Previous Article | Next Article 
Journal of Bacteriology, May 1999, p. 3069-3075, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Role of Quinolinate Phosphoribosyl Transferase in
Degradation of Phthalate by Burkholderia cepacia
DBO1
Hung-Kuang
Chang and
Gerben J.
Zylstra*
Biotechnology Center for Agriculture and the
Environment, Cook College, Rutgers University, New Brunswick, New
Jersey 08901-8520
Received 28 January 1999/Accepted 10 March 1999
 |
ABSTRACT |
Two distinct regions of DNA encode the enzymes needed for phthalate
degradation by Burkholderia cepacia DBO1. A gene coding for
an enzyme (quinolinate phosphoribosyl transferase) involved in the
biosynthesis of NAD+ was identified between these two
regions by sequence analysis and functional assays. Southern
hybridization experiments indicate that DBO1 and other
phthalate-degrading B. cepacia strains have two dissimilar
genes for this enzyme, while non-phthalate-degrading B. cepacia strains have only a single gene. The sequenced gene was
labeled ophE, due to the fact that it is specifically
induced by phthalate as shown by lacZ gene fusions.
Insertional knockout mutants lacking ophE grow noticeably
slower on phthalate while exhibiting normal rates of growth on other
substrates. The fact that elevated levels of quinolinate phosphoribosyl
transferase enhance growth on phthalate stems from the structural
similarities between phthalate and quinolinate: phthalate is a
competitive inhibitor of this enzyme and the phthalate catabolic
pathway cometabolizes quinolinate. The recruitment of this gene for
growth on phthalate thus gives B. cepacia an advantage over
other phthalate-degrading bacteria in the environment.
| |
INTRODUCTION |
Phthalate is a ubiquitous compound
in the environment due to its widespread use not only in the
manufacture of plastics and textiles but also as an ingredient in
pesticide, munitions, and cosmetic formulations (22, 47).
There is some concern over the health effects of phthalate-based
compounds, as they have been shown to be both nervous system
depressants and stimulators, teratogenic, and estrogen mimics (3,
6, 17, 26, 63, 66). Although many different types of
microorganisms have been shown to readily degrade phthalate (31,
44, 50) there have not been many studies on the toxicity of
phthalate to microorganisms. Perhaps the best studied
phthalate-degrading organism is Burkholderia cepacia DBO1,
for which extensive work has been performed on the enzymes and genes
involved in the catabolic pathway (2, 10, 31, 48). DBO1
initiates the degradation of phthalate (Fig. 1) through dioxygenase attack to form
4,5-dihydro-4,5-dihydroxyphthalate (cis-phthalate
dihydrodiol). The enzyme responsible for this initial step is a
two-component enzyme consisting of an oxygenase, which actually
catalyzes the addition of oxygen to phthalate, and a reductase, which
shuttles electrons from NADH to the oxygenase component
(2). Phthalate catabolism continues through the action of a dehydrogenase, which restores the aromatic character of the ring,
and a decarboxylase (48), which removes one of the two carboxyl groups. The resulting compound, protocatechuate, then enters
the central aromatic catabolic 
-ketoadipate pathway (72, 73). The DBO1 genes encoding the enzymes for the conversion of
phthalate to protocatechuate have recently been cloned and sequenced
(10). Interestingly, the four genes are organized into three
operons (Fig. 1) with the two genes for the first catabolic enzymes
situated on the two outside ends, approximately 7 kb from each other.
In between two of the operons is a 4-kb stretch of DNA, which is not
needed for the conversion of phthalate to protocatechuate but is
involved in the ability of this strain to grow on phthalate as
described in this report. The gene organization that is found in
B. cepacia DBO1 is quite different from that described for Pseudomonas putida NMH102-2 (43), in which
similar genes for phthalate degradation are present in an operonic
structure.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 1.
Metabolic pathways for the degradation of phthalate and
the synthesis of nicotinic acid mononucleotide from quinolinate and
phosphoribosyl pyrophosphate. A restriction map and diagram of the
cloned and sequenced genes for phthalate degradation (including the
nadC analogue ophE reported here) from B. cepacia DBO1 are shown at the bottom. A, AatII; B,
BamHI; Bc, BclI; E, EcoRI; N,
NotI; S, SalI; Sp, SphI. TCA,
tricarboxylic acid.
|
|
Aromatic dioxygenases often have a broad substrate range, being able to
attack compounds other than the pathway substrate(s). The best-studied
examples of this are toluene and naphthalene dioxygenase (49,
71). Phthalate dioxygenase is able to transform other
dicarboxylated aromatic compounds to oxygenated products. One example
is the transformation of quinolinate (Fig. 1), a dicarboxylated pyridine (42, 59, 60). Since quinolinate is an intermediate in NAD+ synthesis this could be deleterious to the cell
since quinolinate pools could be depleted and possibly deleterious
dihydroxylated intermediates could form. In addition, because of the
similarity in structure between quinolinate and phthalate, quinolinate
phosphoribosyl transferase (EC 2.4.2.19) is competitively inhibited by
phthalate (4), and thus the pathway for NAD+
synthesis is inhibited. Since phthalate is a negatively charged compound it does not readily diffuse into the bacterial cell, and thus
this competitive inhibition is not normally seen in environments where
phthalate is present. However, bacteria utilizing phthalate as a
carbon source would actively be transporting phthalate into the cell
(10), and the combination of effects of phthalate
dioxygenase on quinolinate and phthalate on quinolinate phosphoribosyl
transferase could result in a loss of competitiveness of the bacterium
in the environment. The present work describes one mechanism devised by
B. cepacia DBO1 to overcome this potential difficulty.
| |
MATERIALS AND METHODS |
Bacterial strains, plasmids, media, and growth of strains.
B. cepacia DBO1 (ATCC 29424) is the wild-type strain capable
of utilizing phthalate as the sole carbon and energy source
(30). B. cepacia ATCC 17616 (57) is a
phthalate-degrading strain isolated by other investigators
independently of DBO1. B. cepacia ATCC 17759 (37)
and ATCC 25416 (51), clinical isolates 715j (38), K56-2 (39), and K63-3 (39), and field isolates D1
and M53 (34a) cannot utilize phthalate. Escherichia
coli DH5
[F
80dlacZ
M15
(lacZY-argF)U169 deoR recA1 endA1
hsdR17(rK mK+)
supE44 thi-1 gyrA96 relA1] (Gibco-BRL, Gaithersburg, Md.)
was used as the recipient strain in the cloning experiments. E. coli WC4546 [nadC8 galT23
IN(rrnD-rrnE)1] (62) was obtained
from the E. coli Genetic Stock Center (Yale University, New
Haven, Conn.). The pGEM series of vectors (Promega, Madison, Wis.) and pRK415 (29) were used for subcloning DNA fragments. pUC4K
with a kanamycin resistance gene cassette was obtained from Pharmacia Biotech (Uppsala, Sweden). pARO180 is a mobilizable narrow-host-range plasmid (46). The promoter probe vector pKRZ-1 has a
promoterless lacZ gene cloned into the broad-host-range
vector pUCD615 (52). Plasmid pRK2013 was used as a helper
strain in mating experiments (19).
L broth (
35) was used as the complete medium. Mineral salts
basal medium (MSB) (
57) was used as the minimal medium.
Basal
medium (BM) (
72) was used for growth curves of
B. cepacia DBO1
mutants. Carbon sources were added to the
medium at concentrations
of 20 mM for succinate, 10 mM for
p-hydroxybenzoate, and 10 mM
for phthalate. Ampicillin and
tetracycline were added at concentrations
of 100 µg/ml and 15 µg/ml, respectively, when needed. Nicotinate
(10 µg/ml) was added
to the minimal medium for culturing
E. coli WC4546.
Burkholderia strains were grown at 30°C, and
E. coli strains
were grown at 37°C.
Molecular techniques.
Genomic and plasmid DNA was prepared
by established procedures (5, 45). Restriction digests,
ligations, transformation, gel electrophoresis, DNA extraction from
gels, probe labeling, Southern hybridizations, and automated DNA
sequencing were performed following standard procedures as described
previously (10, 18, 23, 33, 65). The 0.9-kb probe for the
ophE gene was PCR amplified under standard conditions
(Perkin-Elmer, Inc., Foster City, Calif.) from pGJZ1331 with the SP6
sequencing primer (Promega) and the internal primer
5'-GCGAAATACGGTCCAC-3'. A modification of the
electrotransformation method for Pseudomonas (15)
was used to introduce DNA into B. cepacia. Cells (12 ml)
were grown to mid- to late-log phase, harvested by centrifugation at
5,000 × g at room temperature, washed in an equal
volume of 10% glycerol, and resuspended in 160 µl of the same
buffer. DNA (0.2 to 0.5 µg) was added to 40 µl of cells, the
solution was incubated at room temperature for 5 min, and an electrical
pulse was applied. The electroporator (Gene Pulser; Bio-Rad
Laboratories, Rockville Center, N.Y.) was set at 25 µF, 200 
, and
1.25 kV for 0.1-cm gap cuvettes. SOC solution (0.5 ml) (53)
was added immediately, and the cells were incubated at 30°C for
1 h before being plated on a selective medium.
Construction of an ophE knockout mutant.
The
ophE gene was knocked out with a kanamycin resistance
cassette. Initially a 4.5-kb BamHI fragment containing
ophE from pGJZ1301 (10) was cloned into
pGEM7Z-f(
). A 1.3-kb BamHI kanamycin resistance cassette
derived from pUC4K was inserted into the unique BclI site in
ophE. The BamHI fragment (now 5.8 kb) containing the disrupted ophE gene was moved to the mobilizable suicide
vector pARO180. The resulting construct, designated pGJZ1332, was
transferred by triparental mating into DBO1 with selection on a minimal
medium supplemented with succinate, nicotinate, and kanamycin. Southern hybridization of restriction digested genomic DNA was used to identify
clones which had single-crossover (DBO301) or double-crossover (DBO302)
recombination events or had become spontaneously kanamycin resistant
(DBO303) without the insertion of the resistance gene.
Promoter assays.
The PCR procedure for amplifying the region
upstream of the ophE gene involved a step at 94°C for 1 min, 25 repeated cycles of 15 s at 94°C, 30 s at 50°C,
and 4 min at 60°C, a 10-min step at 72°C, and a step at 4°C until
the tube was removed. Primers HK-A2
(5'-ATTCCGTCGACTCGGGAAG-3') and HK-B
(5'-GCTCTAGAATGTTTCGTCGAACC-3') were utilized.
Primer HK-A2 includes a SalI site (underlined), while a new
XbaI site (underlined) was included in primer HK-B. The PCR
product was cleaved with SalI and XbaI and cloned
into pKRZ-1. The cloned region was sequenced to verify that no changes were introduced by the Taq polymerase. LacZ assays were
performed with cells in mid-log phase. Harvested cells were washed
twice with phosphate buffer (50 mM sodium/potassium phosphate buffer, pH 7.25) and resuspended at 0.1 times the original volume. The cells
were sonicated at 475 W at 4°C for 3 min with intermittent pulsing (1 s on and 3 s off).
A modified
-galactosidase assay (
40) was used to measure
LacZ activity in cell-free extracts. The crude cell extract (30
µl)
was added to 370 µl of buffer Z (60 mM
Na
2HPO
4, 40 mM NaH
2PO
4,
10 mM KCl, 1 mM MgSO
4, and 40 mM 2-mercaptoethanol) in a
1.5-ml
microcentrifuge tube.
o-Nitrophenyl-
-
D-galactopyranoside (100
µl
of a 4 mg/ml stock) in buffer Z was added to initiate the enzyme
reaction, and the mixture was incubated at 37°C for 30 min. The
reaction was stopped by adding 500 µl of 1 M sodium carbonate
to the
mixture. Enzyme activity was calculated as nanomoles of
o-nitrophenol formed per minute per milligram of protein.
The
protein concentration was measured by the Bradford procedure
(
7)
with bovine serum albumin as the
standard.
Nucleotide sequence accession number.
The nucleotide
sequence has been deposited in the GenBank database under accession no.
AF095748.
| |
RESULTS |
Identification of a nadC-like gene associated with the
genes for phthalate degradation.
The four genes coding for the
initial steps in phthalate degradation (Fig. 1) are clustered in two
groups approximately 4 kb apart (10). The intervening region
was sequenced, and two open reading frames were identified (Fig. 1).
One of these open reading frames, designated tnp, shows a
high degree of similarity to known transposases (data not shown). The
enzyme encoded by the second open reading frame shows a high degree of
similarity to quinolinate phosphoribosyl transferase (NadC) from
several different sources (Fig. 2). The
question as to why a gene involved in the synthesis of NAD+
would be physically associated with the genes for phthalate degradation is the subject of this investigation. This nadC-like gene
has been designated ophE, due to its physical association
with the oph genes for the degradation of phthalate and its
role in phthalate degradation as described below.
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 2.
Dendrogram showing the phylogenetic relationship of
different genes coding for quinolinate phosphoribosyl transferase
(nadC or ophE). The nucleotide sequences were
aligned with the pileup program of the Genetics Computer Group package
(16), the alignment was confirmed by visual inspection and
comparison with the deduced amino acid sequence alignments, and the
phylogenetic tree was calculated with the PAUP program by using the
minimal distance method. The nucleotide sequences were obtained from
the following sources: Aquifex aeolicus (13),
Archaeoglobus fulgidus (34), Bacillus
subtilis, (58), E. coli (68),
Helicobacter pylori (61), Homo
sapiens, (20), Methanococcus jannaschii
(8), Mycobacterium leprae (unpublished data;
GenBank accession no. U00010), Methanobacterium
thermoautotrophicum (56), Mycobacterium
tuberculosis (12), N. gonorrhoeae
(51a), P. aeruginosa (47a),
Pyrococcus horikoshi (28), Rhodospirillum
rubrum, (55), Saccharomyces cerevisiae
(41), S. typhimurium (24), and
Synechocystis sp. (27).
|
|
In order to prove that the identified
ophE gene encodes a
protein with NadC activity, mutant complementation experiments were
performed. A 1.2-kb
SphI-
AatII fragment
containing the entire
ophE gene was subcloned into
pGEM5Z-f(
). The resulting plasmid,
designated pGJZ1331, was
introduced into the
nadC E. coli strain
WC4546. This strain
requires nicotinate in order to grow on a
minimal medium due to the
block in its NAD
+ synthesis pathway. However,
WC4546(pGJZ1331) is able to grow
on MSB medium containing succinate
without nicotinate, demonstrating
that
ophE is able to
complement the
nadC mutation and thus that
the encoded
enzyme has quinolinate phosphoribosyl transferase
activity. Control
experiments with WC4546[pGEM5Z-f(
)] showed
no growth on the minimal
medium without
nicotinate.
Two dissimilar copies of nadC-like genes.
The
nadC genes of Salmonella typhimurium and E. coli are not physically linked to other genes for the de novo
synthesis of NAD+ (25, 68). The
ophE/nadC gene identified here is also located in
an isolated area not linked to other genes involved in NAD+
synthesis. It is possible that ophE is really
nadC and that its presence near the genes for phthalate
degradation is a mere coincidence or that there are two
nadC-like genes, one associated with the genes for phthalate
degradation and the other located somewhere else. These alternative
hypotheses were first tested by performing Southern hybridizations with
portions of the ophE gene as probes. Initially, a 0.4-kb
SphI-HindIII fragment from pGJZ1331
containing a portion of the ophE gene coding for the first
116 amino acids of OphE was used as a probe against B. cepacia DBO1 genomic DNA digested separately with either
EcoRI, BamHI, NotI, or
SphI. The results (not shown) indicate that a single
restriction fragment hybridized in each case: 9.2 kb for
EcoRI, 4.5 kb for BamHI, 3.8 kb for
NotI, and 3.3 kb for SphI.
This result suggested that a single
ophE/
nadC
gene exists in
B. cepacia DBO1. However, a detailed analysis
of the amino acid
sequences of all NadC enzymes in the GenBank database
indicates
that the C-terminal half of the protein is more conserved
across
genus and species lines, while the N-terminal half of the
protein
shows more evolutionary divergence. This being the case, a
second
probe was prepared by PCR in order to include most of the
ophE gene. A 0.9-kb fragment of DNA was amplified from
pGJZ1331 with
primers hybridizing to the SP6 promoter region of the
vector and
to a position near the end of the
ophE gene (see
Materials and
Methods). A Southern blot with this fragment as a probe
against
genomic DNA digested with either
BamHI,
EcoRI,
PstI, or
XhoI is
shown in Fig.
3. In every case one strongly hybridizing
band and
one weakly hybridizing band can be seen. The size of the
strongly
hybridizing band corresponds to that predicted for the
ophE gene.
The weakly hybridizing band is most likely the
nadC gene of
B. cepacia DBO1. The fact that the
bands are not of equal intensity
suggests that the
ophE gene
was not recruited by a simple gene
duplication event but actually was
obtained from an evolutionarily
distinct source. In order to
investigate this further Southern
hybridizations were carried out with
BamHI-digested genomic DNA
prepared from phthalate-degrading
and non-phthalate-degrading
B. cepacia strains and the
0.9-kb
ophE gene probe. In every case
(Fig.
3) genomic DNA
from non-phthalate-degrading
B. cepacia strains
showed a
single faintly hybridizing fragment, while phthalate-degrading
B. cepacia DBO1 and ATCC 17616 showed two disparately hybridizing
bands. The
ophE gene is thus only associated with
phthalate-degrading
B. cepacia strains and may be somehow
related to the ability to
metabolize phthalate.
View larger version (88K):
[in this window]
[in a new window]
|
FIG. 3.
Southern hybridization of total genomic DNA from
B. cepacia DBO1 (A) and from different B. cepacia
strains (B) with the 0.9-kb PCR-amplified ophE gene probe
from DBO1. (A) Total DNA from B. cepacia was digested with
BamHI (lane 1), EcoRI (lane 2), PstI
(lane 3), and XhoI (lane 4). The migration distances of the
size standards are indicated on the left. (B) Total DNA from different
B. cepacia strains was digested with BamHI. Lanes
1 through 9 contain DNA from strains DBO1, 382, D1, M53, ATCC 25416, ATCC 17616, k56-2, k63-3, and 715j, respectively. Only DBO1 and ATCC
17616 are phthalate degraders. The deduced size of each band is
indicated.
|
|
OphE enhances growth on phthalate.
In order to verify that
there are two genes (ophE and nadC) in B. cepacia DBO1 coding for quinolinate phosphoribosyl transferase, gene knockout experiments were performed. A mutant strain, designated DBO302, which has a kanamycin resistance cassette inserted into the
BclI site of the ophE gene, was constructed by
double reciprocal recombination as described in Materials and Methods.
DBO302 is able to grow on MSB medium containing succinate, indicating
that a lack of ophE does not impair the ability of the
strain to synthesize NAD+. This verifies that there must be
a functional nadC gene somewhere else in the genome.
Additionally, DBO302 is still able to grow on MSB medium containing
phthalate, indicating that ophE is not absolutely required
for the degradation of phthalate. However, the close physical proximity
of ophE to the genes coding for the enzymes involved in
phthalate degradation led us to believe that it must play some role in
the metabolism of phthalate. This being the case, growth rates of the
ophE knockout mutant DBO302 and the spontaneously
kanamycin-resistant mutant DBO303 were compared on various substrates
(Fig. 4). DBO302 and DBO303 have the same growth characteristics when cultured in BM broth with either succinate or p-hydroxybenzoate as the sole carbon source. The doubling
time in all of these cases was approximately 1.1 h. This indicates that the loss of the ophE gene has no discernible effect on
central metabolism (succinate-grown cells) or on aromatic metabolism
(p-hydroxybenzoate-grown cells). However, DBO302 and
DBO303 show much different growth patterns when cultured in BM
broth with phthalate as the sole carbon source. In this case DBO303 has
a doubling time of 1.4 h, while DBO302 grows at less than half of
this rate with a doubling time of 3.1 h. In order to test whether
this deficiency of growth rate is due to the missing ophE
gene, mutant complementation experiments were performed. The
ophE gene was cloned into a broad-host-range vector by first
subcloning a 2.4-kb XhoI fragment containing the ophE gene into pGEM11Z-f(), then by utilizing restriction
sites that cleave in the vector, a BamHI to EcoRI
fragment was transferred into pRK415, and the resulting clone was
designated pGJZ1333. The growth of DBO302 carrying either pRK415 or
pGJZ1333 was compared on BM broth containing phthalate (Fig.
5). The cloned ophE gene restored the ability of DBO302 to grow on phthalate, increasing the
growth rate above wild-type levels. In fact, the wild-type DBO1 strain
grew slightly faster on phthalate when it carried the cloned
ophE gene (pGJZ1333) than when it carried the vector alone.
These experiments clearly demonstrate that OphE enhances the ability of
DBO1 to grow on phthalate while not being required for the actual
metabolism of phthalate.
View larger version (9K):
[in this window]
[in a new window]
|
FIG. 4.
Growth of DBO302 (ophE knockout) ( ) and
DBO303 (spontaneously kanamycin-resistant mutant of DBO1) ( ) in a
minimal medium with succinate (A), p-hydroxybenzoate (B), or
phthalate (C) as the sole carbon source. OD600, optical
density at 600 nm.
|
|
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 5.
Growth of wild-type DBO1 (circles) and the
ophE knockout mutant DBO302 (squares) in a minimal medium
containing phthalate with either the vector (pRK415) (black) or the
cloned ophE gene (pGJZ1333) (white). Growth is slower than
that shown in Fig. 4 due to the incorporation of tetracycline in the
medium.
|
|
ophE is induced by phthalate.
If ophE
is indeed involved in phthalate degradation by B. cepacia
DBO1 then it stands to reason that the gene should be induced when the
strain is grown in the presence of phthalate. The putative promoter
region (384 bases from within orf2 to within
ophE) was amplified by PCR with primers HK-A2 and HK-B and
cloned into the lacZ promoter-probe plasmid pKRZ-1. The
resulting plasmid, designated pGJZ1334, was introduced into B. cepacia DBO1 by electroporation. -Galactosidase activity was
measured in crude cell extracts following growth in the presence of
various compounds (Fig. 6).
DBO1(pGJZ1334) had minimal LacZ activity when grown in MSB broth with
succinate (<50 nmol/min/mg). However, growth on phthalate as the sole
carbon source resulted in a >16-fold increase in LacZ activity (~800 nmol/min/mg). In order to verify that this increase in LacZ activity is
the result of induction of the ophE gene by phthalate and
not just a generalized stress response due to the inhibition of
quinolinate phosphoribosyl transferase (OphE or NadC) a second
experiment was performed. DBO1(pGJZ1334) was grown on MSB broth
with succinate and 20 mM fructose 1,6-bisphosphate, a known inhibitor
of NadC. In this case no increase of LacZ activity above basal levels
was detected. These experiments clearly indicate that the
ophE gene is induced by phthalate and thus is specific for
growth in the presence of phthalate.
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 6.
Induction of ophE by phthalate. Comparison of
the -galactosidase activities produced by B. cepacia DBO1
containing the promoter-probe construct pGJZ1334 following growth on
succinate, phthalate, or succinate plus fructose 1,6-bisphosphate. LacZ
activity is reported in nanomoles per minute per milligram of protein
in crude cell extracts. The standard deviation (error bar) is the
average of three independent assays.
|
|
| |
DISCUSSION |
Aromatic hydrocarbons are widely found in the environment. It is
not surprising therefore that microorganisms have evolved mechanisms to
utilize these compounds as carbon and energy sources for growth. In
fact, microorganisms are constantly evolving such abilities, and much
literature has been devoted not only to an analysis of the catabolic
pathways but also to the mechanisms by which microorganisms evolve such
capabilities (64). Catabolic genes are often present as
operonic segments that can be recruited or recombined to make metabolic
pathways for new substrates. However, only a few mechanisms have been
reported by which microorganisms overcome the potential toxic effects
of the compounds they are metabolizing. The best known of these
mechanisms is perhaps that of solvent resistance, protecting the cell
against the deleterious effects of hydrophobic solvents on the cell
membrane (32, 67). In the case of growth on phthalate the
cell is exposed to two potentially deleterious effects that act on the
same step in NAD+ synthesis: the conversion of quinolinate
and phosphoribosyl pyrophosphate to nicotinic acid mononucleotide by
quinolinate phosphoribosyl transferase. Growth on phthalate results in
high levels of phthalate dioxygenase that not only has the ability to
oxidize phthalate to a cis-dihydrodiol (Fig. 1) but also is
able to convert quinolinate to oxidized products. This effectively
depletes the intracellular pool of quinolinate and decreases the rate
of synthesis of NAD+. Growth on phthalate also results in
high intracellular levels of phthalate due to the transport of this
negatively charged molecule into the cell (10). Since
phthalate is a known competitive inhibitor of quinolinate
phosphoribosyl transferase (4) this also results in a
negative effect on the same step in NAD+ synthesis.
B. cepacia DBO1 has overcome this potentially deleterious effect of growth on phthalate by recruiting a gene for quinolinate phosphoribosyl transferase and placing it not only under the inducible control of phthalate but also moving it within the cluster of genes
responsible for phthalate degradation. To our knowledge, this is the
first example of the recruitment of a gene for a biosynthetic activity
into a catabolic operon to overcome the possible deleterious effects of
a growth substrate on the cell. The fact that the phthalate-inducible ophE-encoded quinolinate phosphoribosyl transferase confers
an advantage on the cell is evident by the data presented in Fig. 5.
Insertional knockout mutants lacking this gene grow slower on phthalate
than does the wild type. The lack of ophE has no discernable
effect on the growth of DBO1 on other substrates such as succinate or
the related aromatic compound p-hydroxybenzoate.
B. cepacia is one of the more versatile pseudomonads in
terms of its ability to utilize a wide variety of carbon sources found in the environment (57). Part of this ability is due to the large size of its genome (11, 36), but the species' ability to recruit and reorganize DNA also plays a role. For instance, certain
insertion sequences in B. cepacia are known to activate the
expression of foreign genes under selective pressure (9, 21, 54,
69, 70). In these cases gene expression is often constitutive,
driven from a promoter in the insertion sequence. Interestingly, an
insertion sequence is adjacent to the ophE gene in DBO1
(Fig. 1). This insertion sequence may have been involved in the
recruitment of ophE and its association with the
ophA1A2BCD genes for phthalate degradation. However,
ophE is specifically inducible by phthalate and thus has
evolved its own promoter, contrary to other insertion
sequence-recruited genes in B. cepacia where expression is
constitutive. It is interesting to note that the ophA1A2BCD
structural genes for phthalate degradation are arranged into three
operons, on both sides of ophE. ophD codes for a
frame-shifted nonfunctional phthalate permease (10), while the real phthalate transporter is encoded elsewhere on the DBO1 genome
(14). This means that there are at least five
phthalate-inducible operons in B. cepacia DBO1
[ophA1, ophDC, ophE,
ophA2B, and the transport gene(s)]. This gene organization
is not unique to DBO1, as B. cepacia 249, another phthalate
degrader, has a restriction fragment length polymorphism pattern
identical to that of DBO1 for this locus. This is contrary to that
found in P. putida NMH102-2, in which the genes for
phthalate degradation are adjacent to one another and are transcribed
in the same direction (43). No ophE analogue has
been found clustered with the genes for phthalate degradation in
P. putida.
A comparative analysis of ophE and other quinolinate
phosphoribosyl transferase genes (Fig. 2) shows that it is most similar to those from Pseudomonas aeruginosa (59.9% identity with
zero gaps), S. typhimurium (55.0% identity with two gaps),
E. coli (53.0% identity with two gaps), and Neisseria
gonorrhoeae (51.0% identity with three gaps). The G+C content of
ophE (63%) is less than the 68% reported for the B. cepacia species (1) and the 67% reported for the genes
for protocatechuate dioxygenase (73) from DBO1. However, it
is in line with the 62 to 63% G+C content of the other oph
genes. This suggests that ophE and the other oph
genes were recruited from outside of the B. cepacia species. This is backed up by the fact that Southern hybridizations (Fig. 3)
with ophE show a strongly hybridizing band (for itself) and a weakly hybridizing band. The latter is presumably due to the hybridization to the housekeeping gene nadC. The fact that
there is not a strongly hybridizing second band suggests that
ophE was not recruited by a duplication of existing
nadC followed by evolution to become phthalate inducible.
The similarity of ophE to nadC genes from related
gram-negative bacteria indicates that the recruitment was not from an
evolutionarily distant species.
| |
ACKNOWLEDGMENTS |
This work was supported by cooperative agreement CR822634 from
the U.S. Environmental Protection Agency Gulf Breeze Environmental Research Laboratory and a National Science Foundation Young
Investigator Award to G.J.Z.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biotechnology
Center for Agriculture and the Environment, Foran Hall, 59 Dudley Rd., Cook College, Rutgers University, New Brunswick, NJ 08901-8520. Phone:
(732) 932-8165, ext. 320. Fax: (732) 932-0312. E-mail: zylstra{at}aesop.rutgers.edu.
| |
REFERENCES |
| 1.
|
Ballard, R. W.,
N. J. Palleroni,
M. Doudoroff,
R. Y. Stainer, and M. Mandel.
1970.
Taxonomy of the aerobic pseudomonads: P. cepacia, P. marginata, P. alliicola and P. caryophylli.
J. Gen. Microbiol.
60:199-214[Abstract/Free Full Text].
|
| 2.
|
Batie, C. J.,
E. LaHaie, and D. P. Ballou.
1987.
Purification and characterization of phthalate oxygenase and phthalate oxygenase reductase from Pseudomonas cepacia.
J. Biol. Chem.
262:1510-1518[Abstract/Free Full Text].
|
| 3.
|
Beliles, R.,
J. A. Salinas, and W. M. Kluwe.
1989.
A review of di(2-ethylhexyl)phthalate (DEHP) risk assessments. Drug archives of environmental contamination and toxicology.
N.Y. Metab. Rev.
21:3-12.
|
| 4.
|
Bhatia, R., and K. C. Calvo.
1996.
The sequencing, expression, purification, and steady-state kinetic analysis of quinolinate phosphoribosyl transferase from Escherichia coli.
Arch. Biochem. Biophys.
325:270-278[Medline].
|
| 5.
|
Birnboim, H. C., and J. Doly.
1979.
A rapid alkaline extraction procedure for screening recombinant plasmid DNA.
Nucleic Acids Res.
7:1513-1523[Abstract/Free Full Text].
|
| 6.
|
Blom, A.,
E. Ekman,
A. Johannisson,
L. Norrgren, and M. Pesonen.
1998.
Effects of xenoestrogenic environmental pollutants on the proliferation of a human breast cancer cell line (MCF-7).
Arch. Environ. Contam. Toxicol.
34:306-310[Medline].
|
| 7.
|
Bradford, M. M.
1976.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[Medline].
|
| 8.
|
Bult, C. J.,
O. White,
G. J. Olsen,
L. Zhou,
R. D. Fleischmann,
G. G. Sutton,
J. A. Blake,
L. M. FitzGerald,
R. A. Clayton,
J. D. Gocayne,
A. R. Kerlavage,
B. A. Dougherty,
J.-F. Tomb,
M. D. Adams,
C. I. Reich,
R. Overbeek,
E. F. Kirkness,
K. G. Weinstock,
J. M. Merrick,
A. Glodek,
J. L. Scott,
N. S. M. Geoghagen,
J. F. Weidman,
J. L. Fuhrmann,
E. A. Presley,
D. Nguyen,
T. R. Utterback,
J. M. Kelley,
J. D. Peterson,
P. W. Sadow,
M. C. Hanna,
M. D. Cotton,
M. A. Hurst,
K. M. Roberts,
B. P. Kaine,
M. Borodovsky,
H.-P. Klenk,
C. M. Fraser,
H. O. Smith,
C. R. Woese, and J. C. Venter.
1996.
Complete genome sequence of the methanogenic archaeon Methanococcus jannaschii.
Science
273:1058-1073[Abstract].
|
| 9.
|
Byrne, A. M., and T. G. Lessie.
1994.
Characteristics of IS401, a new member of the IS3 family implicated in plasmid rearrangements in Pseudomonas cepacia.
Plasmid
31:138-147[Medline].
|
| 10.
|
Chang, H.-K., and G. J. Zylstra.
1999.
Novel organization of the genes for phthalate degradation from Burkholderia cepacia DBO1.
J. Bacteriol.
180:6529-6537[Abstract/Free Full Text].
|
| 11.
|
Cheng, H. P., and T. G. Lessie.
1994.
Multiple replicons constituting the genome of Pseudomonas cepacia 17616.
J. Bacteriol.
176:4034-4042[Abstract/Free Full Text].
|
| 12.
|
Cole, S. T.,
R. Brosch,
J. Parkhill,
T. Garnier,
C. Churcher,
D. Harris,
S. V. Gordon,
K. Eiglmeier,
S. Gas,
C. E. Barry III,
F. Tekaia,
K. Badcock,
D. Basham,
D. Brown,
T. Chillingworth,
R. Connor,
R. Davies,
K. Devlin,
T. Feltwell,
S. Gentles,
N. Hamlin,
S. Holroyd,
T. Hornsby,
K. Jagels,
B. G. Barrell, et al.
1998.
Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.
Nature
393:537-44[Medline].
|
| 13.
|
Deckert, G.,
P. V. Warren,
T. Gaasterland,
W. G. Young,
A. L. Lenox,
D. E. Graham,
R. Overbeek,
M. A. Snead,
M. Keller,
M. Aujay,
R. Huber,
R. A. Feldman,
J. M. Short,
G. J. Olson, and R. V. Swanson.
1998.
The complete genome of the hyperthermophilic bacterium Aquifex aeolicus.
Nature
392:353-358[Medline].
|
| 14.
| Dennis, J. J., H.-K. Chang, and G. J. Zylstra. Unpublished data.
|
| 15.
|
Dennis, J. J., and P. A. Sokol.
1995.
Electrotransformation of Pseudomonas, p. 125-133.
In
J. A. Nickoloff (ed.), Methods in molecular biology, vol. 47. Electroporation protocols for microorganisms. Humana Press Inc., Totowa, N.J.
|
| 16.
|
Devereux, J.,
P. Haeberli, and O. Smithies.
1984.
A comprehensive set of sequence analysis programs for the VAX.
Nucleic Acids Res.
12:387-395.
|
| 17.
|
Ema, M.,
E. Miyawaki, and K. Kawashima.
1998.
Reproductive effects of butyl benzyl phthalate in pregnant and pseudopregnant rats.
Reprod. Toxicol.
12:127-132[Medline].
|
| 18.
|
Feinberg, A. P., and B. Vogelstein.
1983.
A technique for radiolabeling DNA restriction endonuclease fragments to specific activity.
Anal. Biochem.
132:6-13[Medline].
|
| 19.
|
Figurski, D. H., and D. R. Helinski.
1979.
Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans.
Proc. Natl. Acad. Sci. USA
76:1648-1652[Abstract/Free Full Text].
|
| 20.
|
Fukuoka, S. I.,
C. M. Nyaruhucha, and K. Shibata.
1998.
Characterization and functional expression of the cDNA encoding human brain quinolinate phosphoribosyltransferase.
Biochim. Biophys. Acta
1395:192-201[Medline].
|
| 21.
|
Gaffney, T. D., and T. G. Lessie.
1987.
Insertion-sequence-dependent rearrangements of Pseudomonas cepacia plasmid pTGL1.
J. Bacteriol.
169:224-230[Abstract/Free Full Text].
|
| 22.
|
Graham, P. R.
1973.
Phthalate ester plasticizers why and how they are used.
Environ. Health Perspect.
3:3-12[Medline].
|
| 23.
|
Hanahan, D.
1983.
Studies on transformation of E. coli with plasmids.
J. Mol. Biol.
166:557-580[Medline].
|
| 24.
|
Hughes, K. T.,
A. Dessen,
J. P. Gray, and C. Grubmeyer.
1993.
The Salmonella typhimurium nadC gene: sequence determination by use of Mud-P22 and purification of quinolinate phosphoribosyl transferase.
J. Bacteriol.
175:479-486[Abstract/Free Full Text].
|
| 25.
|
Hughes, K. T.,
J. R. Roth, and B. M. Olivera.
1991.
A genetic characterization of the nadC gene of Salmonella typhimurium.
Genetics
127:657-670[Abstract].
|
| 26.
|
Kamrin, M. A., and G. H. Mayor.
1991.
Diethyl phthalate: a perspective.
J. Clin. Pharmacol.
31:484-489[Medline].
|
| 27.
|
Kaneko, T.,
S. Sato,
H. Kotani,
A. Tanaka,
E. Asamizu,
Y. Nakamura,
N. Miyajima,
M. Hirosawa,
M. Sugiura,
S. Sasamoto,
T. Kimura,
T. Hosouchi,
A. Matsuno,
A. Muraki,
N. Nakazaki,
K. Naruo,
S. Okumura,
S. Shimpo,
C. Takeuchi,
T. Wada,
A. Watanabe,
M. Yamada,
M. Yasuda, and S. Tabata.
1996.
Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions.
DNA Res.
3:109-136[Abstract].
|
| 28.
|
Kawarabayasi, Y.,
M. Sawada,
H. Horikawa,
Y. Haikawa,
Y. Hino,
S. Yamamoto,
M. Sekine,
S. Baba,
H. Kosugi,
A. Hosoyama,
Y. Nagai,
M. Sakai,
K. Ogura,
R. Otsuka,
H. Nakazawa,
M. Takamiya,
Y. Ohfuku,
T. Funahashi,
T. Tanaka,
Y. Kudoh,
J. Yamazaki,
N. Kushida,
A. Oguchi,
K. Aoki, and H. Kikuchi.
1998.
Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3.
DNA Res.
5:55-76[Abstract].
|
| 29.
|
Keen, N. T.,
S. Tamaki,
D. Kobayashi, and D. Trollinger.
1988.
Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria.
Gene
70:191-197[Medline].
|
| 30.
|
Keyser, P.
1974.
Aerobic metabolism of the phthalates by selected pseudomonads. M.S. dissertation.
University of Miami, Miami, Fla.
|
| 31.
|
Keyser, P.,
R. W. Pujar,
R. W. Eaton, and D. W. Ribbons.
1976.
Biodegradation of phthalates and their esters by bacteria.
Environ. Health Perspect.
18:159-166[Medline].
|
| 32.
|
Kieboom, J.,
J. J. Dennis,
J. A. de Bont, and G. J. Zylstra.
1998.
Identification and molecular characterization of an efflux pump involved in Pseudomonas putida S12 solvent tolerance.
J. Biol. Chem.
273:85-91[Abstract/Free Full Text].
|
| 33.
|
Kim, E., and G. J. Zylstra.
1995.
Molecular and biochemical characterization of two meta-cleavage dioxygenases involved in biphenyl and m-xylene degradation by Beijerinckia sp. strain B1.
J. Bacteriol.
177:3095-3103[Abstract/Free Full Text].
|
| 34.
|
Klenk, H. P.,
R. A. Clayton,
J. Tomb,
O. White,
K. E. Nelson,
K. A. Ketchum,
R. J. Dodson,
M. Gwinn,
E. K. Hickey,
J. D. Peterson,
D. L. Richardson,
A. R. Kerlavage,
D. E. Graham,
N. C. Kyrpides,
R. D. Fleischmann,
J. Quackenbush,
N. H. Lee,
G. G. Sutton,
S. Gill,
E. F. Kirkness,
B. A. Dougherty,
K. McKenney,
M. D. Adams,
B. Loftus,
S. Peterson,
C. I. Reich,
L. K. McNeil,
J. H. Badger,
A. Glodek,
L. Zhou,
R. Overbeek,
J. D. Gocayne,
J. F. Weidman,
L. McDonald,
T. Utterback,
M. D. Cotton,
T. Spriggs,
P. Artiach,
B. P. Kaine,
S. M. Sykes,
P. W. Sadow,
K. P. D'Andrea,
C. Bowman,
C. Fujii,
S. A. Garland,
T. M. Mason,
G. J. Olsen,
C. M. Fraser,
H. O. Smith,
C. R. Woese, and J. C. Venter.
1997.
The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.
Nature
390:364-370[Medline].
|
| 34a.
| Kobayashi, D. Personal communication.
|
| 35.
|
Lennox, E. S.
1955.
Transduction of linked genetic characters of the host by bacteriophage P1.
Virology
1:190-206[Medline].
|
| 36.
|
Lessie, T. G.,
W. Hendrickson,
B. D. Manning, and R. Devereux.
1996.
Genomic complexity and plasticity of Burkholderia cepacia.
FEMS Microbiol. Lett.
144:117-128[Medline].
|
| 37.
|
McKenney, D.,
K. E. Brown, and D. G. Allison.
1995.
Influence of Pseudomonas aeruginosa exoproducts on virulence factor production in Burkholderia cepacia: evidence of interspecies communication.
J. Bacteriol.
177:6989-6992[Abstract/Free Full Text].
|
| 38.
|
McKevitt, A. I.,
S. Bajaksouzian,
J. D. Klinger, and D. E. Woods.
1989.
Purification and characterization of an extracellular protease from Pseudomonas cepacia.
Infect. Immun.
41:1099-1104.
|
| 39.
|
McKevitt, A. I.,
M. D. Retzer, and D. E. Woods.
1987.
Development and use of a serotyping scheme for Pseudomonas cepacia.
Serodiagn. Immunother.
1:177-184.
|
| 40.
|
Miller, J. H.
1972.
Experiment 48: assay of -galactosidase, p. 352-355.
In
Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 41.
|
Murakami, Y.,
M. Naitou,
H. Hagiwara,
T. Shibata,
M. Ozawa,
S.-I. Sasanuma,
M. Sasanuma,
Y. Tsuchiya,
E. Soeda,
K. Yokoyama,
M. Yamazaki,
H. Tashiro, and T. Eki.
1995.
Analysis of the nucleotide sequence of chromosome VI from Saccharomyces cerevisiae.
Nat. Genet.
10:261-268[Medline].
|
| 42.
|
Nomura, Y.,
S. Harashima, and Y. Oshima.
1989.
A simple method for detection of enzyme activities involved in the initial step of phthalate degradation in microorganisms.
J. Ferment. Bioeng.
67:291-296.
|
| 43.
|
Nomura, Y.,
M. Nakagawa,
N. Ogawa,
S. Harashima, and Y. Oshima.
1992.
Genes in PHT plasmid encoding the initial degradation pathway of phthalate in Pseudomonas putida.
J. Ferment. Bioeng.
74:333-344.
|
| 44.
|
Nomura, Y.,
N. Takada, and Y. Oshima.
1989.
Isolation and identification of phthalate-utilizing bacteria.
J. Ferment. Bioeng.
67:297-299.
|
| 45.
|
Olsen, R. H.,
G. DeBusscher, and W. R. McCombie.
1982.
Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome.
J. Bacteriol.
150:60-69[Abstract/Free Full Text].
|
| 46.
|
Parke, D.
1990.
Construction of mobilizable vectors derived from plasmids RP4, pUC18, and pUC19.
Gene
93:135-137[Medline].
|
| 47.
|
Peakall, D. B.
1975.
Phthalate esters: occurrence and biological effects.
Residue Rev.
54:1-41[Medline].
|
| 47a.
| Pseudomonas Genome Project. 15 March
1999, revision date. [Online.] http://www.pseudomonas.com. [5 April
1999, last date accessed.]
|
| 48.
|
Pujar, B. G., and D. W. Ribbons.
1985.
Phthalate metabolism in Pseudomonas fluorescens PHK: purification and properties of 4,5-dihydroxyphthalate decarboxylase.
Appl. Environ. Microbiol.
49:374-376[Abstract/Free Full Text].
|
| 49.
|
Resnick, S. M.,
K. Lee, and D. T. Gibson.
1996.
Diverse reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816.
J. Ind. Microbiol. Biotechnol.
17:438-457.
|
| 50.
|
Ribbons, D. W.,
P. Keyser,
D. A. Kunz,
B. F. Taylor,
R. W. Eaton, and B. N. Anderson.
1984.
Microbial degradation of phthalate, p. 371-397.
In
D. T. Gibson (ed.), Microbial degradation of organic compounds. Marcel Dekker, Inc., New York, N.Y.
|
| 51.
|
Rodley, P. D.,
U. Romling, and B. Tummler.
1995.
A physical genome map of the Burkholderia cepacia type strain.
Mol. Microbiol.
17:57-67[Medline].
|
| 51a.
| Roe, B. A., S. P. Lin, L. Song, X. Yuan, S. Clifton, T. Ducey, L. Lewis, and D. W. Dyer. 3 April 1999, revision date.
Gonococcal genome sequencing project. University of Oklahoma.
[Online.] http://www.genome.ou.edu/gono.html. [5 April 1999, last date accessed.]
|
| 52.
|
Rothmel, R. K.,
D. L. Shinabarger,
M. R. Parsek,
T. L. Aldrich, and A. M. Chakrabarty.
1991.
Functional analysis of the Pseudomonas putida regulatory protein CatR: transcriptional studies and determination of the CatR DNA-binding site by hydroxyl-radical footprinting.
J. Bacteriol.
173:4717-4724[Abstract/Free Full Text].
|
| 53.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 54.
|
Scordilis, G. E.,
H. Ree, and T. G. Lessie.
1987.
Identification of transposable elements which activate gene expression in Pseudomonas cepacia.
J. Bacteriol.
169:8-13[Abstract/Free Full Text].
|
| 55.
|
Shelver, D.,
R. L. Kerby,
Y. He, and G. P. Roberts.
1995.
Carbon monoxide-induced activation of gene expression in Rhodospirillum rubrum requires the product of cooA, a member of the cyclic AMP receptor protein family of transcriptional regulators.
J. Bacteriol.
177:2157-2163[Abstract/Free Full Text].
|
| 56.
|
Smith, D. R.,
L. A. Doucette-Stamm,
C. Deloughery,
H. Lee,
J. Dubois,
T. Aldredge,
R. Bashirzadeh,
D. Blakely,
R. Cook,
K. Gilbert,
D. Harrison,
L. Hoang,
P. Keagle,
W. Lumm,
B. Pothier,
D. Qiu,
R. Spadafora,
R. Vicaire,
Y. Wang,
J. Wierzbowski,
R. Gibson,
N. Jiwani,
A. Caruso,
D. Bush,
H. Safer,
D. Patwell,
S. Prabhakar,
S. McDougall,
G. Shimer,
A. Goyal,
S. Pietrovski,
G. M. Church,
C. J. Daniels,
J.-I. Mao,
P. Rice,
J. Nölling, and J. N. Reeve.
1997.
Complete genome sequence of Methanobacterium thermoautotrophicum H: functional analysis and comparative genomics.
J. Bacteriol.
179:7135-7155[Abstract/Free Full Text].
|
| 57.
|
Stanier, R. Y.,
N. J. Palleroni, and M. Duodoroff.
1966.
The aerobic pseudomonads: a taxonomic study.
J. Gen. Microbiol.
43:159-271[Abstract/Free Full Text].
|
| 58.
|
Sun, D., and P. Setlow.
1993.
Cloning, nucleotide sequence, and regulation of the Bacillus subtilis nadB gene and a nifS-like gene, both of which are essential for NAD biosynthesis.
J. Bacteriol.
175:1423-1432[Abstract/Free Full Text].
|
| 59.
|
Taylor, B. F., and J. A. Amador.
1988.
Metabolism of pyridine compounds by phthalate-degrading bacteria.
Appl. Environ. Microbiol.
54:2342-2344[Abstract/Free Full Text].
|
| 60.
|
Taylor, B. F., and C. A. King.
1987.
Phthalic acid and pyridine dicarboxylic acids as catabolic analogs.
FEMS Microbiol. Lett.
44:401-405.
|
| 61.
| Tomb, J.-F., O. White, A. R. Kerlavage,
R. A. Clayton, G. G. Sutton, R. D. Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson,
J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus,
D. Richardson, R. Dodson, H. G. Khalak, A. Glodek, K. McKenney,
L. M. Fitzegerald, N. Lee, M. D. Adams, E. K. Hickey,
D. E. Berg, J. D. Gocayne, T. R. Utterback, J. D. Peterson, J. M. Kelley, M. D. Cotton, J. M. Weidman, C. Fujii, C. Bowman, L. Watthey, E. Wallin, W. S. Hayes, M. Borodovsky, P. D. Karp, H. O. Smith, C. M. Fraser, and
J. C. Venter. The complete genome sequence of the gastric
pathogen Helicobacter pylori. Nature
388:539-547.
|
| 62.
|
Tritz, G. J.,
T. S. Matney,
J. L. Chandler, and R. K. Gholson.
1970.
Chromosomal location of the C gene involved in the biosynthesis of nicotinamide adenine dinucleotide in Escherichia coli K-12.
J. Bacteriol.
104:45-49[Abstract/Free Full Text].
|
| 63.
|
Turner, K. J., and R. M. Sharpe.
1997.
Environmental oestrogenspresent understanding.
Rev. Reprod.
2:69-73[Abstract].
|
| 64.
|
van der Meer, J. R.,
W. M. De Vos,
S. Harayama, and A. J. B. Zehnder.
1992.
Molecular mechanisms of genetic adaptation to xenobiotic compounds.
Microbiol. Rev.
56:677-694[Abstract/Free Full Text].
|
| 65.
|
Vogelstein, B., and D. Gillespie.
1979.
Preparation and analytical purification of DNA from agarose.
Proc. Natl. Acad. Sci. USA
76:615-619[Abstract/Free Full Text].
|
| 66.
|
Wams, T. J.
1987.
Diethylhexylphthalate as an environmental contaminanta review.
Sci. Total Environ.
66:1-16[Medline].
|
| 67.
|
Weber, F. J., and J. A. M. de Bont.
1996.
Adaptation mechanisms of microorganisms to the toxic effects of organic solvents on membranes.
Biochim. Biophys. Acta
1286:225-245[Medline].
|
| 68.
|
Whitchurch, C. B., and J. S. Mattick.
1994.
Escherichia coli contains a set of genes homologous to those involved in protein secretion, DNA uptake and the assembly of type-4 fimbriae in other bacteria.
Gene
150:9-15[Medline].
|
| 69.
|
Wood, M. S.,
A. Byrne, and T. G. Lessie.
1991.
IS406 and IS407, two gene-activating insertion sequences for Pseudomonas cepacia.
Gene
105:101-105[Medline].
|
| 70.
|
Wood, M. S.,
C. Lory, and T. G. Lessie.
1990.
Activation of the lac genes of Tn951 by insertion sequences from Pseudomonas cepacia.
J. Bacteriol.
172:1719-1724[Abstract/Free Full Text].
|
| 71.
|
Zylstra, G. J., and D. T. Gibson.
1991.
Aromatic hydrocarbon degradation: a molecular approach, p. 183-203.
In
J. K. Setlow (ed.), Genetic engineering: principles and methods. Plenum Press, New York, N.Y.
|
| 72.
|
Zylstra, G. J.,
R. H. Olsen, and D. P. Ballou.
1989.
Cloning, expression, and regulation of the Pseudomonas cepacia protocatechuate 3,4-dioxygenase genes.
J. Bacteriol.
171:5907-5914[Abstract/Free Full Text].
|
| 73.
|
Zylstra, G. J.,
R. H. Olsen, and D. P. Ballou.
1989.
Genetic organization and sequence of the Pseudomonas cepacia genes for the alpha and beta subunits of protocatechuate 3,4-dioxygenase.
J. Bacteriol.
171:5915-5921[Abstract/Free Full Text].
|
Journal of Bacteriology, May 1999, p. 3069-3075, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Weaver, V. B., Kolter, R.
(2004). Burkholderia spp. Alter Pseudomonas aeruginosa Physiology through Iron Sequestration. J. Bacteriol.
186: 2376-2384
[Abstract]
[Full Text]
-
Chang, H.-K., Zylstra, G. J.
(1999). Characterization of the Phthalate Permease OphD from Burkholderia cepacia ATCC 17616. J. Bacteriol.
181: 6197-6199
[Abstract]
[Full Text]
Top
Abstract
Introduction
