2001, which is a continuation of U.S.
application Ser. No. 08/936,094, filed Sep. 23, 1997, now U.S. Pat.
No. 6,214,980; which is a continuation-in-part of patent
application Ser. No. 08/883,610, filed Jun. 26, 1997, now U.S. Pat.
No. 6,090,628; which is a continuation-in-part of patent
application Ser. No. 08/717,587, filed Sep. 27, 1996, now U.S. Pat.
No. 5,912,125; which is a continuation-in-part of patent
application Ser. No. 08/493,197, filed Jun. 20, 1995, now U.S. Pat.
No. 5,837,833; which is a continuation-in-part of patent
application Ser. No. 08/262,424, filed Jun. 20, 1994, now U.S. Pat.
No. 5,604,111.
FIELD OF THE INVENTION
[0004] The present invention relates to novel assay methods and
devices for determining the presence or concentration of oxalate in
a sample; Oxalobacter genes encoding enzymes required for the
catabolism of oxalate; and materials and methods for detecting and
identifying Oxalobacter formigenes in a sample.
BACKGROUND OF THE INVENTION
[0005] Oxalic acid (Oxalate) is a highly toxic natural by-product
of catabolism in vertebrate animals and many consumable plants.
Unfortunately, a significant portion of humans are unable to
properly metabolize oxalate, a condition which may result in the
formation of kidney stones in those persons. It is estimated that
70% of all kidney stones are composed of some amount of oxalate.
Approximately 12 percent of the U.S. population will suffer from a
kidney stone at some time in their lives, and the incidence is
rising not only in the United States, but also in Sweden and Japan
(Curhan, 1993). Moreover, although a healthy person breaks down or
excretes sufficient quantities of oxalate to avoid excessive
accumulation of oxalate in the tissues, a number of disease states
are known to be associated with malfunctions of oxalate metabolism,
including pyridoxine deficiency, renal failure and primary
hyperoxaluria, a metabolic genetic disorder that results in the
excessive deposition of oxalate in the kidneys.
[0006] Persons suffering from and at risk for developing kidney
stones, as well as patients with lipid malabsorption problems
(e.g., sprue, pancreatic insufficiency, inflammatory intestinal
disease, bowel resection, etc.), tend to have elevated levels of
urinary oxalate, a fact that has been exploited as a means for
identifying individuals at risk. While elevated levels of oxalate
may be present in urine, detecting elevated levels of oxalate in
serum has not been routine due to the difficulty in detecting the
low levels of oxalate present in serum.
[0007] Most previous methods for measuring oxalate in a biological
sample first require the isolation of the oxalate by precipitation,
solvent extraction, or an ion-exchange absorption (Hodgkinson,
1970). Quantitation of the isolated oxalate may be determined by
any one of several methods including colorimetry, fluorometry,
gas-liquid chromatography or isotope dilution techniques. Because
many of the oxalate isolation techniques used in these analytical
methods are not quantitative, it is normally necessary to correct
for the low recovery of oxalate by adding a .sup.14C-labeled oxalic
acid internal standard, which further complicates the analytical
method. All these methods are laborious, and consequently expensive
because of the amount of skilled laboratory technician time which
must be employed. In addition, isolation of the oxalate may require
relatively large sample volumes for starting material.
[0008] Recently, several advances in the detection and quantitation
of oxalate have been made through the use of (a) oxalate degrading
enzymes and (b) high performance liquid chromatography. One
commercially-available enzymatic test (Sigma Chemical Company, St.
Louis, Mo.) employs oxalate oxidase to oxidize oxalate to carbon
dioxide and hydrogen peroxide. The hydrogen peroxide produced can
then be measured colorimetrically in a second enzymatic reaction in
the presence of peroxidase.
[0009] In another enzymatic method for measuring oxalate, oxalate
decarboxylase is used to convert oxalate to carbon dioxide and
formate. The resultant carbon dioxide can be measured
manometrically, by the pH change in a carbon dioxide trapping
buffer or by the color change in a pH indicator buffer. Whatever
method of carbon dioxide assay is adopted, the time required for
diffusion and equilibration of carbon dioxide is much longer than
is desirable for a rapid analytical method.
[0010] Alternatively, the formate produced by the action of oxalate
decarboxylase can be assayed with formate dehydrogenase in an
NAD/NADH coupled reaction, as described in Costello, 1976 and
Yriberri, 1980. This method is both cumbersome and time-consuming
because oxalate decarboxylase and formate dehydrogenase differ in
their optimum pH requirements, thus necessitating a pH adjustment
during the analysis.
[0011] Another commercially available enzymatic test (Boehringer
Mannheim) cleaves oxalate to formate and carbon dioxide, then
oxidizes the formate to bicarbonate by NAD in the presence of the
enzyme formate dehydrogenase. The amount of NADH is determined by
means of its absorbance at 334, 340, or 365 nm. Another test
("STONE RISK" by Mission Pharmacal) measures oxalate as a part of a
battery of tests for kidney stones.
[0012] Oxalobacter formigenes is a recently discovered,
oxalate-degrading obligately anaerobic bacterium residing primarily
in the intestines of vertebrate animals, including man (Allison et
al., 1986). Although the first isolates of O. formigenes were
cultured from sheep rumen (Dawson et al., 1980), additional strains
have now been isolated from fecal contents of rats, guinea pigs and
pigs (Argenzio et al., 1988, Daniel et al., 1987), fecal samples
from man (Allison et al., 1985), and anaerobic aquatic sediments
(Smith et al., 1985). This bacterium is unique among
oxalate-degrading organisms having evolved a total dependence on
oxalate metabolism for energy (Dawson et al., 1980). Recent
evidence suggests that Oxalobacter formigenes has an important
symbiotic relationship with vertebrate hosts by regulating oxalic
acid absorption in the intestine as well as oxalic acid levels in
the plasma (Hatch and Freel, 1996). Studies by Jensen and Allison
(1994) comparing various O. formigenes isolates revealed only
limited diversity of their cellular fatty acids, proteins, and
nucleic acid fragments. Based on these comparisons, strains of O.
formigenes have been divided into two major subgroups. In general,
group I strains have shown limited intragroup diversity, while
group II strains have shown greater intragroup diversity.
[0013] Special conditions are required to culture O. formigenes and
their detection is based generally on the appearance of zones of
clearance of calcium oxalate crystals surrounding colonies (Allison
et al., 1986). Assays based on the appearance of zones of clearance
of calcium-oxalate crystals surrounding bacterial colonies (Allison
et al., 1985) or degradation of oxalate in culture media measured
by calcium-chloride precipitation (Dawson et al., 1980) fail to
confirm the oxalate-degrading bacteria as Oxalobacter.
[0014] As illustrated above, the currently existing assays for
oxalate suffer from numerous problems, including cost, inaccuracy,
reliability, complexity, and lack of sensitivity. Accordingly, it
is an object of the subject invention to provide a simple,
accurate, and sensitive assay for the detection of low levels of
oxalate in a biological sample.
[0015] The current methods for culturing and identifying the
presence of Oxalobacter formigenes are technically demanding and
time consuming, and therefore, are not suitable for rapid and
specific identification of O. formigenes, particularly for clinical
diagnostics. Accordingly, another object of the subject invention
is to provide a rapid, accurate polynucleotide probe-based assay
for the detection of O. formigenes.
BRIEF SUMMARY OF THE INVENTION
[0016] The subject invention concerns the cloning, sequencing, and
expression of the formyl-CoA transferase (frc) and the oxalyl-CoA
decarboxylase (oxc) genes of Oxalobacter formigenes, and the use of
the enzymes to detect the presence of oxalate in a sample. The
assay of the subject invention provides, for the first time, a
rapid, sensitive method to detect even very low concentrations of
oxalate in biological samples. Advantageously, the biological
samples in which oxalate can be detected include both urine and
serum samples. The enzyme system used according to the subject
invention converts oxalate to carbon dioxide and formate. In a
preferred embodiment of the subject invention, the production of
formate is then measured colorimetrically. This assay provides a
sensitive, accurate and convenient means for detecting oxalate.
[0017] A further aspect of the subject invention is the discovery
of the O. formigenes genes which encode the formyl-CoA transferase
and the oxalyl-CoA decarboxylase enzymes. The discovery of these
genes makes it possible to efficiently produce large quantities of
pure formyl-CoA transferase and oxalyl-CoA decarboxylase for use in
the assay of the subject invention or other appropriate
application.
[0018] The subject invention further concerns a dipstick device for
the detection and quantitation of oxalate in a sample. The dipstick
device comprises the oxalyl-CoA decarboxylase and formyl-CoA
transferase enzymes of the present invention immobilized on a
carrier matrix. A detectable signal is generated on the dipstick if
oxalate is present in the sample.
[0019] The subject invention also provides a means for detecting
the presence of Oxalobacter formigenes organisms in a sample. The
method of detection provided for herein involves polynucleotide
probes which can be used to identify Oxalobacter formigenes.
[0020] The subject invention also concerns the polynucleotide
primers and the use thereof for polymerase chain reaction (PCR)
amplification of Oxalobacter formigenes nucleotide sequences.
Amplified Oxalobacter sequences can then be detected using the
polynucleotide probes of the subject invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIGS. 1A-1E show the detection of varying concentrations of
oxalate in a sample. Colorimetric absorbance for each sample was
plotted over time (minutes). Positive and negative control panels
are also shown.
[0022] FIGS. 2A-2B show the nucleotide sequence of the formyl-CoA
transferase gene and the deduced amino acid sequence of the
formyl-CoA transferase polypeptide from Oxalobacter formigenes.
Bolded letters represent amino acid residues determined by
N-terminal protein sequencing.
[0023] FIGS. 3A-3B show the nucleotide sequence of the oxalyl-CoA
decarboxylase gene and flanking regions from Oxalobacter
formigenes. The consensus ribosome-binding site lies approximately
10 bases upstream (double-underlined letters) from the putative
translation initiation codon (positions 1 to 3). A rho-independent
termination sequence lies at positions 1758 to 1790
(double-underlined letters). A putative TPP-binding site appears
between positions 1351 and 1437.
[0024] FIG. 4 shows an RFLP analysis of O. formigenes, strain OxB
using probes specific for the oxc gene encoding oxalyl-CoA
decarboxylase and the frc gene encoding formyl-CoA transferase.
Genomic DNA isolated from a 14 day culture of O. formigenes strain
OxB was digested with the restriction enzyme HIND III. The digested
DNA was size fractionated by electrophoreses through 0.5% agarose
gels, electroblotted to a nylon membrane, then hybridized with
either probe AP15 (SEQ ID NO. 6) or probe AP34 (SEQ ID NO. 9) to
detect oxc or probe AP273 (SEQ ID NO. 10) to detect frc.
[0025] FIG. 5 shows the sensitivity of detecting the oxc and frc
genes in RFLP of O. formigenes strain OxB versus strain HC-I.
Genomic DNA from each of the two strains was digested with the
restriction enzyme HIND III. Two-fold serial dilutions were made of
the digested DNA and size fractionated by electrophoresis through
0.5% agarose gels (left panels). RFLP analyses were carried out as
described in FIG. 4, except the nylon membranes were hybridized
with a 1:1 mixture of probe AP15 (SEQ ID NO. 6) plus probe AP273
(SEQ ID NO. 10) (right panels).
[0026] FIG. 6 shows the detection of the oxc and frc genes in
various strains of O. formigenes by RFLP analysis. RFLP was carried
out as described in FIG. 5.
[0027] FIG. 7 shows PCR-based amplification of a genetic region of
the oxc gene in various strains of O. formigenes. Using PCR primer
AP15 (SEQ ID NO. 6) and primer AP22 (SEQ ID NO. 11) as PCR primers,
PCR amplification was performed using genomic DNA isolated from
each of the 12 strains of O. formigenes listed in Table 1 as
template. PCR products were size fractionated by electrophoresis
through 1.2% agarose gels and observed visually using ethidium
bromide (EtBr) and UV light.
[0028] FIG. 8 shows a direct analysis of fecal samples for O.
formigenes. Oxalobacter negative stool sample (A & B) was
spiked with 10.sup.2 (C) and 10.sup.4 (D) cfu of OxB or 10.sup.3
(E) and 10.sup.4 (F) cfu of OxK per 0.1 gm. DNA from an unspiked O.
formigenes-positive stool sample diluted 1:25 (G) and 1:50 (H).
[0029] FIGS. 9A-9B show the identification of sequence homologies
within the oxc gene expressed in representative group I and group
II strains of Oxalobacter formigenes to design oligonucleotide
probes. Partial sequences of 5'-end of the oxc gene generated by
PRC amplification of the region bounded by the primer pair,
AP34/AP21. A region of high homology shared by all strains (between
bp 13 and 43) was selected for the genus-specific oligonucleotide
probe, AP286, while regions of high homology shared by only group I
strains (between bp 197 and 214) or shared only by group II strains
(between bp 133 and 150) were selected for group-specific
oligonucleotide probes, HS2 and AP307, respectively.
[0030] FIGS. 10A-10B show the detection of Oxalobacter formigenes
using a genus-specific oligonucleotide probe that hybridizes to the
PCR product of the oxc gene. Using the primer pair AP34/AP21, PCR
amplification was performed using genomic template DNA isolated
from 8 group I and 8 group II strains of O. formigenes. The PCR
products were size fractionated by electrophoresis through 1.2%
agarose gels and the expected 504-508 bp product visualized with
EtBr under UV light (upper panel). The PCR products were
transblotted to nylon membranes and Southern blotted using the
genus-specific oligonucleotide probe, AP286 (lower panel).
[0031] FIGS. 11A-11C show the classification of group I and group
II strains of Oxalobacter formigenes using group-specific
oligonucleotide probes that hybridize with PCR products of the oxc
gene. Using the primer pair AP34/AP21, PCR amplification was
performed using genomic template DNA isolated from 8 group I and 8
group II strains of O. formigenes. The PCR products were size
fractionated by electrophoresis through 1.2% agarose gels and the
expected 504-508 bp product visualized with EtBr under UV light
(upper panel). The PCR products were transblotted to nylon
membranes and Southern blotted using HS2, the group I-specific
(center panel), or AP307, the group II-specific (lower panel),
oligonucleotide probes.
[0032] FIG. 12 shows agarose gels containing oxc amplification
products from quantitative PCR visualized with UV light (FIGS.
12A-12C). FIGS. 12D-12E show Log equivalence plots to determine the
number of oxc molecules in the sample.
BRIEF DESCRIPTION OF THE SEQUENCES
[0033] SEQ ID NO. 1 is a nucleotide sequence for the formyl-CoA
transferase gene (also shown in FIG. 2).
[0034] SEQ ID NO. 2 is a polypeptide encoded by SEQ ID NO. 1, which
can be used according to the subject invention.
[0035] SEQ ID NO. 3 is the nucleotide sequence for the oxalyl-CoA
decarboxylase gene (also shown in FIG. 3).
[0036] SEQ ID NO. 4 is a polypeptide encoded by SEQ ID NO. 3, which
can be used according to the subject invention.
[0037] SEQ ID NO. 5 is an oxalyl-CoA decarboxylase sequence, which
can be used as a probe according to the subject invention.
[0038] SEQ ID NO. 6 is an oxalyl-CoA decarboxylase sequence, which
can be used as a probe or PCR primer according to the subject
invention.
[0039] SEQ ID NO. 7 is an oxalyl-CoA decarboxylase 5'-primer, which
can be used according to the subject invention.
[0040] SEQ ID NO. 8 is an oxalyl-CoA decarboxylase 3'-primer, which
can be used according to the subject invention.
[0041] SEQ ID NO. 9 is an oxalyl-CoA decarboxylase sequence, which
can be used as a probe or primer according to the subject
invention.
[0042] SEQ ID NO. 10 is a formyl-CoA transferase sequence, which
can be used as a probe according to the subject invention.
[0043] SEQ ID NO. 11 is an oxalyl-CoA decarboxylase sequence, which
can be used as a PCR primer according to the subject invention.
[0044] SEQ ID NO. 12 is an oxalyl-CoA decarboxylase sequence, which
can be used as a PCR primer according to the subject invention.
[0045] SEQ ID NO. 13 is an oxalyl-CoA decarboxylase sequence, which
can be used as a PCR primer according to the subject invention.
[0046] SEQ ID NO. 14 is an oxalyl-CoA decarboxylase sequence, which
can be used as a probe according to the subject invention.
[0047] SEQ ID NO. 15 is an oxalyl-CoA decarboxylase sequence, which
can be used as a probe according to the subject invention.
[0048] SEQ ID NO. 16 is an oxalyl-CoA decarboxylase sequence, which
can be used as a probe according to the subject invention.
[0049] SEQ ID NO. 17 is an oxalyl-CoA decarboxylase sequence, which
can be used as a PCR primer according to the subject invention.
[0050] SEQ ID NO. 18 is a formyl-CoA transferase sequence, which
can be used as a PCR primer according to the subject invention.
[0051] SEQ ID NO. 19 is a formyl-CoA transferase sequence, which
can be used as a PCR primer according to the subject invention.
DETAILED DESCRIPTION OF THE INVENTION
[0052] The subject invention provides an accurate, sensitive assay
for oxalate in biological samples such as urine and serum. Elevated
levels of oxalate are correlated with urinary tract stone
formation, as well as other health problems. Early detection of
high levels of oxalate makes it possible to prevent, delay or
reduce adverse health consequences through appropriate medication
and through modulation of diet.
[0053] In the presently described diagnostic system, two enzymes
are used to catabolize oxalate to carbon dioxide and formate.
Specifically, any oxalate that may be present in a sample being
assayed is converted into formate and carbon dioxide (CO.sub.2)
through the combined action of the enzymes oxalyl-CoA decarboxylase
and formyl-CoA transferase. The formate can then be detected using
a variety of techniques known in the art. In a preferred
embodiment, the production of formate is measured colorimetrically
by linking the catabolism of formate with the production of a
detectable color change (for example, the formation of a compound
that absorbs a particular wavelength of light). The production of
formate is directly correlated with the amount of oxalate present
in the sample. Therefore, if a known amount of formate is produced
using the subject enzyme system, then the amount of oxalate present
in the sample can be easily quantitated.
[0054] In a preferred embodiment, the enzymes used in the subject
invention are expressed by genes from the bacterium Oxalobacter
formigenes. The genes encoding both oxalyl-CoA decarboxylase (Lung
et al., 1994) and formyl-CoA transferase enzymes have been cloned
and expressed, thus providing a readily-available source of reagent
material. The subject assay is capable of detecting oxalate levels
in a range as low as 0.00025-0.0005 mM (FIGS. 1A-1E). This level of
sensitivity makes the subject assay capable of direct detection of
oxalate in serum samples consisting of little as 10 .mu.l volume.
The described system can be easily automated with standard systems
known in the art.
[0055] In a preferred embodiment of the subject assay, the
enzymatic reaction can be carried out in the wells of flat-bottomed
96-well microtiter plates and read in an automated plate reader.
Suitable concentrations of the assay reagents oxalyl-CoA
decarboxylase, oxalyl-CoA, .beta.-NAD, formate dehydrogenase, and
the sample to be assayed are added to the microtiter wells. The
reaction is then brought to equilibrium (two minute incubation at
37.degree. C. in the plate reader) to permit degradation of any
residual formate that may be present in the sample. The formyl-CoA
transferase enzyme is then added to the mixture to start the
reaction, and the plate is read at 15 second intervals. Formate
production is determined by measuring the reduction in NAD in the
presence of formate dehydrogenase by detecting changes in
absorbance of the sample at 340 nm (Baetz and Allison, 1989). The
quantity of oxalate is determined by comparison of the unknown
samples with standards having a known amount of oxalate.
[0056] Further, the enzymatic reaction of the subject assay will
not be initiated until the formyl-CoA transferase, oxalyl-CoA
decarboxylase, and oxalyl-CoA are all present within the reaction
mixture. Therefore, initiation of the enzymatic reaction can be
prevented by withholding one of the above reagents from the
reaction mix. Preferably, oxalyl-CoA decarboxylase and oxalyl-CoA
are added first, and the reaction is initiated by the addition of
formyl-CoA transferase to the mix. However, the order of addition
of the three reagents is not material to the function of the assay,
so long as one of the reagents is withheld until just prior to the
desired initiation point of the assay.
[0057] The formyl-CoA transferase and oxalyl-CoA decarboxylase
enzymes used in the subject invention can be obtained and purified
as a natural product of Oxalobacter formigenes (Baetz and Allison,
1989 and 1990). Alternatively, the enzymes can be obtained from
host cells expressing the recombinant polynucleotide molecules of
the subject invention that encode the enzymes. Other reagents used
in the subject assay can be obtained from conventional sources,
such as Sigma Chemical Company, St. Louis, Mo. Further, a person of
ordinary skill in the art can readily determine the optimal
concentrations of the reagents to use in the assay described
herein.
[0058] A further aspect of the subject invention concerns the
cloning, sequencing and expression of the Oxalobacter formigenes
gene which encodes the formyl-CoA transferase used in the assay
that is a subject of the invention. The gene was cloned using
degenerate oligonucleotide probes (based on partial amino acid
sequencing of tryptic peptides) to screen an Oxalobacter genomic
DNA library. The gene encodes a polypeptide having a molecular
weight of approximately 40 kD. The subject invention further
concerns the cloning, sequencing, and expression of the gene which
encodes oxalyl-CoA decarboxylase from Oxalobacter formigenes. The
nucleotide sequence of the cDNA of formyl-CoA transferase and
oxalyl-CoA decarboxylase are shown in FIGS. 2A-2B and 3A-3B,
respectively (SEQ ID NOS. 1 and 3).
[0059] Because of the redundancy of the genetic code, a variety of
different polynucleotide sequences can encode the formyl-CoA
transferase polypeptide disclosed herein. It is well within the
skill of a person trained in the art to create alternative
polynucleotide sequences encoding the same, or essentially the
same, polypeptide of the subject invention. These variant or
alternative polynucleotide sequences are within the scope of the
subject invention. As used herein, references to "essentially the
same" sequence refers to sequences which encode amino acid
substitutions, deletions, additions, or insertions which do not
materially alter the functional enzymatic activity of the encoded
polypeptide. Further, the subject invention contemplates those
polynucleotide molecules having sequences which are sufficiently
homologous with the DNA sequences shown in FIGS. 2A-2B and 3A-3B
(SEQ ID NOS. 1 and 3) so as to permit hybridization with those
sequences under standard high-stringency conditions. Such
hybridization conditions are conventional in the art (see, e.g.,
Maniatis et al., 1989).
[0060] As a person skilled in the art would appreciate, certain
amino acid substitutions within the amino acid sequence of the
polypeptide can be made without altering the functional activity of
the enzyme. For example, amino acids may be placed in the following
classes: non-polar, uncharged polar, basic, and acidic.
Conservative substitutions, whereby an amino acid of one class is
replaced with another amino acid of the same class, fall within the
scope of the subject invention so long as the substitution does not
materially alter the enzymatic reactivity of the polypeptide.
Non-conservative substitutions are also contemplated as long as the
substitution does not significantly alter the functional activity
of the encoded polypeptide.
[0061] The polynucleotides of the subject invention can be used to
express the recombinant formyl-CoA transferase enzyme. They can
also be used as a probe to detect related enzymes. The
polynucleotides can also be used as DNA sizing standards.
[0062] The polypeptides encoded by the polynucleotides can be used
to raise an immunogenic response to the formyl-CoA transferase
enzyme. They can also be used as molecular weight standards, or as
inert protein in an assay. The polypeptides can also be used to
detect the presence of antibodies immunoreactive with the
enzyme.
[0063] The polynucleotide sequences of the subject invention may be
composed of either RNA or DNA. More preferably, the polynucleotide
sequences are composed of DNA. The subject invention also
encompasses those polynucleotides that are complementary in
sequence to the polynucleotide sequences disclosed herein.
[0064] Another aspect of the subject invention pertains to kits for
carrying out the enzyme assay for oxalate. In one embodiment, the
kit comprises, in packaged combination and in relative quantities
to optimize the sensitivity of the described assay method, (a) the
oxalyl-CoA decarboxylase, oxalyl-CoA, .beta.-NAD, and formate
dehydrogenase; and (b) formyl-CoA transferase. The kit may
optionally include other reagents or solutions, such as buffering
and stabilization agents, along with any other reagents that may be
required for a particular signal generation system. Other reagents
such as positive and negative controls can be included in the kit
to provide for convenience and standardization of the assay
method.
[0065] The subject invention further concerns a method for
detecting the presence of Oxalobacter formigenes organisms in a
sample. Specific polynucleotide probes can be prepared based on the
nucleotide sequence of either the oxalyl-CoA decarboxylase or the
formyl-CoA transferase gene sequence of Oxalobacter formigenes. The
polynucleotide probes of the subject invention can be used to
identify Oxalobacter formigenes in a sample, and to classify the
strain of Oxalobacter formigenes detected. The polynucleotide
probes of the subject invention can be used according to standard
procedures and conditions to specifically and selectively detect
polynucleotide sequences that have sufficient homology to hybridize
with the probe. DNA can be isolated from bacterial microorganisms
in a biological specimen (e.g., biopsy, fecal matter, tissue
scrapings, etc.) using standard techniques known in the art and the
isolated DNA screened for hybridization with Oxalobacter oxalyl-CoA
decarboxylase-specific and/or formyl-CoA transferase-specific
polynucleotide probes. Various degrees of stringency can be
employed during the hybridization, depending on the amount of probe
used for hybridization, the level of complementarity (i.e.,
homology) between the probe and target DNA fragment to be detected.
The degree of stringency can be controlled by temperature, ionic
strength, pH, and the presence of denaturing agents such as
formamide during hybridization and washing. Hybridization methods
and conditions are known in the art and are generally described in
Nucleic Acid Hybridization: A Practical Approach (Hames, B. D., S.
J. Higgins, eds.), IRL Press (1985).
[0066] The polynucleotide probes of the subject invention include,
for example, the oxalyl-CoA decarboxylase probe A (SEQ ID NO. 5),
probe AP15 (SEQ ID NO. 6), and probe AP34 (SEQ ID NO. 9), probe AP
286 (SEQ ID NO. 14), probe AP307 (SEQ ID NO. 15), and probe HS-2
(SEQ ID NO. 16), specifically exemplified herein. Probes for
formyl-CoA transferase include, for example, probe AP273 (SEQ ID
NO. 10) specifically exemplified herein. The nucleotide sequences
of the exemplified probes are shown below:
1 Probe A (SEQ ID NO. 5) 5'-GAGCGATACCGATTGGA-3' Probe AP15 (SEQ ID
NO. 6) 5'-GCACAATGCGACGACGA-3' Probe AP34 (SEQ ID NO. 9)
5'-ATACTCGGAATTGACGT-3' Probe AP273 (SEQ ID NO. 10)
5'-TTCATGTCCAGTTCAATCGAACG-3' Probe AP286 (SEQ ID NO. 14)
5'-GACAATGTAGAGTTGACTGATGGCTTTCATG-3' Probe AP307 (SEQ ID NO. 15)
5'-CAGGATGGTCAGAAGTTC-3' Probe HS-2 (SEQ ID NO. 16)
5'-CCGGTTACATCGAAGGA-3'
[0067] The polynucleotide probes contemplated in the subject
invention also include any polynucleotide molecule comprising a
nucleotide sequence capable of specifically hybridizing with
oxalyl-CoA decarboxylase or formyl-CoA transferase polynucleotide
sequence of the present invention. As used herein, reference to
"substantial homology" or "substantially complementary" refers not
only to polynucleotide probes of the subject invention having 100%
homology with the nucleotide sequence of the target polynucleotide,
or fragments thereof, but also to those sequences with sufficient
homology to hybridize with the target polynucleotide. Preferably,
the degree of homology will be 100%; however, the degree of
homology required for detectable hybridization will vary in
accordance with the level of stringency employed in the
hybridization and washes. Thus, probes having less than 100%
homology to the oxalyl-CoA decarboxylase or formyl-CoA transferase
polynucleotide sequences can be used in the subject method under
appropriate conditions of stringency. In a preferred embodiment,
high stringency conditions are used. In addition, analogs of
nucleosides may be substituted for naturally occurring nucleosides
within the polynucleotide probes. Such probes having less than 100%
homology or containing nucleoside analogs are within the scope of
the subject invention. The skilled artisan, having the benefit of
the disclosure contained herein, can readily prepare probes
encompassed by the subject invention.
[0068] In addition, the subject invention also concerns
polynucleotide primers that can be used for polymerase chain
reaction (PCR) amplification of Oxalobacter formigenes nucleotide
sequences. PCR amplification methods are well known in the art and
are described in U.S. Pat. Nos. 4,683,195; 4,683,202; and
4,800,159. The polynucleotide primers and probes of the present
invention can also be used to amplify nucleotide sequences encoding
oxalyl-CoA decarboxylase and formyl-CoA transferase by non-PCR
based amplification methods. The subject primers and probes in any
method where amplification of specific sequences is desired. In a
preferred embodiment, the polynucleotide primers are based on the
oxalyl-CoA decarboxylase or formyl-CoA transferase gene sequence
and can be used to amplify the full length or a portion of the
target gene. The amplified Oxalobacter sequences can be detected
using the probes of the subject invention according to standard
procedures known in the art.
[0069] The polynucleotide primers of the subject invention include,
for example, oxalyl-CoA decarboxylase PCR primer 1 (SEQ ID NO. 7),
PCR primer 2 (SEQ ID NO. 8), PCR primer AP15 (SEQ ID NO. 6), PCR
primer AP22 (SEQ ID NO. 11), PCR primer AP34 (SEQ ID NO. 9), PCR
primer AP21 (SEQ ID NO. 17), OXF6 (SEQ ID NO. 12), and OXF7 (SEQ ID
NO. 13), specifically exemplified herein. The nucleotide sequences
of the exemplified PCR primers are shown below:
2 PCR primer 1 5'-CAGGTTATGCAGCTTCT-3' (SEQ ID NO. 7) PCR primer 2
5'-GGATGGTTGTCAGGCAG-3' (SEQ ID NO. 8) PCR primer AP15
5'-GCACAATGCGACGACGA-3' (SEQ ID NO. 6) PCR primer AP22
5'-GTAGTTCATCATTCCGG-3' (SEQ ID NO. 11) PCR primer AP34
5'-ATACTCGGAATTGACGT-3' (SEQ ID NO. 9) PCR primer AP21
5'-TCCAATCGGTATCGCTC-3' (SEQ ID NO. 17) OXF6
5'-AATGTAGAGTTGACTGA-3' (SEQ ID NO. 12) OXF7
5'-TTGATGCTGTTGATACG-3' (SEQ ID NO. 13)
[0070] The primer pair AP34 and AP21 (derived from oxc sequences
between bp -59 to -41 and by 451 to 435, respectively),
consistently amplifies a 500 bp segment of oxc from all O.
formigenes strains and isolates tested. PCR application of whole
fecal DNA with this genus-specific primer pair, in conjunction with
Southern Blotting using genus and group specific probes, now
provides a rapid diagnostic tool to detect and speciate O.
formigenes. Time-consuming steps, e.g., agarose-gel electrophoresis
and Southern blot hybridizations, can be substituted with newer
technologies such as microtiter-plate based colorimetric or
fluorogenic assays (Jordan et al., 1996).
[0071] Polynucleotide primers contemplated by the subject invention
also include any polynucleotide molecule comprising a nucleotide
sequence capable of specifically priming amplification of
oxalyl-CoA decarboxylase or formyl-CoA transferase polynucleotide
sequences disclosed herein. As used herein, reference to
"substantial homology" or "substantially complementary" refers not
only to polynucleotide primers of the subject invention having 100%
homology with the nucleotide sequence of the target polynucleotide,
or fragments thereof, but also to those sequences with sufficient
homology to hybridize with and prime the amplification of a target
polynucleotide. Preferably, the degree of homology will be equal to
or about 100%. The skilled artisan, having the benefit of the
disclosure contained herein, can readily prepare other primers of
varying nucleotide length and sequence that can be used to amplify
all or portions of the oxalyl-CoA decarboxylase and/or the
formyl-CoA transferase gene.
[0072] The polynucleotide probes and primers of the subject
invention can be chemically synthesized or prepared through
recombinant means using standard methods and equipment. The
polynucleotide probes and primers can be in either single- or
double-stranded form. If the probe or primer is double-stranded,
then single-stranded forms can be prepared from the double-stranded
form. The polynucleotide probes and primers may be comprised of
natural nucleotide bases or known analogs of the natural nucleotide
bases. The probes and primers of the subject invention may also
comprise nucleotides that have been modified to bind labeling
moieties for detecting the probe or primer or amplified gene
fragment.
[0073] The polynucleotide molecules of the subject invention can be
labeled using methods that are known in the art. The
polynucleotides may be radioactively labeled with an isotope such
as .sup.3H, .sup.35S, .sup.14C, or .sup.32P. The polynucleotides
can also be labeled with fluorophores, chemiluminescent compounds,
or enzymes. For example, a polynucleotide molecule could be
conjugated with fluorescein or rhodamine, or luciferin or luminol.
Similarly, the polynucleotide molecule can be conjugated with an
enzyme such as horseradish peroxidase or alkaline phosphatase.
Polynucleotide molecules can also be detected by indirect means.
For example, the polynucleotide may be conjugated with ligands,
haptens, or antigenic determinants. The conjugated polynucleotide
is then contacted with the ligand receptor, with an anti-ligand
molecule that binds to the ligands, or with an antibody that binds
to the hapten/antigenic determinant, respectively. For example, the
polynucleotide can be labeled with digoxygenin and detected with
labeled anti-digoxygenin antibodies. The ligand receptor,
anti-ligand molecule, or antibody may be directly labeled with a
detectable signal system, such as a fluorophore, chemiluminescent
molecule, radioisotope, or enzyme. Methods for preparing and
detecting labeled moieties are known in the art.
[0074] The subject invention also concerns methods for
quantitatively detecting oxalyl-CoA decarboxylase and formyl-CoA
transferase nucleotide sequences and, thereby, determining the
number of Oxalobacter formigenes in a sample. General methods and
techniques for performing quantitative PCR are known in the art
(Tarmuzzer et al., 1996)
[0075] In one embodiment of the present detection method, samples
to be tested for the presence of Oxalobacter formigenes are
obtained from a person or animal, and DNA is isolated from the
specimen using standard techniques known in the art. For example,
cells can be lysed in an alkali solution, the nucleic acid
extracted in phenol:chloroform, and then precipitated with ethanol.
The DNA is then fragmented into various sizes using restriction
endonuclease enzymes or other means known in the art. The DNA
fragments are then electrophoretically separated by size on an
agarose gel. In an alternative embodiment, the DNA fragments are
subjected to PCR amplification using PCR primers of the present
invention prior to gel electrophoresis in order to specifically
amplify portions of the formyl-CoA transferase and oxalyl-CoA
decarboxylase genes.
[0076] After the DNA fragments are separated on the gel, the
size-fractionated DNA fragments are transferred to a membrane
matrix, such as nitrocellulose, nylon, or polyvinylidene difluoride
(PVDF), by Southern blotting. The DNA immobilized on the membrane
matrix is single-stranded. Polynucleotide probes of the subject
invention are then contacted with the membrane and allowed to
hybridize with the DNA immobilized on the membrane. A probe of the
present invention can be labeled with a detectable signal, such as
a radioisotope, or the probe can be labeled with a hapten or
antigen such as digoxigenin. The hybridization can be performed
under conditions known in the art. After hybridization of the probe
with the DNA fragments on the membrane, the membrane is washed to
remove non-hybridized probe. Standard wash conditions are known in
the art, and the stringency and number of washes employed can
vary.
[0077] The membrane is then tested or observed for the presence of
hybridized probe. For example, if the hybridized probe was labeled
with a hapten or antigen, then it can be detected using an antibody
that binds to the conjugated hapten or antigen on the probe. The
antibody can be directly labeled with a detectable fluorophore,
chemiluminescent molecule, radioisotope, enzyme, or other signal
generating system known in the art. Alternatively, the antibody can
be detected using a secondary reagent that binds to the antibody,
such as anti-immunoglobulin, protein A, protein G, and other
antibody binding compositions known in the art. The secondary
reagent can be labeled with a detectable fluorophore,
chemiluminescent molecule, radioisotope, or enzyme. The presence of
a detectable hybridization signal on the membrane indicates the
presence of Oxalobacter formigenes in a test sample.
[0078] The subject invention also concerns a kit for the detection
of Oxalobacter formigenes in a sample. A kit contemplated by the
subject invention may include in one or more containers:
polynucleotide probes, positive and negative control reagents, and
reagents for detecting the probes. The kit may also include
polynucleotide primers for performing PCR amplification of specific
Oxalobacter formigenes genes. In a preferred embodiment, the
polynucleotide probes and primers are specific for the oxalyl-CoA
decarboxylase and formyl-CoA transferase genes of O.
formigenes.
[0079] The subject invention also concerns a dipstick device
comprising the enzymes of the subject invention and dyes and/or
substrates immobilized on a carrier matrix. Any dye or substrate
that yields a detectable product upon exposure to the reaction
products that are produced by the enzymatic reaction of oxalate
with oxalyl-CoA decarboxylase and formyl-CoA transferase as
described herein is contemplated for use with the subject dipstick
device. The carrier matrix of the assay device can be composed of
any substance capable of being impregnated with the enzyme and dye
components of the subject invention, as long as the matrix is
substantially inert with respect to the analyte being assayed for.
For example, the carrier matrix may be composed of paper,
nitrocellulose, PVDF, or plastic materials and the like.
[0080] Incorporation of the enzymes, dye and other components on
the carrier matrix can be accomplished by any method such as
dipping, spreading or spraying. A preferred method is impregnation
of the carrier matrix material by dipping in a reagent solution and
drying to remove solvent. Drying can be accomplished by any means
which will not deleteriously affect the reagents incorporated, and
typically is by means of an air drying oven.
[0081] The dipstick device of the subject invention is dipped in or
contacted with a sample to be tested for the presence or amount of
oxalate. Positive and negative controls can be used in conjunction
with the dipstick device. An appropriate amount of time is allowed
to pass and then the dipstick is assessed for a positive reaction
by visual inspection. If oxalate is present in the sample then a
detectable signal, usually in the form of a color, can be observed
on the dipstick. Typically, the intensity of the color developed in
a fixed time period is proportional to the concentration of oxalate
present in the sample.
[0082] All publications cited herein are incorporated
reference.
[0083] Following are examples which illustrate procedures,
including the best mode, for practicing the invention. These
examples should not be construed as limiting. All percentages are
by weight and all solvent mixture proportions are by volume unless
otherwise noted.
EXAMPLE 1
Determination of Level of Sensitivity of Enzyme Assay System
[0084] Samples containing oxalate at concentrations ranging from
0.004 mM to 0.00025 mM were prepared in 10 .mu.l volumes. The
samples were then assayed using the enzyme system of the subject
invention in 96-well microtiter plates. Reagents were then added at
the following concentrations: KH.sub.2PO.sub.4 (pH 6.7), 50 mM;
MgCl.sub.2, 5 mM; thiamine PPi (TPP), 2 mM; oxalyl-CoA, 0.375 mM;
.beta.-NAD, 1.0 mM; formate dehydrogenase, 0.25 IU; and oxalyl-CoA
decarboxylase, 0.03 U. The reaction mixture was then incubated at
37.degree. C. for 2 minutes in order to permit the degradation of
any residual formate that may be present in the sample mixture. The
reaction was then initiated by the addition of formyl-CoA
transferase to the sample mixture. Changes in A.sub.340 were
measured every 15 seconds at 37.degree. C. (FIGS. 1A-1E).
Appropriate positive and negative controls were run simultaneously
with the assay.
EXAMPLE 2
Detection of Oxalobacter Formigenes in a Sample
[0085] Strains of Oxalobacter formigenes used in the following
methods are listed in Table 1 below.
3TABLE 1 Description of the Oxalobacter formigenes strains Group
Classification of Source of O. formigenes strains.sup.a Strain
Isolate Group I OxB Sheep rumen OxWR Wild rat cecum SOx-C
Freshwater lake sediment SOx-6 Freshwater lake sediment POxC Pig
cecum HC-1 Human feces Group II BA-1 Human feces OxK Human feces
HOxBLS Human feces HOxRW Human feces OxCR Lab rat cecum OxGP Guinea
pig cecum
[0086] All Oxalobacter formigenes strains were grown in medium B
containing 30 mM oxalate, as described in Allison et al. (1985).
Human fecal samples (approximately 60 mg) were inoculated
anaerobically into vials containing 9 ml of media B, then
sequentially transferred through 10.sup.-8 dilutions. Cultures were
incubated at 37.degree. C. for 10 days and biochemically tested for
the catabolic consumption of oxalate by CaCl.sub.2 precipitation
(50 .mu.l media, 100 .mu.l 1% CaCl.sub.2, and 2.7 ml dH.sub.2O) and
spectrophotometric analyses (600 nm).
[0087] Cultures (10-15 ml) of O. formigenes were centrifuged at
10,000.times.g, the bacterial pellet was resuspended in 567 .mu.l
TE buffer (10 mM Tris-Cl, pH 7.5 plus 1 mM EDTA, pH 8.0), 30 .mu.l
10% sodium dodecyl sulfate (SDS) and 3 .mu.l of proteinase K (20
mg/ml), and the mixture incubated 5 hr at 37.degree. C. to ensure
bacterial cell lysis. Nucleic acids were extracted from the lysates
using phenol/chloroform/isoamylalcohol (25:24:1). Chromosomal DNA
was precipitated from the aqueous phase by adding 1/2 volume of 7.5
M ammonium acetate and 2 volumes of 100% ethanol. DNA was recovered
by centrifugation (12,000.times.g), washed once with 70% ethanol,
and the pellet resuspended in 15-20 .mu.l H.sub.2O. Bacterial DNA
was also isolated directly from fresh human stool samples following
lysis with chaotropic salt and guanidine thiocyanate, then binding
to glass matrix (GlasPac, National Scientific Supply, San Rafael,
Calif.) (Stacy-Phips et al., 1995).
[0088] Bacterial DNA was digested with the restriction endonuclease
Hind III (Life Technologies, Inc., Gaithersburg, Md.). The
restriction-enzyme generated fragments were size separated by gel
electrophoresis through 0.5% agarose, stained with ethidium bromide
(EtBr), illuminated with UV light, and photographed to document
proper digestion. Digested DNA was then transferred from the
agarose gels to positively-charged nylon membranes
(Boehringer-Mannheim GmBH, Indianapolis, Ind.) by positive pressure
blotting and UV crosslinking (Stratagene, LaJolla, Calif.).
Hybridizations were carried out using internal sequence
oligonucleotide probes. Oligonucleotides were synthesized in the
University of Florida ICBR Oligonucleotide Synthesis Laboratory
(Gainesville, Fla.) and have the sequences:
4 AP15 5'-GCACAATGCGACGACGA-3' (SEQ ID NO. 6) AP22
5'-GTAGTTCATCATTCCGG-3' (SEQ ID NO. 11) AP34 5'ATACTCGGAATTGACGT-3'
(SEQ ID NO. 9) AP273 5'-TTCATGTCCAGTTCAATCGAACG-3'. (SEQ ID NO.
10)
[0089] Each oligonucleotide was end-labeled with digoxigenin in a
reaction using terminal transferase. The digoxigenin-labeled
oligonucleotide probes were hybridized to the immobilized DNA
fragments and hybridization detected colorimetrically by
enzyme-linked immunoassay (ELISA) using an anti-digoxigenin
alkaline phosphatase conjugate according to the manufacturer's
protocol provided with the GENIUS III detection system
(Boehringer-Mannheim).
[0090] All PCRs were performed according to protocols described in
Anderson et al. (1993). Briefly, 50 .mu.l reactions contained 1.5
mM MgCl.sub.2, 200 .mu.M dNTP, 1.25 U Taq polymerase (GIBCO-BRL,
Bethesda, Md.), 1 .mu.g template DNA and 1 .mu.M each of a 5' and
3' primer. A preferred reaction profile proved to be 94.degree. C.
for 5 min, then 45 cycles of 94.degree. C. for 1 min of
denaturation, 55.degree. C. for 2 min of annealing and 72.degree.
C. for 3 min of primer extension. PCR products were size separated
by gel electrophoresis in 1.2% agarose containing EtBr and
photographed in UV light. PCR primer AP15 (SEQ ID NO. 6) and primer
AP22 (SEQ ID NO. 11) were used as primers.
[0091] Previous studies by Lung et al. (1994) showed that genomic
DNA of O. formigenes, strain OxB, could be digested with the
restriction enzyme Hind III and that a limited number of enzyme
cleavage sites existed near or within the oxalyl-CoA decarboxylase
(oxc) gene. A RFLP analysis of Hind III digested OxB genomic DNA
using either probe AP15 (SEQ ID NO. 6), a probe homologous to an
internal sequence of the oxc gene, probe AP34 (SEQ ID NO. 9), a
probe homologous to a 5'-end sequence of the oxc gene but separated
from the probe AP15 (SEQ ID NO. 6) sequence by a Hind III site, or
probe AP273 (SEQ ID NO. 10), a probe homologous to an internal
sequence of the formyl-CoA transferase (frc) gene, is shown in FIG.
4. Using probe AP15 (SEQ ID NO. 6), a fragment of approximately 7
kb containing a portion of the oxc gene was detected, while
fragments of approximately 3 kb were detected using either probe
AP34 (SEQ ID NO. 9) or probe AP273 (SEQ ID NO. 10). The 3 kb
fragment identified by probe AP34 (SEQ ID NO. 9) is distinct from
the 3 kb fragment detected by probe AP273 (SEQ ID NO. 10).
[0092] As shown in FIG. 5, the oxalyl-CoA decarboxylase and
formyl-CoA transferase genes were consistently detected in samples
containing as little as 0.06 to 0.20 .mu.g of O. formigenes, strain
OxB, DNA or approximately 0.20 to 0.40 .mu.g of O. formigenes DNA
from other group I strains, such as HC-1. The 23-bp probe AP273
(SEQ ID NO. 10) can detect the frc gene in DNA samples containing
only one-fourth the amount of DNA required for the 13 bp probe AP15
(SEQ ID NO. 6) to detect the oxc gene (FIG. 5, upper panel). These
probes are highly specific for O. formigenes since they fail to
bind to other bacterial DNA, including Escherichia coli,
Alcaligenes oxalaticus, and fecal bacteroides.
[0093] Protein, lipid and genetic studies of several isolates of O.
formigenes have provided the basis for dividing this genus into two
major subgroupings (Jensen et al., 1994). When RFLP analyses were
performed on genomic DNA isolated from various Oxalobacter
formigenes strains, probes AP15 (SEQ ID NO. 6) and AP273 (SEQ ID
NO. 10) were able to distinguish group I strains from group II
strains on the Southern blot hybridizations (FIG. 6). All strains
of O. formigenes belonging to group I (to which OxB is assigned)
hybridized with both probe AP15 (SEQ ID NO. 6) and probe AP273 (SEQ
ID NO. 10). Due to a characteristic slow growth of strain HC-1,
only faint bands appeared in this experiment. In contrast, none of
the O. formigenes strains assigned to group II hybridized with
probe AP273 (SEQ ID NO. 10) and only BA-1 hybridized with probe
AP15 (SEQ ID NO. 6). These data indicate a highly conserved
homology of oxc and frc within group I strains and a less conserved
homology within group II strains.
[0094] To increase the sensitivity of detecting O. formigenes, PCR
was used to amplify that portion of oxc which by RFLP appeared to
differentiate the group I and group II strains. Using primer AP15
(SEQ ID NO. 6) and primer AP22 (SEQ ID NO. 11) as PCR primers to
amplify a DNA segment in the carboxy-terminal region of oxc,
strains assigned to group I (i.e., OxB, HC-1, OxWR, POxC, SOx-4 and
SOx-6) exhibited a common band at 452 bp (FIG. 7). In contrast, the
other six strains, all belonging to group II, showed variable
amplification patterns, but all showed a dominant PCR band of
approximately 630 bp, with a weaker 452 bp band. Sequence analysis
of this 630 bp band from strain OxK has revealed the presence of
the 452 bp sequence present in the 630 bp PCR product. Close
analysis of the group II strains suggest that their PCR
amplification profiles are highly reproducible, suggesting group II
strains may fall into three (sub)groupings: HOxBLS and HOxRW
(subgroup 1), OxCR and OxGP (subgroup 2), and BA-1 and OxK
(subgroup 3).
[0095] The use of PCR-based detection of the oxc gene to identify
O. formigenes in clinical specimens was examined by comparing PCR
and biochemical methods of detection. Specimen 1, known to be
positive for O. formigenes, gave ambiguous results in biochemical
testing for oxalate depletion, but exhibited the presence of the
450 bp PCR product indicative of an O. formigenes group I strain.
Specimen 2, known to be negative for O. formigenes, proved negative
using both PCR-based and biochemical testing. Specimen 3, known to
be positive for O. formigenes, showed depletion of oxalate in all
dilutions and revealed a PCR pattern suggestive of an O. formigenes
group II strain. PCR amplification was not observed in the original
culture or the first dilution due to the presence of inhibitors of
PCR e.g., bile salts, bilirubin, etc.) which copurify with DNA.
[0096] To circumvent the inhibition of the PCR by factors
co-purifying with the bacterial DNA, DNA isolation was performed by
lysing fresh stool samples with guanidine thiocyanate followed by
adsorption to and elution from glass matrices. Using this method,
PCR-based detection of O. formigenes can be performed using fecal
DNA diluted only 1:25 to 1:50 to eliminate PCR inhibitors.
Sensitivity experiments using different stool samples spiked with
strains OxB or OxK in the range of 10.sup.1 to 10.sup.7 cfu per 0.1
g of sample showed that as few as 10.sup.2 to 10.sup.3 cfu of O.
formigenes per 0.1 g sample could be detected (FIG. 8). Again,
PCR-based analyses of DNA isolated directly from a stool sample
known to be positive for O. formigenes by culture methods, showed
amplification patterns indicative of a group II strain (FIG. 8,
lanes F & G).
EXAMPLE 3
Detection and Classification of Oxalobacter Formigenes
Bacterial Strains
[0097] O. formigenes strains used included OxB (isolated from sheep
rumen) and HC1, OxK, BA1, HOxBLS, HOxRW, HOxRA, HOxCC13, and
HOxHM18 (isolated from human feces). In addition, several new
purified cultures, including HOxUK5, HOxUK88, HOxUK90, and HOxHS
(grown from human feces), were also used. All strains and isolates
were grown in media B containing 30 mM potassium oxalate, as
described elsewhere (Allison et al., 1985), and maintained under
strict anaerobic conditions until used.
Preparation of Genomic DNA from O. Formigenes Cultures
[0098] Fifteen ml cultures of O. formigenes were centrifuged at
10,000.times.g, the bacterial pellet resuspended in 567 .mu.l of TE
buffer (10 mM Tris-HCl, pH 7.5, plus 1 mM EDTA, pH 8.0), 30 .mu.l
of 10% sodium dodecyl sulfate plus 3 .mu.l of proteinase K (20
mg/ml), and this mixture incubated for 5 hours at 37.degree. C. to
ensure bacterial cell lysis. Nucleic acids were extracted from the
lysates with phenol:chloroform:isoamyl alcohol (25:24:1).
Chromosomal DNA was precipitated by adding 1/2 volume of 7.5 M
ammonium acetate and 2 volumes of 100% ethanol. DNA was recovered
by centrifugation (12,000.times.g) and washed once in 70% ethanol.
The final DNA precipitation was resuspended in 20 .mu.l
H.sub.2O.
Sequence Analysis of the oxc Genes
[0099] The primer pair,
[0100] 5'-ATACTCGGAATTGACGT-3' (a 5'-primer designated AP34) (SEQ
ID NO. 9) and
[0101] 5'-TCCAATCGGTATCGCTC-3' (a 3'-primer designated AP21) (SEQ
ID NO. 17)
[0102] homologous to sequences within the 5'-end of the oxc gene
present in strain OxB (Lung et al., 1994), was used to amplify a
500 bp DNA fragment from genomic DNA isolated from each of twelve
human O. formigenes strains. Amplifications were performed in 50
.mu.l PCR reactions containing 1.5 mM MgCl.sub.2, 200 .mu.M
deoxynucleoside triphosphate, 1.25 U of Taq polymerase (Gibco-BRL,
Bethesda, Md.), 1 .mu.g of genomic DNA and 1 .mu.M each of 5'- and
3'-primer. PCR were carried out for 35 cycles and included an
initial 5 minute denaturation step at 94.degree. C., 1 minute
annealing (with a temperature stepdown from 60.degree. C. to
55.degree. C.), 1 minute extension at 72.degree. C. and a final 8
minute extension at 72.degree. C. The PCR products were size
fractionated by electrophoreses through 1.2% agarose gels
containing ethidium bromide for visualization of the bands in UV
light. Each 500 bp PCR product was cloned into the TA cloning
system, pCR-2.1 (Invitrogen, Inc., San Diego, Calif.). Competent
DH5.varies. E. coli bacteria were transfected with the recombinant
plasmid and transformed bacteria selected on LB agar plates
containing 10 .mu.l/ml of ampicillin and 20 mg/ml of X-Gal. DNA
from appropriate clones was isolated, checked for the presence of
an insert of correct size by digestion with the restriction enzyme,
Eco RI. Inserts of recombinant plasmids were sequenced using
M13-forward and M13-reverse primers.
Clinical Samples
[0103] Fecal samples of 100 generally healthy children of either
sex ranging in age from 0 to 12 years were examined for the
presence of O. formigenes. All fecal samples were collected in
Dzerzhinsk, a city in the Donetsk region of the Ukraine. This
particular population was selected due to the fact that these
children have had limited use of antibiotics, that might influence
bacterial colonization of the intestinal tract, in treatment of
childhood diseases. Approximately 25 mg sample of fresh stool
(within 3-4 hours of collection), was inoculated into vials
containing 10 ml of anaerobically sealed media B supplemented to 30
mM with potassium-oxalate. The vials were analyzed at the
University of Florida, Gainesville, Fla. After incubation at
37.degree. C. for one week, the loss of oxalate from each fecal
culture was determined using a calcium-chloride precipitation
method in which 50 .mu.l culture media is mixed with 100 .mu.l 0.1%
CaCl.sub.2 plus 3.0 ml dH.sub.2O and the absorbance of each mixture
determined spectrophotometrically (600 nm). The calcium
precipitation test for loss of oxalate has been repeatedly verified
as reliable by other methods (e.g., gas chromatography and butyl
esters) for detection of oxalate. Typically, cultures not showing
catabolism of oxalate generally have O.D. readings of about 0.1,
whereas cultures with oxalate degradation have O.D. readings less
than about 0.02.
PCR-Based Detection and Identification of O. Formigenes
[0104] DNA was isolated from individual fecal cultures by the
method of Phipps et al. (Stacy-Phipps et al., 1995) using guanidine
thiocyanate as a chaotropic agent and glass-matrix for DNA binding.
One .mu.l of each DNA sample was used as template in a 50 .mu.l PCR
reaction as described above. The amplified PCR products were size
separated by electrophoresis through 1.2% agarose gels containing
ethidium bromide and visualized with UV light. Each reaction was
controlled using a reaction containing all the components of the
PCR with the exception of template DNA.
Southern Blot Analysis
[0105] Southern blots were carried out as previously detailed in
Example 2. Briefly, the size separated PCR products were
transferred to positively charged nylon membranes (Boehringer
Mannheim GmBH, Indianapolis, Ind.) by positive pressure blotting
and UV-crosslinking. The oxc derived genus specific (AP286), group
I specific (HS-2) and group II specific (AP307) oligonucleotides
were synthesized in the University of Florida ICBR DNA Synthesis
Laboratory (University of Florida, Gainesville, Fla.) and
end-labeled with digoxigenin in a reaction using terminal
transferase. The digoxigenin labeled oligonucleotides were
hybridized to the immobilized PCR products under conditions of high
stringency (5.times.SSC and 68.degree. C.). Hybridization was
detected colorimetrically by enzyme-linked immunosorbent assay
(ELISA) with an anti-digoxigenin alkaline phosphatase conjugate
according to the manufacturer's protocol provided with the GENIUS
III kit (Boehringer Mannheim GmBH).
Generation of Genus-Specific and Group-Specific Probes
[0106] Preliminary studies looking at the efficacy of various
oligonucleotide pairs to amplify portions of the oxc gene present
in various O. formigenes strains revealed that the PCR primer pair
AP34 (5'-primer)/Ap21 (3'-primer) amplified a 500 bp DNA fragment
in both group I and group II strains. To determine the degree of
sequence homology within the 5'-end of the oxc gene between various
strains of O. formigenes, genomic DNA was prepared from 5 group I
and 7 group II strains isolated from human fecal samples for use as
template in PCR with AP34 and AP21. Each PCR amplified an expected
500 bp product that was subsequently cloned into the pCR-2.1 vector
system and sequenced. A comparison of the 5'-end sequences of the
oxc gene from these 12 human isolates with the OxB gene is shown in
part in FIGS. 9A-9B. The 5'-end of the oxc gene appears to be
relatively conserved for a bacterial gene, with most bp changes
occurring in the wobble base such that the codon translation is not
altered. Nevertheless, there were enough sequence differences to
demarcate group I strains from group II strains, thus permitting
selection of regions that are conserved within strains of a
specific group, but differ significantly from strains of the other
group. Based on these conserved regions, genus-specific
oligonucleotide probes (for example, probe AP286, homologous to the
region between bp 13 and 43 of the open-reading frame), as well as
group I-specific (for example, probe HS2, homologous to the region
between bp 197 and 214 of the open-reading frame) and group
II-specific (for example, probe AP307, homologous to the region
between bp 133 and 150 of the open-reading frame) probes were
prepared.
Specificity of the Genus-Specific and Group-Specific
Oligonucleotide Probes
[0107] The specificity of probes AP286, AP307, and HS2 in detecting
and classifying O. formigenes was examined using genomic DNA
prepared from a number of known strains and isolates. PCR
amplifications with the genus-specific primer pair AP34 and AP21
resulted in the 500 bp amplification product in all cultures tested
(FIG. 10A, top panel). On Southern blotting, this 500 bp fragment
hybridized with a genus-specific probe, AP286 (FIG. 10B, bottom
panel).
[0108] In a separate experiment, the amplified 500 bp PCR product
was hybridized with either the group I-specific probe, HS2, (FIG.
11B, middle panel) or the group II-specific probe, AP307, (FIG.
11C, bottom panel). Results clearly show a group specificity in the
binding of these group-specific probes and their ability to
identify subgroups of O. formigenes.
Application of a PCR-Based Detection System for O. formigenes
[0109] In a double-blinded study, 100 fecal samples were collected
from children ranging in age from newborn to 12 years and tested
for the presence of O. formigenes using both an oxalate degradation
system and our PCR-based assay system. The aim of this study was to
determine the age at which children become naturally colonized with
this intestinal anaerobic bacterium. Of the 100 fecal samples
examined, 72 samples tested positive for O. formigenes by PCR, 59
of which also exhibited oxalate degradation in an oxalate
degradation assay. Interestingly, of the 72 positive samples, 68
were group II strains while only 4 were group I strains. All fecal
cultures exhibiting degradation of oxalate tested positive for O.
formigenes by PCR. Although there were 13 cultures that failed to
degrade oxalate that proved positive for O. formigenes by PCR, the
majority of the samples that failed to degrade oxalate also failed
to exhibit amplification of a product in the PCR-reaction. These
data show that the PCR-based assay is probably more sensitive than
the biochemical (calcium chloride precipitation) test, yet highly
specific.
[0110] When the data were unblinded, a clear pattern for the
natural colonization of children became evident. O. formigenes
could not be detected in infants less than 6-9 months of age. O.
formigenes began appearing in the intestinal tracts of children
around 1 year of age, and by 3-4 years of age, all children showed
signs of being colonized. Although the sample size is small, the
number of children colonized with O. formigenes declined between
8-12 years of age, reaching the colonization frequency of 70-80%
estimated for adult populations (Doane et al., 1989, Kleinschmidt
et al., 1993, Allison et al., 1986, and Goldkin et al., 1985).
EXAMPLE 4
Quantitative DNA-Based Detection and Identification of Oxalobacter
Formigenes
[0111] Because the original PCR primer pair AP34/AP21 was based on
the nucleotide sequence encoding oxalyl-CoA decarboxylase in strain
OxB (isolated from sheep), a PCR primer pair based on conserved
sequences found in human strains was used. This primer pair, OXF6
and OXF7, amplifies a 416 bp product. The nucleotide sequences of
OXF6 and OXF7 are shown below:
[0112] OXF6 5'-AATGTAGAGTTGACTGA-3' (SEQ ID NO. 12)
[0113] OXF7 5'-TTGATGCTGTTGATACG-3' (SEQ ID NO. 13)
[0114] To develop a competitive DNA template for use as an internal
control for QC-PCR, a 227 bp fragment of the oxalyl-CoA
decarboxylase gene (oxc) flanked by sequences homologous for the
OXF6/OXF7 primer pair and containing the genus-specific, group
I-specific and group II-specific probe sites was generated. To
accomplish this, a PCR reaction was performed using the OXF6
5'-primer plus a modified OXF7 3'-primer. The modified 3'-primer
consisted of two portions: a 5'-end which contained the 3'-primer
sequence within the oxc gene plus a 3'-end which annealed at a site
located approximately 200 bp downstream of the 5'-primer site. The
PCR using the primer pair OXF6/modified-OXF7 amplified the 210 bp
segment and synthesized the 17 bp OXF7 primer site at the 3'-end.
This PCR fragment was purified and ligated into pCR-2.1
(Invitrogen, Inc., San Diego, Calif.). A recombinant pCR-2.1
plasmid with the proper insert (confirmed by sequencing) was
selected for use as the internal competitive template.
[0115] Quantitative PCR (QC-PCR) were performed as described below.
Competitive template diluted from 1.times.10.sup.10 to
1.times.10.sup.2 copies/PCR were used to establish standard curves.
Experimental PCRs were established containing both experimental O.
formigenes DNA and dilutions of the synthetic template. The PCR
products were size separated by electrophoresis through 1.5%
agarose gels visualized with UV light and photographed (FIGS.
12A-12C). Photographs were scanned for relative band intensities,
normalized for differences in molecular mass, and plotted against
the log of the copy number of synthetic template added per
reaction.
[0116] Quantitation of the number of oxc genes, thereby the number
of bacteria in a sample, revealed the accuracy of this QC-PCR
detection system. Assuming the genome of O. formigenes is similar
in size to that of E. coli (4.7.times.10.sup.3 Kb), then 1 .mu.g of
genomic DNA would contain 1.8.times.10.sup.8 molecules (or gene
copies). Genomic DNA prepared from O. formigenes OxB had a
concentration by spectrophotometric analysis of 1.126 .mu.g
DNA/.mu.l or 2.times.10.sup.8 molecules/.mu.l. Two dilutions,
10.sup.-4 (20,000 molecules) and 10.sup.-6 (200 molecules) of this
DNA were used as template in the QC-PCR with dilutions of
competitive template ranging from 50 to 250,000 molecules. As shown
in FIGS. 12D-12E, the Log equivalence revealed that the number of
molecules of O. formigenes OxB in the reaction were estimated
between 19,900-25,100 and 126-158.
[0117] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims.
REFERENCES
[0118] Allison, M. J., H. M. Cook, D. B. Milne, S. Gallaher, R. V.
Clayman (1986) "Oxalate degradation by gastrointestinal bacteria
from humans," J Nutr 116:455-460.
[0119] Allison, M. J., K. A. Dawson, W. R. Mayberry, J. G. Foss
(1985) "Oxalobacter formigenes gen. nov., sp. nov.: oxalate
degrading bacteria that inhabit the gastrointestinal tract," Arch
Microbiol. 141:1-7.
[0120] Anderson, J. T., J. G. Cornellius, A. J. Jarpe, W. E.
Winter, A. B. Peck (1993) "Insulin-dependent diabetes in the NOD
mouse model. II. .beta. cell destruction in autoimmune diabetes is
a T.sub.H1 mediated event," Autoimmunity 15:113-122.
[0121] Argenzio, R. A., J. A. Liacos, M. J. Allison (1988)
"Intestinal oxalate degrading bacteria reduce oxalate absorption
and toxicity in guinea pigs," J Nutr 118:787-791.
[0122] Baetz, A. L., M. J. Allison (1989) "Purification and
Characterization of Oxalyl-Coenzyme A Decarboxylase from
Oxalobacter formigenes," J. Bacteriol. 171:2605-2608.
[0123] Baetz, A. L., M. J. Allison (1990) "Purification and
Characterization of Formyl-Coenzyme A Transferase from Oxalobacter
formigenes," J. Bacteriol. 172:3537-3540.
[0124] Costello, J., M. Hatch, E. Bourke (1976) "An enzymic method
for the spectrophotometric determination of oxalic acid," J. Lab.
Clin. Med. 87(5):903-908.
[0125] Curhan, et al. (1993) "A Prospective study of dietary
calcium and other nutrients and the risk of symptomatic kidney
stones," N.E.J. Med. 328:833-838.
[0126] Daniel, S. L., P. A. Hartman, M. J. Allison (1987)
"Microbial degradation of oxalate in the gastrointestinal tracts of
rats," Appl Environ Microbiol 53:957-964.
[0127] Dawson, K. A., M. J. Allison, P. A. Hartman (1980)
"Characteristics of anerobic oxalate-degrading bacteria from the
rumen," Applied Microbiol. 22:522-529.
[0128] Dawson, K. A., M. J. Allison, P. A. Hartman (1980)
"Isolation and some characteristics of anaerobic oxalate-degrading
bacteria from ruman" Appl. Environ. Microbiol. 40:833-839.
[0129] Doane, L. A., M. Liebman, D. R. Caldwell (1989) "Microbial
oxalata degradation: effects on oxalate and calcium balance in
humans," Nutrition Res 9:957-964.
[0130] Goldkin, L., D. R. Cave, B. Jaffin, W. Robinson, C. M. Bliss
(1985) "A new factor in enteric hyperoxaluria: Oxalobacter
formigenes" AM J Gastro 80:860.
[0131] Hatch, M., R. W. Freel (1996) "Oxalate transport across
intestinal and renal epithelia" Calcium Oxalate in Biological
Systems, pages 217-238, CRC Press, Boca Raton, Fla.
[0132] Hodgkinson, A. (1970) "Determination of Oxalic acid in
Biological Material," Clin. Chem. 16(7):547-557.
[0133] Jensen, N. S., M. J. Allison (1994) "Studies on the
diversity among anaerobic oxalate-degrading bacteria now in the
species Oxalobacter formigenes" Abst. Ann. Mtg. Amer. Soc.
Microbial., pages 1-29.
[0134] Jordan, J. A., M. B. Durso (1996) "Rapid speciation of the
five most medically relevant candida species using PCR
amplification and a microtitre plate-based detection system," Mol
Diagnosis 1:51-58.
[0135] Kleinschmidt K., A. Mahlmann, R. Hautmann (1993) "Anaerobic
oxalate-degrading bacteria in the gut decrease faecal and urinary
oxalate concentrations in stone formers," In R. Ryall, R. Bais, V.
R. Marshall, A. M. Rofe, L. H. Smith, V. R. Walker Urolithiasis 2,
Plenum Press, New York, pp. 439-441.
[0136] Lung, H., A. L. Baetz, A. B. Peck (1994) "Molecular Cloning,
DNA Sequence and Gene Expression of the Oxalyl-CoA Decarboxylase
Gene, oxc, from the Bacterium Oxalobacter formigenes," J.
Bacteriol. 176(8):2468-2472.
[0137] Maniatis, T., E. F. Fritsch, J. Sambrook (1989) Molecular
Cloning: A Laboratory Manual, 2d Edition, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.
[0138] Smith, R. L., F. E. Strohmaier, R. S. Oremland (1985)
"Isolation of anaerobic oxalate-degrading bacteria from fresh water
lake sediments," Arch Microbiol 141:8-13.
[0139] Stacy-Phips, S., J. J. Mecca, J. B. Weiss (1995) "Multiplex
PCR assay and simple preparation method for stool specimens detect
enterotoxigenic E. coli DNA during course of infection," J. Clin.
Microbiol. 33:1054-1059.
[0140] Tarmuzzer, R. W., S. P. Macauley, W. Farmerie, S. Cabellaro,
M. R. Ghassemifar, C. Robinson, M. B. Grant, M. G. Humphreys-Beher,
L. Franzen, A. B. Peck, and G. S. Schultz (1996) "Competitive RNA
templates for detection and quantitation of growth factors,
cytokines, extracellular matrix components and matrix
metalloproteinases by RT-PCR," Biotechnology 20:670-674.
[0141] Yriberri, J., L. S. Posten (1980) "A semi-automatic enzymic
method for estimating urinary oxalate," Clin. Chem. 26(7):881-884.
Sequence CWU 1
1
19 1 1577 DNA Oxalobacter formigenes 1 aagcttgctt cattttgaga
tgttatgcga agtgttagca acccaagtta gtacccttca 60 gccctttggg
cgaagttttt ctttcttggc agttcctttc ggggaaacag cacagagaat 120
aaaaaccaaa agttgtacca acgacaagga aatgagaaat tatgactaaa ccattagatg
180 gaattaatgt gcttgacttt acccacgtcc aggcaggtcc tgcctgtaca
cagatgatgg 240 gtttcttggg cgcaaacgtc atcaagattg aaagacgtgg
ttccggagat atgactcgtg 300 gatggctgca ggacaaacca aatgttgatt
ccctgtattt cacgatgttc aactgtaaca 360 aacgttcgat tgaactggac
atgaaaaccc cggaaggcaa agagcttctg gaacagatga 420 tcaagaaagc
cgacgtcatg gtcgaaaact tcggaccagg cgcactggac cgtatgggct 480
ttacttggga atacattcag gaactgaatc cacgcgtcat tctggcttcc gttaaaggct
540 atgcagaagg ccacgccaac gaacacctga aagtttatga aaacgttgca
cagtgttccg 600 gcggtgctgc agctaccacc ggtttctggg atggtcctcc
aaccgtttcc ggcgctgctc 660 tgggtgactc caactccggt atgcacctga
tgatcggtat tctggccgct ctggaaatgc 720 gtcacaaaac cggccgtggt
cagaaagttg ccgtcgctat gcaggacgct gttctgaatc 780 tggttcgtat
caaactgcgt gaccagcaac gtctggaaag aaccggcatt ctggctgaat 840
acccacaggc tcagcctaac tttgccttcg acagagacgg taacccactg tccttcgaca
900 acatcacttc cgttccacgt ggtggtaacg caggtggcgg cggccagcca
ggctggatgc 960 tgaaatgtaa aggttgggaa accgatgcgg actcctacgt
ttacttcacc atcgctgcaa 1020 acatgtggcc acagatctgc gacatgatcg
acaagccaga atggaaagac gacccagcct 1080 acaacacatt cgaaggtcgt
gttgacaagc tgatggacat cttctccttc atcgaaacca 1140 agttcgctga
caaggacaaa ttcgaagtta ccgaatgggc tgcccagtac ggcattcctt 1200
gcggtccggt catgtccatg aaagaactgg ctcacgatcc ttccctgcag aaagttggta
1260 ccgtcgttga agttgtcgac gaaattcgtg gtaaccacct gaccgttggc
gcaccgttca 1320 aattctccgg attccagccg gaaattaccc gtgctccgct
gttgggcgaa cataccgacg 1380 aagttctgaa agaactgggt cttgacgatg
ccaagatcaa ggaactgcat gcaaaacagg 1440 tagtttgatc cgtcagactt
tctgggcaaa acggcactct ccggagtgcc gttttttgtc 1500 acacgaaacc
taatcaaaca agcacgtgca atgattccac atcattgcgg ccacattcat 1560
ccttcgggtc attactg 1577 2 428 PRT Oxalobacter formigenes 2 Met Thr
Lys Pro Leu Asp Gly Ile Asn Val Leu Asp Phe Thr His Val 1 5 10 15
Gln Ala Gly Pro Ala Cys Thr Gln Met Met Gly Phe Leu Gly Ala Asn 20
25 30 Val Ile Lys Ile Glu Arg Arg Gly Ser Gly Asn Met Thr Arg Gly
Trp 35 40 45 Leu Gln Asp Lys Pro Asn Val Asp Ser Leu Tyr Phe Thr
Met Phe Asn 50 55 60 Cys Asn Lys Arg Ser Ile Glu Leu Asp Met Lys
Thr Pro Glu Gly Lys 65 70 75 80 Glu Leu Leu Glu Gln Met Ile Lys Lys
Ala Asp Val Met Val Glu Asn 85 90 95 Phe Gly Pro Gly Ala Leu Asp
Arg Met Gly Phe Thr Trp Glu Tyr Ile 100 105 110 Gln Glu Leu Asn Pro
Arg Val Ile Leu Ala Ser Val Lys Gly Tyr Ala 115 120 125 Glu Gly His
Ala Asn Glu His Leu Lys Val Tyr Glu Asn Val Ala Gln 130 135 140 Cys
Ser Gly Gly Ala Ala Ala Thr Thr Gly Phe Trp Asp Gly Pro Pro 145 150
155 160 Thr Val Ser Gly Ala Ala Leu Gly Asp Ser Asn Ser Gly Met His
Leu 165 170 175 Met Ile Gly Ile Leu Ala Ala Leu Glu Met Arg His Lys
Thr Gly Arg 180 185 190 Gly Gln Lys Val Ala Val Ala Met Gln Asp Ala
Val Leu Asn Leu Val 195 200 205 Arg Ile Lys Leu Arg Asp Gln Gln Arg
Leu Glu Arg Thr Gly Ile Leu 210 215 220 Ala Glu Tyr Pro Gln Ala Gln
Pro Asn Phe Ala Phe Asp Arg Asp Gly 225 230 235 240 Asn Pro Leu Ser
Phe Asn Asn Ile Thr Ser Val Pro Arg Gly Gly Asn 245 250 255 Ala Gly
Gly Gly Gly Glu Pro Gly Trp Met Leu Lys Cys Lys Gly Trp 260 265 270
Glu Thr Asp Ala Asp Ser Tyr Val Tyr Phe Thr Ile Ala Ala Asn Met 275
280 285 Trp Pro Gln Ile Cys Asn Met Ile Asp Lys Pro Glu Trp Lys Asp
Asp 290 295 300 Pro Ala Tyr Asn Thr Phe Glu Gly Arg Val Asp Lys Leu
Met Asp Ile 305 310 315 320 Phe Ser Phe Ile Glu Thr Lys Phe Ala Asp
Lys Asp Lys Phe Glu Val 325 330 335 Thr Glu Trp Ala Ala Gln Tyr Gly
Ile Pro Cys Gly Pro Val Met Ser 340 345 350 Met Lys Glu Leu Ala His
Asp Pro Ser Leu Gln Lys Val Gly Thr Val 355 360 365 Val Glu Val Val
Asp Glu Ile Arg Gly Asn His Leu Thr Val Gly Ala 370 375 380 Pro Phe
Lys Phe Ser Gly Phe Gln Pro Glu Ile Thr Arg Ala Pro Leu 385 390 395
400 Leu Gly Glu His Thr Asp Glu Val Leu Lys Glu Leu Gly Leu Asp Asp
405 410 415 Ala Lys Ile Lys Glu Leu His Ala Lys Gln Val Val 420 425
3 2088 DNA Oxalobacter formigenes 3 atttgtttaa attgacctga
atcaatattg ccggattgat ctaggtcaat gaatgcaaat 60 tgacttatgt
caatggtgcc aaattgacct aggtcaacgg gatttttaaa gggtatgcgg 120
catactcgga attgacgtta aacaacgttt atcaaaacca accaaagaaa ggtattactc
180 atgagtaacg acgacaatgt agagttgact gatggctttc atgttttgat
cgatgccctg 240 aaaatgaatg acatcgatac catgtatggt gttgtcggca
ttcctatcac gaacctggct 300 cgtatgtggc aagatgacgg tcagcgtttt
tacagcttcc gtcacgaaca acacgcaggt 360 tatgcagctt ctatcgccgg
ttacatcgaa ggaaaacctg gcgtttgctt gaccgtttcc 420 gcccctggct
tcctgaacgg cgtgacttcc ctggctcatg caaccaccaa ctgcttccca 480
atgatcctgt tgagcggttc cagtgaacgt gaaatcgtcg atttccaaga cggcgattac
540 gaagaaatgg atcagatgaa tgttgcacgt ccacactgca aagcttcttt
ccgtatcaac 600 agcatcaaag acattccaat cggtatcgct cgtgcagttc
gcaccgctgt atccggacgt 660 ccaggtggtg tttacgttga cttcccagca
aaactgttcg gtcagaccat ttctgtagaa 720 gaagctaaca aactgctctt
caaaccaatc gatccagctc cggcacagat tcttgctgaa 780 gacgctatcg
ctcgcgctgc tgacctgatc aagaacgcca aacgtccagt tatcatgctg 840
ggtaaaggcg ctgcatacgc acaatgcgac gacgaaatcc gcgcactggt tgaagaaacc
900 ggcatcccat tcctgccaat gggtatggct aaaggcctgc tgcctgacaa
ccatccacaa 960 tccgctgctg caacccgtgc tttcgcactg gcacagtgtg
acgtttgcgt actgatcggc 1020 gctcgtctga actggctgat gcagcacggt
aaaggcaaaa cctggggcga cgaactgaag 1080 aaatacgttc agatcgacat
ccaggctaac gaaatggaca gcaaccagcc tatcgctgca 1140 ccagttgttg
gtgacatcaa gtccgccgtt tccctgctcc gcaaagcact gaaaggcgct 1200
ccaaaagctg acgctgaatg gaccggcgct ctgaaagcca aagttgacgg caacaaagcc
1260 aaactggctg gcaagatgac tgccgaaacc ccatccggaa tgatgaacta
ctccaattcc 1320 ctgggcgttg ttcgtgactt catgctggca aatccggata
tttccctggt taacgaaggc 1380 gctaatgcac tcgacaacac tcgtatgatt
gttgacatgc tgaaaccacg caaacgtctt 1440 gactccggta cctggggtgt
tatgggtatt ggtatgggct actgcgttgc tgcagctgct 1500 gttaccggca
aaccggttat cgctgttgaa ggcgatagcg cattcggttt ctccggtatg 1560
gaactggaaa ccatctgccg ttacaacctg ccagttaccg ttatcatcat gaacaatggt
1620 ggtatctata aaggtaacga agcagatcca caaccaggcg ttatctcctg
tacccgtctg 1680 acccgtggtc gttacgacat gatgatggaa gcatttggcg
gtaaaggtta tgttgccaat 1740 actccagcag aactgaaagc tgctctggaa
gaagctgttg cttccggcaa accatgcctg 1800 atcaacgcga tgatcgatcc
agacgctggt gtcgaatctg gccgtatcaa gagcctgaac 1860 gttgtaagta
aagttggcaa gaaataatta gcccaacttt gatgaccggt tacgaccggt 1920
cacataaagt gttcgaatgc ccttcaagtt tacttgaagg gcattttttt accttgcagt
1980 ttataaacag gaaaaattgt attcagagcg gaaaagcaga tttaagccac
gagaaacatt 2040 cttttttatt gaaaattgcc ataaacacat ttttaaagct
ggcttttt 2088 4 568 PRT Oxalobacter formigenes 4 Met Ser Asn Asp
Asp Asn Val Glu Leu Thr Asp Gly Phe His Val Leu 1 5 10 15 Ile Asp
Ala Leu Lys Met Asn Asp Ile Asp Thr Met Tyr Gly Val Val 20 25 30
Gly Ile Pro Ile Thr Asn Leu Ala Arg Met Trp Gln Asp Asp Gly Gln 35
40 45 Arg Phe Tyr Ser Phe Arg His Glu Gln His Ala Gly Tyr Ala Ala
Ser 50 55 60 Ile Ala Gly Tyr Ile Glu Gly Lys Pro Gly Val Cys Leu
Thr Val Ser 65 70 75 80 Ala Pro Gly Phe Leu Asn Gly Val Thr Ser Leu
Ala His Ala Thr Thr 85 90 95 Asn Cys Phe Pro Met Ile Leu Leu Ser
Gly Ser Ser Glu Arg Glu Ile 100 105 110 Val Asp Leu Gln Gln Gly Asp
Tyr Glu Glu Met Asp Gln Met Asn Val 115 120 125 Ala Arg Pro His Cys
Lys Ala Ser Phe Arg Ile Asn Ser Ile Lys Asp 130 135 140 Ile Pro Ile
Gly Ile Ala Arg Ala Val Arg Thr Ala Val Ser Gly Arg 145 150 155 160
Pro Gly Gly Val Tyr Val Asp Leu Pro Ala Lys Leu Phe Gly Gln Thr 165
170 175 Ile Ser Val Glu Glu Ala Asn Lys Leu Leu Phe Lys Pro Ile Asp
Pro 180 185 190 Ala Pro Ala Gln Ile Pro Ala Glu Asp Ala Ile Ala Arg
Ala Ala Asp 195 200 205 Leu Ile Lys Asn Ala Lys Arg Pro Val Ile Met
Leu Gly Lys Gly Ala 210 215 220 Ala Tyr Ala Gln Cys Asp Asp Glu Ile
Arg Ala Leu Val Glu Glu Thr 225 230 235 240 Gly Ile Pro Phe Leu Pro
Met Gly Met Ala Lys Gly Leu Leu Pro Asp 245 250 255 Asn His Pro Gln
Ser Ala Ala Ala Thr Arg Ala Phe Ala Leu Ala Gln 260 265 270 Cys Asp
Val Cys Val Leu Ile Gly Ala Arg Leu Asn Trp Leu Met Gln 275 280 285
His Gly Lys Gly Lys Thr Trp Gly Asp Glu Leu Lys Lys Tyr Val Gln 290
295 300 Ile Asp Ile Gln Ala Asn Glu Met Asp Ser Asn Gln Pro Ile Ala
Ala 305 310 315 320 Pro Val Val Gly Asp Ile Lys Ser Ala Val Ser Leu
Leu Arg Lys Ala 325 330 335 Leu Lys Gly Ala Pro Lys Ala Asp Ala Glu
Trp Thr Gly Ala Leu Lys 340 345 350 Ala Lys Val Asp Gly Asn Lys Ala
Lys Leu Ala Gly Lys Met Thr Ala 355 360 365 Glu Thr Pro Ser Gly Met
Met Asn Tyr Ser Asn Ser Leu Gly Val Val 370 375 380 Arg Asp Phe Met
Leu Ala Asn Pro Asp Ile Ser Leu Val Asn Glu Gly 385 390 395 400 Ala
Asn Ala Leu Asp Asn Thr Arg Met Ile Val Asp Met Leu Lys Pro 405 410
415 Arg Lys Arg Leu Asp Ser Gly Thr Trp Gly Val Met Gly Ile Gly Met
420 425 430 Gly Tyr Cys Val Ala Ala Ala Ala Val Thr Gly Lys Pro Val
Ile Ala 435 440 445 Val Glu Gly Asp Ser Ala Phe Gly Phe Ser Gly Met
Glu Leu Glu Thr 450 455 460 Ile Cys Arg Tyr Asn Leu Pro Val Thr Val
Ile Ile Met Asn Asn Gly 465 470 475 480 Gly Ile Tyr Lys Gly Asn Glu
Ala Asp Pro Gln Pro Gly Val Ile Ser 485 490 495 Cys Thr Arg Leu Thr
Arg Gly Arg Tyr Asp Met Met Met Glu Ala Phe 500 505 510 Gly Gly Lys
Gly Tyr Val Ala Asn Thr Pro Ala Glu Leu Lys Ala Ala 515 520 525 Leu
Glu Glu Ala Val Ala Ser Gly Lys Pro Cys Leu Ile Asn Ala Met 530 535
540 Ile Asp Pro Asp Ala Gly Val Gly Ser Gly Arg Ile Lys Ser Leu Asn
545 550 555 560 Val Val Ser Lys Val Gly Lys Lys 565 5 17 DNA
Oxalobacter formigenes 5 gagcgatacc gattgga 17 6 17 DNA Oxalobacter
formigenes 6 gcacaatgcg acgacga 17 7 17 DNA Oxalobacter formigenes
7 caggttatgc agcttct 17 8 17 DNA Oxalobacter formigenes 8
ggatggttgt caggcag 17 9 17 DNA Oxalobacter formigenes 9 atactcggaa
ttgacgt 17 10 23 DNA Oxalobacter formigenes 10 ttcatgtcca
gttcaatcga acg 23 11 17 DNA Oxalobacter formigenes 11 gtagttcatc
attccgg 17 12 17 DNA Oxalobacter formigenes 12 aatgtagagt tgactga
17 13 17 DNA Oxalobacter formigenes 13 ttgatgctgt tgatacg 17 14 31
DNA Oxalobacter formigenes 14 gacaatgtag agttgactga tggctttcat g 31
15 18 DNA Oxalobacter formigenes 15 caggatggtc agaagttc 18 16 17
DNA Oxalobacter formigenes 16 ccggttacat cgaagga 17 17 17 DNA
Oxalobacter formigenes 17 tccaatcggt atcgctc 17 18 21 DNA
Oxalobacter formigenes 18 tttacccacg tccaggcagg t 21 19 23 DNA
Oxalobacter formigenes 19 ttcatgtcca gttcaatcga acg 23
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