A bacterial strain capable of utilizing n-alkanes with chain lengths ranging from decane (C10H22) to tetracontane (C40H82) as a sole carbon source was isolated using a system for screening microorganisms able to grow on paraffin (mixed long-chain n-alkanes). The isolate, identified according to its 16S rRNA sequence as Acinetobacter venetianus, was designated A. venetianus 6A2. Two DNA fragments encoding parts of AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, were polymerase chain reaction-amplified from the genome of A. venetianus 6A2. To study the roles of these two alkM paralogues in n-alkane utilization in A. venetianus 6A2, we constructed alkMa, alkMb, and alkMa/alkMb disruption mutants. Studies on the growth patterns of the disruption mutants using n-alkanes with different chain lengths as sole carbon source demonstrated central roles for the alkMa and alkMb genes in utilization of C10 to C18 n-alkanes. Comparative analysis of these patterns also suggested different substrate preferences for AlkMa and AlkMb in n-alkane utilization. Because both single and double mutants were able to grow on n-alkanes with chain lengths of C20 and longer, we concluded that yet another enzyme(s) for the utilization of these n-alkanes must exist in A. venetianus 6A2.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Baptist JN, Gholson RK, Coon MJ (1963) Hydrocarbon oxidation by a bacterial enzyme system: I. Products of octane oxidation. Biochim Biophys Acta 69:40–47
Brautaset T, Borgos SEF, Sletta H, Ellingsen TE, Zotchev SB (2003) Site-specific mutagenesis and domain substitution in the loading module of the nystatin polyketide synthase and their effects on nystatin biosynthesis in Streptomyces noursei. J Biol Chem 278:14913–14919
Chakrabarty AM, Chou G, Gunsalus JC (1973) Genetic regulation of the octane dissimilation plasmid in Pseudomonas. PNAS 70:1137–1140
de Lorenzo V, Eltis L, Kessler B, Timmis KN (1993) Analysis of Pseudomonas gene products using lacI q/Ptrp-lac plasmids and transposons that confer conditional phenotypes. Gene 123:17–24
Di Cello F, Pepi M, Baldi F, Fani R (1997) Molecular characterization of an n-alkane-degrading bacterial community and identification of a new species, Acinetobacter venetianus. Res Microbiol 148:237–249
Eggink G, Lageveen RG, Altenburg B, Witholt B (1987a) Controlled and functional expression of the Pseudomonas oleovorans alkane utilizing system in Pseudomonas putida and Escherichia coli. J Biol Chem 262:17712–17718
Eggink G, van Lelyveld PH, Arnberg A, Arfman N, Witteveen C, Witholt B (1987b) Structure of the Pseudomonas putida alkBAC operon. J Biol Chem 262:6400–6406
Hamamura N, Yeager CM, Arp DJ (2001) Two distinct monooxygenases for alkane oxidation in Nocardioides sp. strain CF8. Appl Environ Microbiol 67:4992–4998
Hara A, Baik S-H, Syutsubo K, Misawa N, Smits THM, van Beilen JB, Harayama S (2004) Cloning and functional analysis of alkB genes in Alcanivorax borkumensis SK2. Environ Microbiol 6:191–197
Kok M, Oldenhuis R, van der Linden MPG, Raatjes P, Kingma J, van Lelyveld PH, Witholt B (1989) Pseudomonas oleovorans alkane hydroxylase gene. Sequence and expression. J Biol Chem 264:5435–5441
Maeng JH, Sakai Y, Tani Y, Kato N (1996) Isolation and characterization of a novel oxygenase that catalyses the first step of n-alkane oxidation in Acinetobacter sp. strain M-1. J Bacteriol 178:3695–3700
Maier T, Förster H-H, Asperger O, Hahn U (2001) Molecular characterization of the 56-kDA CYP153 from Acinetobacter sp. EB104. Biochem Biophys Res Commun 286:652–658
Minas W, Gutnick DL (1993) Isolation, characterization and sequence analysis of cryptic plasmids from Acinetobacter calcoaceticus and their use in construction of Escherichia coli shuttle plasmids. Appl Environ Microbiol 59:2807–2816
Rosenberg M, Bayer EA, Delarea J, Rosenberg E (1982) Role of thin fimbriae in adherence and growth of Acinetobacter calcoaceticus RAG-1 on hexadecane. Appl Environ Microbiol 44:929–937
Sakai Y, Maeng JH, Tani Y, Kato N (1994) Use of long-chain n-alkanes (C13-C44) by an isolate, Acinetobacter sp. M-1. Biosci Biotechnol Biochem 58:2128–2130
Smits THM, Röthlisberger M, Witholt B, van Beilen JB (1999) Molecular screening for alkane hydroxylase genes in Gram-negative and Gram-positive strains. Environ Microbiol 1:307–317
Smits THM, Balada SB, Witholt B, van Beilen JB (2002) Functional analysis of alkane hydroxylases from Gram-negative and Gram-positive bacteria. J Bacteriol 184:1733–1742
Tani A, Ishige T, Sakai Y, Kato N (2001) Gene structure and regulation of the alkane hydroxylase complex in Acinetobacter sp. Strain M-1. J Bacteriol 183:1819–1823
van Beilen JB, Panke S, Lucchini S, Franchini AG, Röthlisberger M, Witholt B (2001) Analysis of the Pseudomonas putida alkane-degradation gene cluster and flanking insertion sequences: evolution and regulation of the alk genes. Microbiology 147:1621–1630
van Beilen JB, Neuenschwander M, Smits THM, Roth C, Balada SB, Witholt B (2002a) Rubredoxins involved in alkane oxidation. J Bacteriol 184:1722–1732
van Beilen JB, Smits THM, Whyte LG, Schorcht S, Röthlisberger M, Plaggemeier T, Engesser K-H, Witholt B (2002b) Alkane hydroxylase homologues in Gram-positive strains. Environ Microbiol 4:676–682
van Beilen JB, Li Z, Duetz WA, Smits THM, Witholt B (2003) Diversity of alkane hydroxylase systems in the environment. Oil Gas Sci Technol 58:427–440
van Beilen JB, Marín MM, Smits THM, Röthlisberger M, Franchini AG, Witholt B, Rojo F (2004) Characterization of two alkane hydroxylase genes from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis. Environ Microbiol 6:264–273
van Beilen, JB, Smits THM, Roos FF, Brunner T, Balada SB, Röthlisberger M, Witholt B (2005) Identification of an amino acid position that determines the substrate range of integral membrane alkane hydroxylases. J Bacteriol 187:85–91
Vaneechoutte M, Tjernberg I, Baldi F, Pepi M, Fani R, Sullivan ER, van der Toorn J, Dijkshoorn L (1999) Oil-degrading Acinetobacter strain RAG-1 and strains described as ‘Acinetobacter venetianus sp. nov.’ belong to the same genomic species. Res Microbiol 150:69–73
Van Hamme JD, Singh A, Ward OP (2003) Recent advances in petroleum microbiology. Microbiol Mol Biol Rev 67:503–549
Vomberg A, Klinner U (2000) Distribution of alkB genes within n-alkane-degrading bacteria. J Appl Microbiol 89:339–348
Whyte LG, Smits THM, Labbe D, Witholt B, Greer CW, van Beilen JB (2002) Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531. Appl Environ Microbiol 68:5933–5942
Zotchev SB, Haugan K, Sekurova O, Sletta H, Ellingsen TE, Valla S (2000) Identification of a gene cluster for antibacterial polyketide-derived antibiotic biosythesis in the nystatin producer Streptomyces noursei ATCC 11455. Microbiology 146:611–619
This project was supported by the VISTA foundation and Statoil AS.
About this article
Cite this article
Throne-Holst, M., Markussen, S., Winnberg, A. et al. Utilization of n-alkanes by a newly isolated strain of Acinetobacter venetianus: the role of two AlkB-type alkane hydroxylases. Appl Microbiol Biotechnol 72, 353–360 (2006). https://doi.org/10.1007/s00253-005-0262-9
- Sole Carbon Source
- Disruption Mutant
- alkB Gene
- Solid Droplet