Identification of novel genes involved in long-chain n-alkane degradation by Acinetobacter sp. strain DSM 17874.
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 degrees 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.
Throne-Holst
Mimmi
M
SINTEF Materials and Chemistry, Department of Biotechnology, Norwegian University of Science and Technology, Sem Saelandsvei 2A, 7465 Trondheim, Norway. Mimmi.Throne-Holst@sintef.no
Wentzel
Alexander
A
Ellingsen
Trond E
TE
Kotlar
Hans-Kristian
HK
Zotchev
Sergey B
SB
D000150:Acinetobacter / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D000473:Alkanes / Q000378:metabolism*; D001673:Biodegradation, Environmental; D002244:Carbon / Q000378:metabolism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000473:Alkanes; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D002244:Carbon; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)