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Applied and Environmental Microbiology, October 2007, p. 6551-6556, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.00493-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Membrane-Bound, 2-Keto-D-Gluconate-Yielding D-Gluconate Dehydrogenase from "Gluconobacter dioxyacetonicus" IFO 3271: Molecular Properties and Gene Disruption{triangledown}

Hirohide Toyama,1,2* Naoko Furuya,1 Ittipon Saichana,1 Yoshitaka Ano,1 Osao Adachi,1 and Kazunobu Matsushita1

Department of Biological Chemistry, Faculty of Agriculture,1 Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan2

Received 4 March 2007/ Accepted 14 August 2007

Most Gluconobacter species produce and accumulate 2-keto-D-gluconate (2KGA) and 5KGA simultaneously from D-glucose via GA in culture medium. 2KGA is produced by membrane-bound flavin adenine dinucleotide-containing GA 2-dehydrogenase (FAD-GADH). FAD-GADH was purified from "Gluconobacter dioxyacetonicus" IFO 3271, and N-terminal sequences of the three subunits were analyzed. PCR primers were designed from the N-terminal sequences, and part of the FAD-GADH genes was cloned as a PCR product. Using this PCR product, gene fragments containing whole FAD-GADH genes were obtained, and finally the nucleotide sequence of 9,696 bp was determined. The cloned sequence had three open reading frames (ORFs), gndS, gndL, and gndC, corresponding to small, large, and cytochrome c subunits of FAD-GADH, respectively. Seven other ORFs were also found, one of which showed identity to glucono-{delta}-lactonase, which might be involved directly in 2KGA production. Three mutant strains defective in either gndL or sldA (the gene responsible for 5KGA production) or both were constructed. Ferricyanide-reductase activity with GA in the membrane fraction of the gndL-defective strain decreased by about 60% of that of the wild-type strain, while in the sldA-defective strain, activity with GA did not decrease and activities with glycerol, D-arabitol, and D-sorbitol disappeared. Unexpectedly, the strain defective in both gndL and sldA (double mutant) still showed activity with GA. Moreover, 2KGA production was still observed in gndL and double mutant strains. 5KGA production was not observed at all in sldA and double mutant strains. Thus, it seems that "G. dioxyacetonicus" IFO 3271 has another membrane-bound enzyme that reacts with GA, producing 2KGA.

* Corresponding author. Present address: Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, Okinawa 903-0213, Japan. Phone and fax: 81-98-895-8805. E-mail: toyama{at}agr.u-ryukyu.ac.jp

{triangledown} Published ahead of print on 24 August 2007.

Applied and Environmental Microbiology, October 2007, p. 6551-6556, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.00493-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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