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. 2000 Apr;182(8):2230-7.
doi: 10.1128/JB.182.8.2230-2237.2000.

Genetic and biochemical characterization of the pathway in Pantoea citrea leading to pink disease of pineapple

C J Pujol  1 , C I Kado
Affiliations

Genetic and biochemical characterization of the pathway in Pantoea citrea leading to pink disease of pineapple

C J Pujol et al. J Bacteriol. 2000 Apr.

Abstract

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.

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Figures

FIG. 1
FIG. 1
Involvement of sugars in pink disease color. P. citrea 1056R cells were grown for 3 days in MGY supplemented with 50 mM sugar substrates from the Entner-Doudoroff pathway as described in Materials and Methods. The growth medium was then autoclaved for 5 min at 121°C, cells were removed by centrifugation, and the relative OD (inoculated medium versus noninoculated medium treated under the same conditions) was measured at 420 nm. Each bar corresponds to the mean of three independent measurements with standard deviation.
FIG. 2
FIG. 2
Phenotypes of various P. citrea mutants unable to induce pink disease of pineapple. Strains were grown for 3 days in pineapple juice at 30°C and then autoclaved for 5 min at 121°C. The P. citrea wild-type strain (1056R) was able to color pineapple juice, while mutants CP6C8, CP15E7, 105D2, 103C11, CP9D9, and CP9G10 were not . Pineapple juice medium that was not inoculated with bacteria remained yellow.
FIG. 3
FIG. 3
Schematic representation of the genes inactivated by transposition mutagenesis. Thin arrows represent the Tn10 insertion with the name of the mutant. Genes inactivated are designated by arrowed boxes; other genes are marked by dark arrows. Coordinates of genes are indicated. (A) Mutant CP9D9, CP9G10, and 105D2. Tn10 is inserted at nt 1858 (in opposite orientation in CP9D9 and CP9G10) and 2703, respectively. The sequence of the ABC transporter gene is only partial. (B) Mutant CP6C8. Tn10 is inserted at nt 1186. (C) Mutants CP15E7 and 103C11. Tn10 is inserted, in opposite orientation, at nt 2595 and 2604, respectively.
FIG. 4
FIG. 4
Qualitative detection of d-Gln, 2-KDG, and 2,5-DKG by TLC. P. citrea wild-type (1056R) and mutant strains were grown in MGY medium supplemented with 50 mM -d-glucose, d-Gln, or 2-KDG; 5μl of the culture supernatant were deposited on a Silica Gel 60 TLC plate and analyzed as described in Materials and Methods. Controls correspond to 5 μl of MGY supplemented with the corresponding sugar. The wild-type strain was able to accumulate 2,5-DKG when grown in the presence of d-glucose, d-Gln, or 2-KDG. (A) 1056R and mutants strains CP6C8 (dsbD) and CP15E7 (ccmC). Mutants CP6C8 and CP15E7 were able to produce only d-Gln. (B) 1056R and mutants strains CP9D9 and CP9G10 (orfB). These mutants are able to oxidize d-Gln to 2-KDG but are unable to accumulate 2,5-DKG. Arrows indicate the position of the running front of each sugar.
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