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Journal of Bacteriology, April 2000, p. 2230-2237, Vol. 182, No. 8
Department of Plant Pathology, University of
California, Davis, California 95616
Received 15 September 1999/Accepted 30 December 1999
Pink disease of pineapple, caused by Pantoea citrea, is
characterized by a dark coloration on fruit slices after autoclaving. This coloration is initiated by the oxidation of glucose to gluconate, which is followed by further oxidation of gluconate to as yet unknown
chromogenic compounds. To elucidate the biochemical pathway leading to
pink disease, we generated six coloration-defective mutants of P. citrea that were still able to oxidize glucose into gluconate.
Three mutants were found to be affected in genes involved in the
biogenesis of c-type cytochromes, which are known for their role as specific electron acceptors linked to dehydrogenase activities. Three additional mutants were affected in different genes within an
operon that probably encodes a 2-ketogluconate dehydrogenase protein. These six mutants were found to be unable to oxidize gluconate
or 2-ketogluconate, resulting in an inability to produce the
compound 2,5-diketogluconate (2,5-DKG). Thus, the production of
2,5-DKG by P. citrea appears to be responsible for the dark color characteristic of the pink disease of pineapple.
Pink disease of pineapple is
characterized by the production of distinct dark orange-brown color
produced in the fruit tissue after the heating process of canning
(27). Since the fruits remain superficially symptomless in
the field, eliminating the disease by culling fruits either in the
field or at postharvest is highly problematic and therefore of economic
importance. Pink disease was first observed in Hawaii and was later
found in Australia and in the Philippines (22, 24, 27). The
incidence of seasonal factors, such as rainfall and temperature, were
analyzed to reveal that flowering during wet weather preceded by dry
periods increases pink disease occurrence (18). Confusion on
the cause of pink disease remained for many years because four
different bacteria were thought to be responsible for the disease:
Erwinia herbicola, Gluconobacter oxydans,
Enterobacter agglomerans, and Acetobacter aceti
(28). However, recent molecular studies of the disease has
identified Pantoea citrea as the causal agent of pink
disease (8).
P. citrea, a member of the Enterobacteriaceae
family commonly isolated from fruit and soil samples, was shown to
effectively produce 2,5-diketo-D-gluconic acid from
D-gluconic acid and 2-keto-D-gluconic acid
(20, 37). To identify the bacterial genes involved in formation of the chromogenic compound(s) characteristic of pink disease, we used the virulent strain 1056R and developed an assay that
enabled us to reproduce the disease under laboratory conditions (7). We then generated and analyzed various chemically
induced mutants of P. citrea that were unable to induce pink
disease. This allowed us to show that one of the key steps leading to
the coloration of pineapple juice was the oxidation of glucose to gluconate (7). In P. citrea, this process is
carried out by two highly similar, membrane-bound
quinoprotein glucose dehydrogenases, GdhA and GdhB (7, 26).
gdhA is constitutively expressed at very low levels, while
gdhB is fully expressed to produce gluconate from glucose
(26). Mutants affected in the ability to oxidize glucose
were able to carry out coloration of pineapple when supplemented with
gluconate in the growth medium, indicating that production of gluconate
is the first step in the pathway leading to the formation of the
chromogenic compound(s) (7, 26).
To further characterize additional steps involved in color formation,
we used transpositional mutagenesis to generate mutant strains of
P. citrea that, although still able to oxidize glucose to
gluconate, are unable to color pineapple juice. Six independent mutants
were isolated. Their biochemical and genetic characterization revealed
that the genes implicated in pink disease are involved in the
conversion of gluconate to 2-keto-D-gluconate (2-KDG) and 2,5-diketo gluconate (2,5-DKG). We also identified several open reading
frames (ORFs) for genes required for the biogenesis of c-type cytochromes that are used as electron acceptors in
the oxidation reactions in P. citrea. Taken together, our
data show that accumulation of 2,5-DKG is directly responsible for the
intense coloration characteristic of pink disease of pineapple.
Bacterial strains, media, growth conditions, and
antibiotics.
All bacterial strains used in this study are listed
in Table 1. Escherichia coli
strains were grown at 37°C in LB medium (29). P. citrea strains were grown at 30°C either in Luria-Bertani (LB)
medium containing rifampin, in mannitol-glutamate-yeast (MGY) medium
(8), or in pineapple juice (8). All media were
solidified with 1.5% (wt/vol) Bacto Agar (Difco Laboratories).
E. coli and P. citrea competent cells were
transformed by electroporation as previously described (7).
After transformation with pBluescript SKII(
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Genetic and Biochemical Characterization of the
Pathway in Pantoea citrea Leading to Pink Disease of
Pineapple
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) [pBSSKII()]
derivatives, E. coli transformants were selected on LB agar
plates containing ampicillin (100 µg/ml),
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal;
40 µg/ml; Denville), and 0.1 mM
isopropyl--D-thiogalactopyranoside (IPTG; Gibco-BRL Life
Technologies). Antibiotics (Sigma) were used at the following
concentrations (micrograms per milliliter): rifampin, 100; kanamycin,
15; tetracycline, 10 and 15 for P. citrea and E. coli, respectively; and ampicillin, 250 and 100 for P. citrea and E. coli, respectively.
TABLE 1.
Bacterial strains and plasmids used
Pink test and coloration assay. The Pink test was performed in pineapple juice as previously described (7). For the coloration assay, bacteria were grown in MGY supplemented with various carbon sources (see below) for 3 days at 30°C with gentle shaking (150 rpm). The culture was then autoclaved for 5 min at 121°C, and the cells were removed by centrifugation (5,000 × g, 5 min). The absorbance of the supernatant was determined spectrophotometrically at 420 nm in a Shimadzu spectrophotometer.
Carbon sources. The carbon sources D-arabinose, D-cellobiose, fructose, D-fucose, D-galactose, D-lactose, maltose, D-melibiose, raffinose, L-rhamnose, D-ribose, saccharose, D-trehalose, and D-xylose were used at 100 mM. D-Gluconic acid, D-glucose, 2-keto-D-gluconic acid, 5-keto-D-gluconic acid, 2,5-DKG, and gulonic acid were used at 50 mM.
Plasmids and DNA manipulations. Cloning vectors and plasmids used and constructed in this study are listed in Table 1. Plasmid screening was carried out by the alkaline lysis method of Birnboim (4). Large-scale plasmid DNAs were prepared using a Qiagen plasmid kit. Total DNA extraction was performed by the method of te Riele et al. (39). DNAs were cleaved with restriction endonucleases as specified by the suppliers (Boehringer Mannheim and New England Biolabs). Ligations were performed with T4 DNA ligase (Boehringer Mannheim) according to standard protocols (29).
Transposition mutagenesis.
Tn10 insertion mutants
were isolated by using the Tn10 derivative pBSL346
(1). pBSL346 was introduced into P. citrea 1056R and 1058RC by electroporation; the mutants generated were selected on
solid LB medium supplemented with rifampin (100 µg/ml) and tetracycline (15 µg/ml) and tested for the ability to color pineapple juice. Total DNA from a 5-ml overnight culture of the putative mutants
was purified, digested with convenient restriction enzymes, analyzed by
agarose gel electrophoresis, and transferred onto a Hybond N+ nylon
membrane (Amersham). The bound DNA was then hybridized with an
-32P-labeled probe corresponding to the 1.3-kb
EcoRI-StyI fragment containing the tet
gene from plasmid pBR322 to detect transposon Tn10.
Fragments with sizes ranging from 3 to 9 kb were then purified from
agarose gel with a Wizard purification kit (Promega), ligated into
convenient unique sites of pBSSKII() or pUCKm vector, and transformed
into E. coli DH5 competent cells with selection for tetracycline resistance (Tcr). Among the Tcr
transformants, those containing plasmids of the expected size (vector
plus insert) were selected for nucleotide sequencing.
), leading to plasmid pUCD5071. To
extend the size of the region, a 9-kb EcoRI DNA fragment
containing the transposon was isolated from CP9G10 and was cloned into
the EcoRI site of pBSSKII(), yielding plasmid pUCD6718.
The sequence of a 4,349-bp DNA contig was then determined and analyzed.
In mutant CP6C8, the Tcr marker of transposon
Tn10 was localized on a 4-kb PvuII chromosomal
fragment. This fragment was cloned into the unique EcoRV
site of pBSSKII(), leading to plasmid pUCD5066.
In mutant 103C11, the Tn10 Tcr marker was
localized on a 2.8-kb AvaI chromosomal fragment, which was
cloned into the unique XmaI site of pUCKm, leading to
plasmid pUCD5074. In mutant CP15E7, the marker was localized on a 10- to 12-kb PvuII chromosomal fragment which, upon cloning into
the EcoRV site of pBSSKII(), led to plasmid pUCD5079.
Nucleotide sequences of pUCD5074 and pUCD5079 revealed that the 2.8-kb
AvaI DNA fragment and the larger PvuII DNA
fragment were overlapping.
DNA hybridization, sequencing, and PCRs.
Southern blot
hybridizations were performed as previously described (29)
and were carried out at 65°C with Rapid-Hyb buffer according to the
directions of the supplier (Amersham Life Science). Probes were
prepared by nick translation (Boehringer Mannheim) using
[-32P]dCTP as the radioactive nucleotide (Dupont-NEN
Products). Double-stranded DNA sequences were determined by the
dideoxy-chain termination method (30) using a Sequenase
version 2.0 kit (USB Amersham) with the M13 universal primers or
convenient primers (Gibco-BRL Life Technologies) and
[-35S]dATP as the radioactive nucleotide (DuPont-NEN
Products). The sequences were run on a GenomyxLR sequencer (Genomyx
Co.). Sequences were compiled and comparative analyses were made with
the Wisconsin Package version 9.0 (Genetics Computer Group, University
of Wisconsin, Madison) and the BlastN and BlastX programs developed by
the National Center for Biotechnology Information. All PCRs were
carried out in a GeneAmp PCR System 2400 apparatus (Perkin-Elmer),
using the recombinant Pfu DNA polymerase (Stratagene) and
convenient primers (Gibco-BRL Life Technologies). Nucleotide sequences
of the constructs were verified to ensure that the inability to
complement the mutant was not due to mutations introduced during the
PCRs. A 2,169-bp fragment containing the orfB gene was
amplified using primers 5070-11 (5'-AACGGGACAATCAGTTCAGC-3'
nucleotides [nt] 889 to 908) and 5071-R1
(5'-AACGCTGTCTTACTGCCG-3'; nt 3058 to 3041) along with total
DNA from strain 1056R as the template. The amplified fragments were
then cloned into the EcoRV site of pBSSKII(), yielding
plasmid pUCD5089. A 3,848-bp DNA fragment containing orfA,
orfB, and orfC was amplified using primers
StartEcoRI (5'-AGCAGAATTCGCGCGTTGTAACACTCCACCGC-3'; nt 483 to 512) and EndBamHI
(5'-TCTGGTGGATCCTTCAGAGTTCAGTGATGTTAAGC-3'; nt
4331 to 4298) (the underlined nucleotides represent restriction sites
for EcoRI and BamHI, respectively). After
digestion of the PCR product with the EcoRI and
BamHI, the fragment was cloned into pBSSKII(), previously
digested with the same enzymes.
),
yielding plasmid pUCD5077.
The ccmC gene was amplified using total DNA from strain
1056R as the template and primers hem1
(5'-TGGCACCTTTTGCCACAGCCGCAGCGG-3'; nt 2081 to 2107) and
hem2 (5'-GCCAGACATAGAAGGCATAACCTCCC-3'; nt 2977 to 2952).
The resulting 896-bp fragment was cloned into the EcoRV site
of pBSSKII(), to yield plasmid pUCD5094.
Dehydrogenase assays. In vivo glucose dehydrogenase activity was detected on solid MGY supplemented with 2% (wt/vol) glucose and eosin yellow-methylene blue as previously described (26).
Detection of gluconate dehydrogenase activity was adapted from the method of Bouvet et al. (5). Bacteria were grown overnight in 5 ml of MGY supplemented with 50 mM D-gluconic acid and then collected by centrifugation (2,700 × g for 3 min). The pellet was rinsed once with fresh sterile MGY and resuspended in 50 µl of sterile water. Then 10-µl aliquots of bacterial suspensions were dispensed into the wells of a microtiter plate, each well containing 100 µl of solution A (0.2 M acetate buffer [pH 5.2], 1% [vol/vol] Triton X-100, 2.5 mM MgSO4, 50 mM gluconate, 1 mM iodoacetate). The microtiter plate was incubated at 30°C for 20 min with gentle shaking (100 rpm). Then 10 µl of 0.1 M potassium ferricyanide was added to each well, and the plates were incubated at 30°C for 40 min with shaking (100 rpm). Finally, 50 µl of solution B [0.6 g of Fe2(SO4)3, 0.36 g of sodium dodecyl sulfate, 11.4 ml of 85% phosphoric acid, distilled water to 100 ml) was added to the wells. Wells were examined for the development of a green to blue color within 15 min at room temperature. The color of the negative control (MGY medium without bacteria) remained yellow. The 2-ketogluconate dehydrogenase assay was the same as described above, except that the cells were grown in MGY supplemented with 50 mM 2-keto-D-gluconate and solution A contained 50 mM 2-keto-D-gluconate instead of D-gluconate.TLC. Qualitative detection of D-gluconate (D-Gln), 2-KDG, and 2,5-DKG was done by thin-layer chromatography (TLC) according to Joveva et al. (19). The three acids were separated on Silica Gel 60 plates (20 by 20 cm; Whatman) impregnated with 5% metaphosphoric acid, using a two-step migration. The first migration was done in solvent A (isopropanol-pyridine-water-acetic acid [8:8:1:4]) until the front of the solvent was 12 cm away from the start. The plate was then air dried, and the second migration took place in solvent B (isopropanol-pyridine-water-acetic acid [8:8:3:4]) until the front of the solvent was 18 cm away from the start. The plate was then air dried before spraying freshly prepared reactive C (p-anisaldehyde 0.5% [Sigma] in methanol-sulfuric acid-acetic acid [9:5:5]). Finally the plate was dried under vacuum at 120°C for 10 to 15 min. The retention front (Rf) values corresponding to D-glucose, D-Gln, 2-KDG, and 2,5-DKG are 0.56, 0.36, 0.36, and 0.28, respectively. Despite the fact that D-Gln and 2-KDG show the same Rf, the two compounds are easily distinguishable since D-Gln gives a light blue spot, while the spot corresponding to 2-KDG is dark brown.
Nucleotide sequence accession numbers. The nucleotide sequence of the dsbD gene, the ccm operon, and the putative 2-ketogluconate dehydrogenase operon are deposited in GenBank with accession no. AF102175, AF103874, and AF131202, respectively.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Glucose catabolism is responsible for development of pink disease. We previously showed that the oxidation of D-glucose to D-Gln is the first step leading to pink disease of pineapple (7, 26). Since glucose dehydrogenases can oxidize glucose and other aldoses to their corresponding acids (9, 17), we wanted to determine more precisely which sugars can be used as substrates to produce the coloration of pineapple juice by P. citrea. For that purpose, P. citrea 1056R was grown for 3 days in MGY medium containing 100 mM carbon source (as described in Materials and Methods). The cultures were autoclaved, and the relative (before and after bacterial growth) turbidity (optical density [OD]) of the supernatant was measured at 420 nm. A substantial increase of the OD is observed when glucose is present in the medium, indicating that glucose is the key substrate leading to the dark coloration of pineapple juice.
In bacteria which possess a direct glucose oxidation metabolism, D-glucose is oxidized to D-Gln, which can be further oxidized into 2-KDG or 5-KDG by membrane-bound gluconate dehydrogenases 55). Furthermore, a number of Acetobacter, Erwinia, and Gluconobacter species are able to oxidize 2-KDG to 2,5-DKG by membrane bound 2-ketogluconate dehydrogenases (5, 43). We therefore decided to test these different compounds for the ability to be used as a substrate for the dark coloration characteristic of pink disease. We supplemented liquid MGY medium with 50 mM D-glucose, D-Gln, 2-KDG, 5-KDG, or 2,5-DKG and measured the relative absorbance of the clarified medium supernatant after 3 days of growth, as described above. As shown in Fig. 1, coloration of the medium was observed with three of the sugars, with the most intense coloration obtained with 2-KDG. Interestingly, addition of 5-KDG or 2,5-DKG to the medium appears to have no effect on the absorbance after growth (Fig. 1). In the case of 2,5-DKG, this is because when put into solution, this sugar already forms the dark orange color characteristic of pink disease (data not shown). Therefore, although no increase in the intensity of the color was observed after growth of P. citrea, the results suggest that pink disease is due to the accumulation of 2,5-DKG.
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Isolation of color-deficient mutants.
We recently
characterized color-deficient mutants of P. citrea that are
affected in the ability to oxidize glucose to gluconate, allowing the
identification of the first step of the pathway leading to pink disease
(7, 26). To identify additional genes involved in the
disease, and to confirm our physiological studies using various
derivatives of D-glucose, we used Tn10
transposon mutagenesis to generate mutant strains of P. citrea that are unable to color pineapple juice but can still
carry out the first oxidation step. Plasmid pBSL346 (1)
containing transposon Tn10 was introduced into P. citrea 1056R as described in Materials and Methods. Of the 2,578 independent Tcr mutants obtained, 8 were unable to color
pineapple juice; of these 8, 6 were still able to oxidize glucose into
gluconate. The phenotypes of these six mutants (CP6C8, 103C11, CP15E7,
CP9D9, CP9G10, and 105D2) regarding the ability to color pineapple
juice are shown in Fig. 2 and Table
2. The genes affected in these mutants
were cloned and sequenced, and their functions were analyzed as
described below (Fig. 3).
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Identification of a three-gene operon encoding a dehydrogenase
complex involved in pink disease formation.
Sequencing of the
CP9D9, CP9G10, and 105D2 insertion sites revealed that the transposon
was inserted at the same locus, orfB, in all three cases
(Fig. 3A). Further sequencing of the
region flanking this locus indicates that it contains two additional genes, orfA, and orfC (GenBank accession no.
AF131202). orfA, orfB, and orfC are
likely to be organized as an operon, since no transcription termination
signal could be detected between them. Intriguingly, no obvious
expression signals (35 and 10 boxes, ribosome-binding sequence)
were detected upstream of the operon. However, upstream of
orfA in the contig is a partial ORF (nt 1 to 443) showing
62% similarity at the amino acid level with a hypothetical ABC
transporter ATP-binding protein of Methanococcus jannaschii
(6) (data not shown) (Fig. 3A). This partial ORF is followed
by a potential rho-independent transcriptional terminator 79 bp
downstream of the stop codon (nt 524 to 537), indicating that it
belongs to a separate transcriptional unit.
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). We introduced these plasmids into strains CP9D9, CP9G10,
and 105D2 and tested four independent transformants in complementation
assays. Results indicated that none of the transformants restored the ability of the strain to induce the coloration of pineapple juice (data
not shown). This result could simply indicate that orfB was
not expressed in our construct, or that the defect in the three mutant
strains is due to a polar effect of the transposon insertion on
orfC. To test these hypotheses, we cloned orfC
alone, as well as a fragment containing both orfB and
orfC or orfB and orfA (data not
shown). However, none of these constructs could complement the mutants,
strongly suggesting that the three genes are necessary for
complementation. We could rule out a polar effect on an ORF downstream
of orfC since no significant ORF (longer than 60 aa) could
be detected downstream of orfC (data not shown). We then
amplified a 3,848-bp DNA fragment containing the three ORFs, as
described in Materials and Methods, and added the ligation mixture to E. coli DH5 competent cells.
However, none of the transformants contained an insert of the correct
size: all of the plasmids recovered after transformation carried
deletions, suggesting that the operon might encode a product that is
toxic to E. coli. Our attempts to clone the operon
directly in P. citrea CP9D9 or in a lower-copy-number vector
(such as pACYC184) were also unsuccessful (the plasmids recovered after
transformation also carried deletions), again suggesting a toxic effect
results when the entire operon is expressed.
Biogenesis of c-type cytrochrome is required for pink
disease formation.
Isolation and sequencing of the CP6C8 insertion
site demonstrated that the Tn10 was inserted in 1,734-bp ORF
that would encode a 578-aa protein (nt 150 to 1884) (Fig. 3B). Homology
searches revealed that this putative protein is 80% similar to the
E. coli DsbD protein, a protein involved in the biogenesis
of c-type cytochromes (11). By analogy, the
putative protein of P. citrea was called DsbD. A thioredoxin
family active site characteristic of thiol-disulfide interchange
proteins was also found between aa 484 and 512 at the C terminus of
DsbD. Analysis of the sequences flanking dsbD revealed that
it is surrounded by two genes showing more than 70% homology with
cutA (upstream) and cutA3 (downstream) that are
involved in copper tolerance in E. coli (15). No
transcriptional terminator could be detected between the three genes,
suggesting that they form an operon structure. To confirm that
alteration of DsbD function is responsible for the pink
phenotype of mutant CP6C8, we introduced a plasmid containing the gene,
pUCD5077, into strain CP6C8 and randomly selected four independent
transformants. These transformants were tested for the ability to
induce pink disease and were all able to color pineapple juice, albeit
at 75% of the wild-type strain level (data not shown), showing that
the defect in CP6C8 can be complemented solely by dshD.
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The pink mutants are deficient for gluconate or 2-KDG
dehydrogenase activity.
The pathway forming 2,5-DKG from
D-glucose oxidation appears to be responsible for the
coloration of pineapple juice. To determine directly at which
step of the pathway the mutants were affected, CP6C8 (dsbD),
CP15E7 (ccmC), and CP9D9 and CP9G10 (orfB) cells were assayed for gluconate dehydrogenase and 2-KDG
dehydrogenase activities as described in Materials and Methods.
No gluconate dehydrogenase activity was detected in mutant CP6C8
or CP15E7, while mutants CP9D9 and CP9G10 were able to oxidize
D-Gln. However, mutants CP6C8 and CP15E7 were able to
oxidize 2-KDG, while mutants CP9D9 and CP9G10 were not (data not
shown). These results were confirmed by direct deletion of 2-KDG and
2,5-DKG by TLC. P. citrea wild-type strain 1056R and the
above mutants were grown for 3 days in MGY medium supplemented
with 50 mM D-glucose, D-Gln, or 2-KDG, and
culture supernatants were analyzed by TLC (see Materials and Methods).
As shown in Fig. 4, P. citrea 1056R accumulates 2,5-DKG when
grown in the presence of D-glucose, D-Gln, or
2-KDG. When mutant strains CP6C8 and CP15E7 were grown in the presence of glucose or gluconate, only a weak light blue spot corresponding to
gluconate was detected (data not shown); no spot characteristic of
2-KDG was detected. However, when these strains were grown in presence
of 2-KDG, a spot corresponding to 2,5-DKG was visible, although to a
lesser intensity than with the wild-type strain (data not shown; see
Discussion). These results indicate that strains CP6C8 and CP15E7,
deficient for dsbD and ccmC, respectively, are
affected in their gluconate dehydrogenase activity and, to a
lesser extent, in the ability to oxidize 2-KDG. In contrast, mutant
strains CP9D9 and CP9G10, deficient in orfB, were able to product 2-KDG when grown in presence of glucose or gluconate but
could not convert this compound to 2,5-DKG (Fig. 4B), confirming that
they lack 2-KDG dehydrogenase activity. Taken together, these results
demonstrate that production of 2,5-DKG by this pathway in P. citrea is responsible for pink disease of pineapple.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This study provides evidence for the presence of an oxidative pathway beginning with D-glucose and ending with 2,5-DKG in P. citrea. The end product, 2,5-DKG, is a highly chromogenic compound that turns intensely rusty red when heated and appears to be the primary contributor of the pink-to-red coloration associated with pink disease of pineapple.
The rationale behind this work was to identify the complete pathway leading to the economically important pink disease of pineapple at the molecular level, using transposon mutagenesis and biochemical assays. Previously, we showed that oxidation of D-glucose to D-Gln was required for the disease (7, 26). However, addition of -D-Gln in the medium could bypass the need for glucose dehydrogenase activity, indicating that the complete pathway included at least one additional step. In Erwinia sp., D-Gln can be converted to 2-keto gluconate or 5-keto gluconate by membrane-bound dehydrogenases linked to the cytochrome chain that release the products in the periplasmic space (33, 34, 37). We showed that sugars of the Entner-Doudoroff pathway, 5-DKG, L-gulonic acid, and sugars of the pentose phosphate pathway were not involved in the dark coloration by P. citrea (Fig. 1 and data not shown). However, 2-KDG allowed for an intense coloration of the medium by P. citrea, demonstrating that conversion of D-gluconate to 2-KDG was a second important step in the pathway (26). 2-KDG can then be further oxidized to 2,5-DKG. Interestingly, the addition of 2,5-DKG to the culture medium produced the red color characteristic of pink disease, even in the absence of bacterial growth, strongly suggesting that accumulation of 2,5-DKG by P. citrea is responsible for the coloration of pineapple juice (data not shown).
This scenario received strong support from our genetic screen of P. citrea mutants which are deficient in the ability to induce the disease. The first series of mutants, corresponding to strains CP6C8, 103C11, and CP15E7, are unable to oxidize D-Gln to 2-KDG and are therefore affected in their D-gluconate dehydrogenase activity (Fig. 4B). The second series of mutants, corresponding to strains CP9D9, CP9G10, and 105D2, are clearly deficient in the ability to convert 2-KDG to 2,5-DKG. The reason behind these deficiencies were, however, multiple and therefore allowed us to gain insight regarding the catabolism of glucose in P. citrea and the use of cytochromes c. c-type cytochromes are found in many respiratory chains and are usually located in the periplasm or attached to the periplasmic side of the cytoplasmic membrane (13, 14, 25). They serve as specific electron acceptors linked to various oxidation reactions, including those resulting from dehydrogenase activities (10, 12, 43).
Mutant strain CP6C8 (Fig. 3B) is inactivated in a gene potentially encoding a 578-aa protein that is very (70 to 80%) similar to the disulfide bond isomerase proteins DsbD and DipZ of E. coli and Haemophilus influenzae, respectively (data not shown). In E. coli, DsbD is involved in the biogenesis of c-type cytochromes by maintaining cytochrome c apoproteins in the correct conformation for the covalent attachment of heme (11). In E. coli, DsbD is a cytoplasmic membrane protein with a thioredoxin-like domain at its C terminus, a domain that is also found in the DsbD protein of P. citrea (aa 484 to 512) (23). Mutant strains 103C11 and CP15E7 (Fig. 3C) are defective in a 738-bp gene showing 80% identity with the ccmC gene of E. coli. This gene is part of an eight-gene operon which shares significant homology, and a conserved structure, with the ccm operon of E. coli (Table 3). This operon encodes eight membrane-associated proteins required for cytochrome c maturation in E. coli (for a review, see reference 40). More specifically, CcmC is required for the transfer of the heme prosthetic group of c-type cytochrome to the newly synthesized apocytochrome c (31, 32). Taken together, these three mutant trains are affected in genes involved in the biogenesis of c-type cytochromes.
In P. citrea, a defect in the biogenesis of c-type cytochromes is responsible for a failure of gluconate dehydrogenase activity. It is worth mentioning that when 2-KDG was added to the growth medium of these mutant cells, only a small amount of 2,5-DKG was detected, indicating that c-type cytochromes might also be involved in 2-KDG dehydrogenase activity (data not shown). In contrast, D-glucose dehydrogenase activity was not affected in these mutants, in agreement with our previous results showing that this reaction uses pyroloquinoline quinone as an electron acceptor (26).
Mutant strains CP9D9, CP9G10, and 105D2 are affected in a putative 1,659-bp gene (orfB) that appears to be part of a three-gene operon (Fig. 3A and 4). The corresponding protein, OrfB, shared 34% similarity with the E. cypripedii membrane-bound gluconate dehydrogenase. In this organism, the gluconate dehydrogenase is thought to be composed of three subunits corresponding to the dehydrogenase itself, a cytochrome c subunit, and a third small subunit of unknown function (43). A similar organization has also been reported for Gluconobacter dioxyacetonicus (34). Interestingly, the product of the third gene of the P. citrea operon, OrfC, shows 60% similarity to the cytochrome c subunit of the E. cypripedii gluconate dehydrogenase complex (43), as well as to the A. aceti alcohol dehydrogenase (38). This finding, together with our results regarding the phenotype of the orfB mutations, allows us to suggest that the P. citrea operon encodes for a three-subunit, 2-keto-D-gluconate dehydrogenase. This enzyme therefore carries out the last step of the pathway leading to the accumulation of 2,5-DKG, the likely cause of the dark coloration of pineapple.
A way to confirm the hypothesis that the accumulation of 2,5-DKG by P. citrea is the cause of the pink coloration is to use a 2,5-DKG reductase that would catalyze conversion of 2,5-DKG to 2-keto-L-gulonic acid (2-KLG), as is observed in a number of organisms such as Corynebacterium sp. (3, 36). 2-KLG can then be further reduced to gulonic acid, which is not a substrate for coloring the medium (data not shown). For this purpose, we introduced plasmid pTrp1-35 containing the 2,5-DKG dehydrogenase from Corynebacterium sp. (3, 21) into P. citrea and found that transformants still retained the ability to color pineapple juice (data not shown). This negative result can be interpreted in several ways. One possibility is that the Corynebacterium sp. 2,5-DKG reductase does not efficiently reduce 2,5-DKG to 2-KLG in P. citrea. In support of this possibility, the 2,5-DKG reductase used here, 2,5-DKG reductase I, has a specific activity 33 times lower than that of the 2,5-DKG reductase II from Corynebacterium sp. used by Grindley et al. to convert glucose to 2-KLG in a recombinant strain of P. citrea (16). This might explain why 2,5-DKG was not efficiently reduced in our experiments. A second possibility is that P. citrea possesses enzymes that efficiently reduce 2-KLG to idonate and 2-KDG to gluconate, which in effect would produce even more substrate for the 2-KDG dehydrogenase. Indeed, some 2-KDG reductases from acetic acid bacteria are able to catalyze the reduction of 2-KLG to L-idonate and are also able to reduce 2-KDG to D-gluconate (2, 44). In support of this possibility, kinetics experiments showed that 2,5-DKG accumulated even faster in the presence of plasmid pTrp1-35 (data not shown). Therefore, the failure of our attempt to eliminate accumulation of 2,5-DKG does not disprove our initial suggestion.
In conclusion, we have described new genes in P. citrea that are involved in the coloration of pineapple juice. These genes allowed the identification of the biochemical pathway leading to the production of 2,5-DKG, which we now identify as the chromogenic compound responsible for the dark color characteristic of pink disease of pineapple.
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ACKNOWLEDGMENTS |
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We thank M. Alexeyev and L. Thöny-Meyer for kindly providing plasmids pBSL346 and pEC2, respectively. We appreciate Stephen Anderson for providing plasmid pTrp135-A. We thank Peter Pujic for supplying 2,5-DKG and gulonic acid and for helpful discussion. We are grateful to Frédéric Chédin and Timothy Durfee for critical reading of the manuscript. We thank Natalie Macher and Viet Pham for technical help.
This work was sponsored by Dole Philippines, Inc.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Plant Pathology, University of California, One Shields Avenue, Davis, CA 95616. Phone: (530) 752-0325. Fax: (530) 752-5674. E-mail: cikado{at}ucdavis.edu.
Present address: Plant and Microbial Biology Department, University
of California, Berkeley, CA 94720-3102.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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