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Journal of Bacteriology, March 2002, p. 1733-1742, Vol. 184, No. 6
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.6.1733-1742.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Functional Analysis of Alkane Hydroxylases from Gram-Negative and Gram-Positive Bacteria

Theo H. M. Smits,,{dagger} Stefanie B. Balada, Bernard Witholt, and Jan B. van Beilen*

Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Hönggerberg, CH-8093 Zürich, Switzerland

Received 2 November 2001/ Accepted 20 December 2001

We have cloned homologs of the Pseudomonas putida GPo1 alkane hydroxylase from Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens CHA0, Alcanivorax borkumensis AP1, Mycobacterium tuberculosis H37Rv, and Prauserella rugosa NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts, Escherichia coli GEc137 and P. putida GPo12, were equipped with pGEc47{Delta}B, which encodes all proteins necessary for growth on medium-chain-length alkanes (C6 to C12), except a functional alkane hydroxylase. The third host was an alkB knockout derivative of P. fluorescens CHA0, which is no longer able to grow on C12 to C16 alkanes. All alkane hydroxylase homologs, except the Acinetobacter sp. ADP1 AlkM, allowed at least one of the three hosts to grow on n-alkanes.

* Corresponding author. Mailing address: Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Hönggerberg, CH-8093 Zürich, Switzerland. Phone: 41-1-633 34 44. Fax: 41-1-633 10 51. E-mail: vanbeilen{at}biotech.biol.ethz.ch.

{dagger} Present address: ENAC/LBE, EPFL, CH-1015 Lausanne, Switzerland.

Journal of Bacteriology, March 2002, p. 1733-1742, Vol. 184, No. 6
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.6.1733-1742.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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