Department of Microbiology and Immunology,
The Brody School of Medicine, East Carolina University, Greenville,
North Carolina 27858-4354
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INTRODUCTION |
A cyclic version of the
Entner-Doudoroff pathway is used by Pseudomonas aeruginosa
to metabolize carbohydrates (Fig. 1)
(9, 19, 36). The members of the pathway that are responsible
for the metabolism of glucose to glyceraldehyde 3-phosphate and
pyruvate are coordinately regulated and induced by growth on
glycerol, fructose, mannitol, glucose, and gluconate. They are
clustered in at least three operons near 39 min on the chromosome and
are referred to as the hex regulon. They are under the
control of the recently identified repressor hexR
(29; W. D. Proctor, P. W. Hager, and
P. V. Phibbs, Jr., Abstr. 98th Annu. Meet. Am. Soc. Microbiol.
1998, abstr. K-135).
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FIG. 1.
Cyclic Entner-Doudoroff pathway of P. aeruginosa. In this pathway, the catabolism of mannitol occurs via
glucose-6-phosphate and requires the activity of glucose-6-phosphate
dehydrogenase (Zwf, EC 1.1.1.49), while the catabolism of gluconate
does not require Zwf. Additional enzymatic activities are abbreviated
as follows: Eda, 2-keto-3-deoxy-6-phosphogluconate aldolase (EC
4.1.2.14); Edd, 6-phosphogluconate dehydratase (EC 4.2.1.12); Fbp,
fructose-1,6-bisphosphatase (EC 3.1.3.11); Fba, fructose bisphosphate
aldolase (EC 4.1.2.13); Frk, fructokinase (EC 2.7.1.4); GnuK,
gluconokinase (EC 2.7.1.12); Mdh, mannitol dehydrogenase (EC 1.1.1.67);
Pgi, phosphoglucoisomerase (EC 5.3.1.9); Pgl, 6-phosphogluconolactonase
(EC 3.1.1.31); Tpi, triose phosphate isomerase (EC 5.3.1.1).
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The DNA sequence of the P. aeruginosa zwf gene encoding the
unique glucose 6-phosphate dehydrogenase of P. aeruginosa
was recently reported (22). Zwf catalyzes the oxidation of
glucose-6-phosphate to 6-phosphogluconolactone using either NAD or NADP
as a cofactor. While 6-phosphogluconolactone can be hydrolyzed
nonenzymatically to 6-phosphogluconate, the enzymatic activity (EC
3.1.1.31) was described some time ago (6). A
phosphogluconolactonase activity has been identified and partially
purified from Pseudomonas fluorescens (16) but,
to our knowledge, has not been identified in P. aeruginosa.
Purification of 6-phosphogluconolactonase has been achieved from
Zymomonas mobilis (33), bovine erythrocytes (3), and bass liver (26), and in each case the
enzyme appears to be a monomer of 26 to 30 kDa. Escherichia
coli contains a 6-phosphogluconolactonase which is necessary for
optimal growth when using the pentose phosphate shunt. Hence, pgi
pgl double mutants, which cannot convert glucose-6-phosphate to
fructose-6-phosphate, grow very slowly on glucose (18). The pgl gene locus was tightly linked by transductional analysis
to att-
, located at 15 min on the E. coli K-10
chromosome (17).
The isolation and molecular characterization of a pgl
structural gene were first reported only recently (GenBank accession no. AF029673) (P. W. Hager, M. W. Calfee, and P. V. Phibbs, Abstr. 99th Annu. Meet. Am. Soc. Microbiol. 1999, abstr.
K-148). An open reading frame with homology to devB, a
putative "developmentally regulated" glucose-6-phosphate
dehydrogenase, was identified immediately downstream of the
P. aeruginosa Zwf coding sequence. Insertional inactivation of that devB homolog resulted in a slow-growth
phenotype on mannitol and the loss of 6-phosphogluconolactonase
activity. Normal growth on mannitol and 6-phosphogluconolactonase
activity were restored by a plasmid containing the subcloned open
reading frame, identifying the P. aeruginosa devB homolog as
pgl. The expression of Pgl activity was positively regulated
by growth on carbohydrates and negatively regulated by growth on
succinate, a phenotype that is consistent with this gene's being a
member of the hex regulon. Subsequently, a human cDNA with
homology to the P. aeruginosa pgl and other devB
homologs was reported to encode Pgl (8).
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
The strains and plasmids
used are listed in Table 1.
Genetic techniques.
The eda gene was subcloned
from plasmid pPZ300 in two steps. First, both orientations of a 2.9-kb
BamHI fragment from pPZ300 were cloned into pGEM-3Zf(+),
creating pPZ474 and pPZ475. Second, a PstI deletion of
pPZ475 (removing 1.8 kb) resulted in pPZ505. The remaining 1.1-kb
fragment containing eda from pPZ475 was cut out with
PstI and BamHI and ligated into the
Pseudomonas-compatible vector pUCP18, creating pPZ502, in
which the eda gene is in the sense orientation following the
lac promoter. The antisense orientation of the
eda gene relative to the lac promoter was created
by moving the 1.1-kb eda fragment into the
Pseudomonas-compatible vector pPZ375, creating pPZ571.
The pgl knockout strain PAO8029 and the pgl
merodiploid PAO8033 were constructed as follows. The 2-kb
XhoI-ApaI fragment from plasmid pPZ303,
containing the pgl gene, was cloned into the XhoI site of pGEM-7Z(+), creating pPZ594. The BamHI site within
the polylinker of pPZ594 was removed by digestion of pPZ594 with
EcoRI and SacI with ligation of the product,
creating pPZ595. The 0.9-kb SmaI fragment containing the
gentamicin resistance cassette (aacC1) from pUCGM
(32) was then cloned into the single remaining
BamHI site of pPZ595 (after making the BamHI ends
flush with T4 DNA polymerase), resulting in a disruption of the
pgl coding sequence. Both orientations of aacC1
into pgl were obtained as plasmids pPZ596 and pPZ602. DNA
from pPZ596 or pPZ602 was electroporated into P. aeruginosa
PAO1 using the method of Enderle and Farwell (11). Three
putative recombinants were isolated on L agar containing gentamicin.
The characterization of two of these mutants as PAO8029 and PAO8033 is
described in the Results section.
Southern blots.
Genomic DNA was isolated from strains PAO1,
PAO8029, and PAO8033 using hexadecyltrimethyl ammonium bromide (CTAB)
followed by equilibrium centrifugation in a CsCl gradient
(2). The genomic DNA was digested with BamHI, and
1 µg of each digest was loaded on a 1% agarose gel, electrophoresed,
and transferred to a nylon membrane by capillary action (2).
Digestion of pPZ595 with ApaI and XhoI yielded
DNA fragments of 2 and 3 kb, representing the pgl gene and
the vector, respectively. The fragments were isolated from a 1%
agarose gel using Qiaex resin (Qiagen, Chatsworth, Calif.) and labeled
with [
-32P]dATP using the RadPrime DNA labeling system
(Life Technologies, Rockville, Md.) as described by the manufacturers.
Duplicate blots were probed with approximately 107 cpm of
either the pgl or the vector-derived probe, washed, and exposed to X-ray film for 24 to 48 h.
Preparation of 6-phosphogluconolactone and assays of
6-phosphogluconolactonase and glucose-6-phosphate dehydrogenase
activity.
A qualitative assay of 6-phosphogluconolactonase,
including the preparation of 6-phosphogluconolactone (by lyophilization of 6-phosphogluconate) and its quantitation using hydroxylamine and
ferric chloride, has been previously described (17). The extinction coefficient of the ferric hydroxymate of
6-phosphogluconolactone was assumed to be equal to that determined for
-gluconolactone (E530 = 580 M
1). For the determination of the specific activity of
6-phosphogluconolactonase, a method based on that of Beutler et al. was
employed (4). Briefly, the production of 6-phosphogluconate
was followed using 6-phosphogluconate dehydrogenase. Assays were done
at 28°C in 0.1 M Tris-Cl buffer (pH 7.5) with 0.3 mM NADP, 0.1 mM
6-phosphogluconolactone, 1 mM dithiothreitol, and 0.5 U of yeast
6-phosphogluconate dehydrogenase. Glucose-6-phosphate dehydrogenase
assays were done at 28°C; otherwise, the assay was as previously
described (15). For the preparation of extracts for
enzymatic assays, cells were harvested in mid-log-phase growth
following growth in minimal medium (21) with a carbon source. Cells were washed with cold 0.9% NaCl, and the cell pellets were suspended in 5% of the original culture volume of 50 mM
Tris-Cl-1 mM EDTA-1 mM dithiothreitol (pH 8) and broken in a French
press at 16,000 lb/in2. Extracts were clarified by
centrifugation at 175,000 × g for 30 min (Sorvall
T-1270, 45,000 rpm). Protein was measured by the method of Bradford
(5) using bovine serum albumin as the standard. Chemicals
and coupling enzymes were obtained from Sigma Chemical Co. (St. Louis,
Mo.), and Bradford reagent was obtained from Bio-Rad (Hercules,
Calif.).
Growth rates.
Strains were initially isolated on selective
Luria-Bertani agar plates and subsequently grown overnight in liquid
minimal medium containing 10 mM succinate. Fresh minimal medium
cultures containing 2 mM succinate and 20 mM gluconate or 2 mM
succinate and 20 mM mannitol were started with a 10% (vol/vol)
inoculum from the overnight culture. For the determination of growth
rates, the turbidity was measured with a Klett-Summerson colorimeter with a red no. 66 filter. Under these conditions, P. aeruginosa utilizes the available succinate for growth before
shifting to metabolism of the gluconate or mannitol (23).
Hence, the gluconate- and mannitol-dependent growth rates were
determined from the second phase of the growth curves.
DNA sequencing.
The DNA sequences of the cloned fragments in
pPZ505, pPZ149, and pPZ595 were determined by Commonwealth
Biotechnologies, Inc. (Richmond, Va.). Plasmid pPZ149 contains a 1.2-kb
BamHI fragment derived from pPZ300 cloned into pTZ19R.
Computer analysis of sequences.
Comparisons between
individual sequences were carried out using the Genetics Computer Group
software analysis package (Madison, Wis.), while comparisons to
databases were carried out using the Basic Local Alignment Search Tool
(BLAST) network service at the National Center for Biotechnology
Information (NCBI), National Institutes of Health (1).
Searches for promoter sequences were carried out using the program NNPP
(www-hgc.lbl.gov/projects/promoter.html) (30).
Nucleotide sequence accession number.
The DNA sequences of
the pgl and eda genes were updated in GenBank
(accession no. AF029673, 2 April 1999).
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RESULTS AND DISCUSSION |
Nucleotide sequence encoding eda and
pgl/devB.
The cloning and DNA sequence of the
glucose-6-phosphate dehydrogenase gene (zwf) of P. aeruginosa were reported recently (22). Continuing
downstream from zwf, two additional open reading frames were
found (summarized in Fig. 2). Earlier
work established that the Entner-Doudoroff aldolase gene
(eda) was closely linked to zwf (10,
31), and both genes were subsequently cloned on an 11-kb fragment
of P. aeruginosa DNA (34). The smallest subclones able to complement the eda mutant PAO1838 for growth on
mannitol, glucose, and gluconate contained the two orientations of a
1.1-kb BamHI-PstI DNA fragment 3' to
zwf (in plasmids pPZ502 and pPZ571). The DNA sequence of
this region showed the eda gene to be 723 bp downstream of
and in the same orientation as zwf (Fig.
3). The DNA sequence predicts that Eda
contains 220 amino acids sharing 42% identity with its E. coli homolog. It is preceded by a Shine-Dalgarno sequence (GGAG)
and followed by a potential transcription terminator. Although a
promoter sequence between zwf and eda has not
been identified, we presume that the 1.1-kb eda fragment
does contains a promoter, since both orientations of the cloned
eda gene are able to complement PAO1838.
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FIG. 2.
Summary of the organization of the zwf,
pgl, and eda genes and plasmids derived from
pPZ300. Plasmid pPZ300 contains 11 kb of PAO1-derived DNA, of which
only 3 kb is indicated here. Plasmid pPZ502 complements the
eda mutant PAO1838. It was constructed by subcloning a 1-kb
BamHI-PstI fragment from pPZ505 into the
broad-host-range vector pUCP18. pPZ595 and pPZ603 contain the same 2-kb
XhoI-SalI fragment, and pPZ603 complements the
pgl mutant PAO8029. pPZ596 is a Pseudomonas
suicide vector, constructed from pPZ595. It contains a gentamicin
resistance cassette, aacC1, inserted into the
BamHI site within pgl/devB. pPZ602 (not shown) is
identical to pPZ596 except for the orientation of the aacC1
cassette.
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FIG. 3.
Nucleotide and deduced amino acid sequences of the
pgl and eda genes. The coding sequence of Pgl
begins within the 3' end of the Zwf coding sequence at nucleotide 76. There is an alternative initiation codon for Pgl at nucleotide 61 (indicated in upper case). The coding sequence for Eda is separated
from the end of Pgl by only 17 bp. Indicated are potential
Shine-Dalgarno (wavy underlining) and rho-independent transcriptional
termination sequences (underlined).
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The 723-bp region between zwf and eda has one
open reading frame in the same orientation as Zwf and Eda with two
potential ATG start codons located within the last eight codons
of the Zwf sequence (Fig. 2). The potential initiation codons are
in frame with each other, separated by only 5 codons, and both have
potential Shine-Dalgarno sequences (GGUGG and GGAGG). The second
initiation codon appears to be the true start site, as a P. aeruginosa-derived protein with the predicted N-terminal amino
acid sequence (AISELKLPAGVGLQV) has been identified
(Dennis Ohman, personal communication). This open reading frame
encoding 238 amino acids has similarity to devB (from
Anabaena) and SOL (from Saccharomyces
cerevisiae) (see below). DevB has been proposed to be a
"developmentally regulated" glucose-6-phosphate dehydrogenase
(GenBank accession no. U14553). While P. fluorescens and
Pseudomonas cepacia (reclassified as Burkholderia
cepacia) have two glucose-6-phosphate dehydrogenases that function
with either NAD+ (in a catabolic role) or NADP+
(in an anabolic role) (7, 20, 25), it is clear that
P. aeruginosa has a single glucose-6-phosphate
dehydrogenase activity (Zwf) capable of using either NAD+
or NADP+ (22, 28). One interpretation consistent
with a single zwf gene is that the open reading frame with
homology to devB/SOL represents a 6-phosphogluconolactonase.
To test this hypothesis, the devB open reading frame was
interrupted by insertion of the aacC1 gentamicin
resistance cassette at the BamHI site. The related suicide
plasmids pPZ596 and pPZ602 contain the two orientations of
aacC1 (Fig. 2). After electroporation of pPZ596 and pPZ602
into P. aeruginosa PAO1, three gentamicin-resistant colonies were isolated. Two of these strains were gentamicin resistant but carbenicillin sensitive, suggesting gene replacement by
recombination of the plasmid into the chromosome via double crossovers.
One of these isolates was named PAO8029 and studied further. The third isolate, PAO8033, had both carbenicillin and gentamicin resistance, suggesting recombination via a single crossover. Southern blot analysis
of BamHI-digested DNA isolated from PAO1, PAO8029, and PAO8033 showed that these interpretations were correct (Fig.
4). BamHI-digested PAO1 DNA
contains two fragments (1.3 and 2.7 kb) that hybridized to the
devB-derived probe. For PAO8029, the BamHI site
within the devB gene has been replaced with the 0.8-kb
aacC1 cassette, resulting in a single band of 4.8 kb. For
PAO8033 there are two bands that hybridized to the devB
probe, 1.3 kb and 7.7 kb (Fig. 4A). When a duplicate blot was probed
with DNA derived from the plasmid vector, only PAO8033 and the plasmid
controls showed hybridization (Fig. 4B). Hence, for PAO8033, the
results were consistent with a single crossover of the plasmid into the amino-terminal side of the gene.
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FIG. 4.
Southern blots of genomic DNA digested with
BamHI. Duplicate blots were prepared as follows: lane 1, PAO1-derived DNA; lane 2, PAO8026-derived DNA; lane 3, PAO8033-derived
DNA. (A) Blot hybridized with pgl-specific probe. (B) Blot
hybridized with plasmid vector-specific probe.
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Identification of devB as pg1.
In P. aeruginosa, Zwf activity is absolutely required for growth on
mannitol but not for growth on gluconate (Fig. 1). Compared to PAO1,
PAO8029 grew at a reduced rate on gluconate (50%), as did the
zwf mutant strain PAO9010 (82%). However, PAO8029 grew very
slowly on mannitol, with a doubling time of approximately 30 h, or
9% of the wild-type rate (Table 2).
While this is the expected phenotype of a pgl mutant (see
below), it could represent a leaky zwf phenotype, so we
tested for complementation using various plasmids. The phenotype of
very slow growth on mannitol was not complemented by a plasmid vector
(pPZ375) or the vector containing zwf (pPZ524) (data not
shown), but was complemented by pPZ603 containing the devB
homology region (Table 2). This confirmed our supposition that this
devB homolog is not a glucose-6-phosphate dehydrogenase;
instead, it represents a unique gene.
The phenotype of very slow growth on mannitol for PAO8029
is reminiscent of E. coli phosphogluconolactonase mutants
(17, 18). Hence, we assayed for 6-phosphogluconolactonase
after growing P. aeruginosa PAO1, PAO9010 (zwf),
PAO8029, and PAO8029 containing pPZ603 with gluconate as the carbon
source. The ferric hydroxymate assay of 6-phosphogluconolactonase
activity that measures the disappearance of the substrate is
essentially qualitative because of a relatively high nonenzymatic rate
and the presence of the 
and 6-phosphogluconolactones in the
substrate (17). Nevertheless, it is clear from the data in
Fig. 5 that extracts from PAO1 and PAO9010 had phosphogluconolactonase activity, while the PAO8029 extract
was essentially identical to the control without extract protein. An
extract of PAO8029 containing plasmid pPZ603 also had
phosphogluconolactonase activity, demonstrating complementation of the
gene. Hence, the P. aeruginosa devB homolog was identified as pgl.
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FIG. 5.
Extracts of strain PAO8029 lack
6-phosphogluconolactonase. Assays contained 6-phosphogluconolactone and
either water ( ) or 100 µg of cell-free extract protein derived
from strain PAO1 ( ), PAO8029 ( ), PAO8029 containing plasmid
pPZ603 ( ), or PAO9010 (zwf) ( ). At the indicated
times, the reactions were terminated, and remaining
6-phosphogluconolactone was converted to a ferric hydroxymate and
quantified colorimetrically. Strains were grown on minimal medium
containing 20 mM gluconate as the sole carbon source, and cell extracts
were prepared as described in the text.
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Given the nonenzymatic rate of hydrolysis for 6-phosphogluconolactone
(with a t1/2 of approximately 50 min; see Fig.
5), the dramatic reduction in growth rate for the pgl mutant
strains grown on mannitol might seem surprising. However, this
phenotype is consistent with the normally short metabolic lifetime of
6-phosphogluconolactone, as pointed out by Scopes (33).
However, this would not account for the reduced rate of growth by
strain PAO8029 on gluconate (compared to PAO1 and PAO9010 in Table 2).
Since the cyclic Entner-Doudoroff pathway in P. aeruginosa
would allow the synthesis of 6-phosphogluconolactone, we suggest that
the substrate for Pgl, 6-phosphogluconolactone, is toxic.
It seems essential that there should be similar amounts of
6-phosphogluconolactonase and glucose-6-phosphate dehydrogenase activity in the cell in order to maintain a balanced flux through this
metabolic pathway. A quantitative assay for
6-phosphogluconolactonase, in which the 6-phosphogluconolactonase is
coupled to a 6-phosphogluconate dehydrogenase, showed that there are
similar levels of these activities in extracts from PAO1 cells grown on
gluconate (353 ± 26 mIU/mg for glucose-6-phosphate dehydrogenase
and 388 ± 41 mIU/mg for 6-phosphogluconolactonase).
Conservation within the Pgl family.
A search of the NCBI
databases using the BLAST program (1) with the deduced amino
acid sequence for Pgl as a query turned up a large number of family
members, previously identified as DevB or SOL homologs. Alignment of
several such members revealed important regions of conservation
(motifs) that may include the active-site residues of
6-phosphogluconolactonase (Fig. 6). The two most conserved regions include IALSGGSTP and LGMG-DGHTASLFPH. Note
that the genomic sequence of the Haemophilus influenzae
6-phosphogluconolactonase homolog (devB; HIO556) is
truncated prior to this first motif (12). However, the
upstream sequence of H. influenzae does encode this motif
with an appropriate ATG start codon (underlined in Fig. 6), as well
as two internal stop codons (indicated as x in Fig. 6). We suggest
either that the stop codons are sequencing artifacts or that
H. influenzae has a mechanism for translating through them.
A cDNA encoding a human cytosolic Pgl has recently been cloned and
expressed in E. coli and shown to have Pgl activity (8). In addition to observing its homology with P. aeruginosa Pgl, the bacterial DevB, and the yeast SOL proteins,
these authors also identified a less conserved homology with
glucosamine-6-phosphate isomerase.
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FIG. 6.
Alignment of amino acid sequences for representative
homologs of the P. aeruginosa phosphogluconolactonase. The
homologs were identified using the BLAST program and were aligned with
Genetics Computer Group programs. The consensus sequence was generated
using a plurality of five. The sequences include the PAO-derived
sequence reported here, and ones from Helicobacter pylori
(GenBank accession no. AE000616), Anabaena (Swiss-Prot.
accession no. P46016), Synechocystis sp. strain PCC6083
(DDBJ accession no. D90916), Actinobacillus
actinomycetemcomitans (DDBJ accession no. D88189), H. influenzae (Swiss-Prot. accession no. Q57039), Treponema
pallidum (GenBank accession no. AAC65464), and S. cerevisiae SOL1 (Swiss-Prot. accession no. P50278). Sequence
numbers refer to the S. cerevisiae SOL1 amino acid sequence
(residues 1 to 30 not shown). Additional N-terminal sequence for the
H. influenzae homolog (underlined) was deduced from the DNA
sequence (12).
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The overlapping nature of Zwf and Pgl in P. aeruginosa
suggests a very tight translational control, perhaps necessary to
balance their enzymatic activities. In general, the prokaryote-derived members of the pgl family are found next to zwf
(24), with the notable exception of E. coli.
Since pgl was originally identified and mapped in E. coli K-10 (an HfrC derivative of K-12), we were surprised when
searches using BLAST failed to identify an obvious homolog in E. coli. The closest might be nagB (8) (GenBank accession no. M19284), whose map position is 1 min from the E. coli pgl gene.
In general, glucose-6-phosphate dehydrogenases are highly conserved
proteins of about 500 amino acids. However, the rabbit microsomal
glucose-6-phosphate dehydrogenase, derived from the lumen of the
endoplasmic reticulum, contains 763 amino acids, with a "noncatalytic
domain" in the middle of the enzyme (27). The
"noncatalytic domain" contains the second Pgl motif (residues 399 to 411 of the rabbit microsomal glucose-6-phosphate
dehydrogenase are GMGtDGHTASLFP). Likewise, the
amino acid sequence of the unusual glucose-6-phosphate dehydrogenase of
the malaria parasite, Plasmodium falciparum, begins with a
Pgl domain (33a). The recently described hexose-6-phosphate
dehydrogenase from human bone marrow also contains a Pgl domain,
located in the carboxy-terminal third of the gene (8, 24).
Thus, it appears that these animals have evolved a simple solution for
efficient metabolic flux through glucose-6-phosphate dehydrogenase and
6-phosphogluconolactonase by combining both activities into a single protein.
pg1 is a member of the hex regulon.
Since pgl follows zwf directly, both in the
arrangement of the genes and in metabolic pathway function, it seemed
likely that they would be coordinately regulated. Growth with gluconate
as the carbon source induced glucose-6-phosphate dehydrogenase
(Zwf), while growth on succinate did not (340 ± 18 mIU/mg
versus 3 ± 3 mIU/mg). Similarly, we observed that PAO1 had
6-phosphogluconolactonase activity when grown on gluconate but not when
grown on succinate (Fig. 7). Recently,
the repressor for the hex regulon has been identified
(29; Procter et al., abstr. K-135). The HexR protein represses the expression of all the enzymes in the hex
regulon by binding to DNA sequences upstream of each operon.
Inactivation of HexR by insertion of a gentamicin resistance cassette
into the chromosomal copy of hexR results in constitutive
expression of the hex regulon (29;
Procter et al., abstr.). In the hexR mutant strain PAO8026,
6-phosphogluconolactonase activity was present at equivalent levels
when the strain was grown on either gluconate or succinate (Fig. 7).
Thus, pg1 is under the control of HexR and is demonstrated
to be another member of the hex regulon.
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FIG. 7.
pg1 is a member of the hex
regulon. Assays contained 6-phosphogluconolactone and either water
() or 100 µg of cell extract protein derived from strain PAO1
grown with succinate () or gluconate () as the sole carbon source
or PAO8026 (hexR) grown with succinate () or gluconate
() as the sole carbon source. At the indicated times, reactions were
terminated, and the remaining 6-phosphogluconolactone was quantified
colorimetrically.
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We thank Ann Covert-Rinaldi for technical assistance and Dan
Martin, Jeff Smith, and Dennis Ohman for their critical comments.
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Abstract
Introduction
