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Appl. Environ. Microbiol. doi:10.1128/AEM.00064-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Identification of novel genes involved in long-chain n-alkane degradation by Acinetobacter sp. DSM 17874

Department of Biotechnology, NTNU, 7491 Trondheim, Norway; Department of Biotechnology, SINTEF Materials & Chemistry, 7465 Trondheim, Norway; Statoil ASA, R&D, Trondheim, Norway

^* To whom correspondence should be addressed. Email: Mimmi.Throne-Holst{at}sintef.no.

	Abstract

Acinetobacter sp. DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from decane (C₁₀H₂₂) to 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 from C10 to C20 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 a chain length longer than C20, which are solid at 30^oC, the optimal growth temperature for Acinetobacter sp. DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. 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 on 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 C32 and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane degrading Acinetobacter strains.