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Journal of Bacteriology, September 2000, p. 4744-4751, Vol. 182, No. 17
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Genetic Analysis of a Gene Cluster for Cyclohexanol Oxidation in Acinetobacter sp. Strain SE19 by In Vitro Transposition

Qiong Cheng,1,* Stuart M. Thomas,1 Kristy Kostichka,1 James R. Valentine,2 and Vasantha Nagarajan1

Biological and Chemical Sciences and Engineering1 and Corporate Center for Analytical Sciences,2 Central Research and Development, E. I. DuPont de Nemours Inc., Wilmington, Delaware 19880-0328

Received 15 February 2000/Accepted 6 June 2000

Biological oxidation of cyclic alcohols normally results in formation of the corresponding dicarboxylic acids, which are further metabolized and enter the central carbon metabolism in the cell. We isolated an Acinetobacter sp. from an industrial wastewater bioreactor that utilized cyclohexanol as a sole carbon source. A cosmid library was constructed from Acinetobacter sp. strain SE19, and oxidation of cyclohexanol to adipic acid was demonstrated in recombinant Escherichia coli carrying a SE19 DNA segment. A region that was essential for cyclohexanol oxidation was localized to a 14-kb fragment on the cosmid DNA. Several putative open reading frames (ORFs) that were expected to encode enzymes catalyzing the conversion of cyclohexanol to adipic acid were identified. Whereas one ORF showed high homology to cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871, most of the ORFs showed only moderate homology to proteins in GenBank. In order to assign functions of the various ORFs, in vitro transposon mutagenesis was performed using the cosmid DNA as a target. A set of transposon mutants with a single insertion in each of the ORFs was screened for cyclohexanol oxidation in E. coli. Several of the transposon mutants accumulated a variety of cyclohexanol oxidation intermediates. The in vitro transposon mutagenesis technique was shown to be a powerful tool for rapidly assigning gene functions to all ORFs in the pathway.

* Corresponding author. Mailing address: E. I. DuPont de Nemours Inc., Experimental Station, E328/B48, Wilmington, DE 19880-0328. Phone: (302) 695-9952. Fax: (302) 695-1829. E-mail: qiong.cheng{at}usa.dupont.com.

Journal of Bacteriology, September 2000, p. 4744-4751, Vol. 182, No. 17
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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