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Applied and Environmental Microbiology, May 2007, p. 3327-3332, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.00064-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Novel Genes Involved in Long-Chain n-Alkane Degradation by Acinetobacter sp. Strain DSM 17874{triangledown}

Mimmi Throne-Holst,1,2* Alexander Wentzel,1 Trond E. Ellingsen,2 Hans-Kristian Kotlar,3 and Sergey B. Zotchev1

Department of Biotechnology, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway,1 Department of Biotechnology, SINTEF Materials & Chemistry, 7465 Trondheim, Norway,2 Statoil ASA, R&D, Trondheim, Norway3

Received 11 January 2007/ Accepted 20 March 2007

Acinetobacter sp. strain DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from that of decane (C10H22) to that of tetracontane (C40H82) as a sole carbon source. Two genes encoding AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, have been shown to be involved in the degradation of n-alkanes with chain lengths of from 10 to 20 C atoms in this strain. Here, we describe a novel high-throughput screening method and the screening of a transposon mutant library to identify genes involved in the degradation of n-alkanes with C chain lengths longer than 20, which are solid at 30°C, the optimal growth temperature for Acinetobacter sp. strain DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. strain DSM 17874 transposon mutants was constructed and screened for mutants unable to grow on dotriacontane (C32H66) while simultaneously showing wild-type growth characteristics on shorter-chain n-alkanes. For 23 such mutants isolated, the genes inactivated by transposon insertion were identified. Targeted inactivation and complementation studies of one of these genes, designated almA and encoding a putative flavin-binding monooxygenase, confirmed its involvement in the strain's metabolism of long-chain n-alkanes. To our knowledge, almA represents the first cloned gene shown to be involved in the bacterial degradation of long-chain n-alkanes of 32 C's and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane-degrading Acinetobacter strains.

* Corresponding author. Mailing address: SINTEF Materials and Chemistry, Department of Biotechnology, Sem Saelandsvei 2A, 7465 Trondheim, Norway. Phone: 47 93092913. Fax: 47 73596995. E-mail: Mimmi.Throne-Holst{at}sintef.no

{triangledown} Published ahead of print on 30 March 2007.

Applied and Environmental Microbiology, May 2007, p. 3327-3332, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.00064-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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