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Appl Environ Microbiol, May 1998, p. 1612-1619, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

A Gene System for Glucitol Transport and Metabolism in Clostridium beijerinckii NCIMB 8052

Martin Tangney,1 John K. Brehm,2 Nigel P. Minton,2 and Wilfrid J. Mitchell1,*

Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS,1 and Department of Molecular Microbiology, Centre for Applied Microbiology and Research, Porton Down, Salisbury SP4 0JG,2 United Kingdom

Received 25 November 1997/Accepted 19 February 1998

The gutD gene of Clostridium beijerinckii NCIMB 8052 encoding glucitol 6-phosphate dehydrogenase was cloned on a 5.7-kbp chromosomal DNA fragment by complementing an Escherichia coli gutD mutant strain and selecting for growth on glucitol. Five open reading frames (ORFs) in the order gutA1 gutA2 orfX gutB gutD were identified in a 4.0-kbp region of the cloned DNA. The deduced products of four of these ORFs were homologous to components of the glucitol phosphotransferase system (PTS) and glucitol 6-phosphate dehydrogenase from E. coli, while the remaining ORF (orfX) encoded an enzyme which had similarities to members of a family of transaldolases. A strain in which gutD was inactivated by targeted integration lacked glucitol 6-phosphate dehydrogenase activity. The gutA1 and gutA2 genes encoded two polypeptides forming enzyme IIBC of the glucitol PTS comprising three domains in the order CBC. Domain IIA of the glucitol PTS was encoded by gutB. Glucitol phosphorylation assays in which soluble and membrane fractions of cells grown on glucose (which did not synthesize the glucitol PTS) or cells grown on glucitol were used confirmed that there is a separate, soluble, glucitol-specific PTS component, which is the product of the gutB gene. The gut genes were regulated at the level of transcription and were induced in the presence of glucitol. Cells grown in the presence of glucose and glucitol utilized glucose preferentially. Following depletion of glucose, the glucitol PTS and glucitol 6-phosphate dehydrogenase were synthesized, and glucitol was removed from the culture medium. RNA analysis showed that the gut genes were not expressed until glucose was depleted.


* Corresponding author. Mailing address: Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, United Kingdom. Phone: 44 131 451 3459. Fax: 44 131 451 3009. E-mail: w.j.mitchell{at}hw.ac.uk.


Appl Environ Microbiol, May 1998, p. 1612-1619, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.



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