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Applied and Environmental Microbiology, January 2003, p. 334-342, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.334-342.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

mRNA Differential Display in a Microbial Enrichment Culture: Simultaneous Identification of Three Cyclohexanone Monooxygenases from Three Species

Patricia C. Brzostowicz, Dana M. Walters, Stuart M. Thomas, Vasantha Nagarajan, and Pierre E. Rouvière*

DuPont Central Research and Development, DuPont Experimental Station, Wilmington, Delaware 19880-0328

Received 9 July 2002/ Accepted 1 October 2002

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.

* Corresponding author. Mailing address: DuPont Central Research and Development, DuPont Experimental Station, P.O. Box 80328, Wilmington, DE 19880-0328. Phone: (302) 695-1782. Fax: (302) 695-1829. E-mail: pierre.e.rouviere{at}usa.dupont.com.

Applied and Environmental Microbiology, January 2003, p. 334-342, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.334-342.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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