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Journal of Bacteriology, November 2001, p. 6662-6666, Vol. 183, No. 22
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.22.6662-6666.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

The TOL Plasmid pWW0 xylN Gene Product from Pseudomonas putida Is Involved in m-Xylene Uptake

Yuki Kasai, Jun Inoue, and Shigeaki Harayama*

Marine Biotechnology Institute, Heita, Kamaishi, Iwate, Japan

Received 23 July 2001/Accepted 17 August 2001

The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives. The last gene of the upper operon, xylN, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to FadL, an outer membrane protein involved in fatty acid transport in Escherichia coli. To analyze the role of the xylN gene product, xylN on TOL plasmid pWW0 was disrupted by inserting a kanamycin resistance gene, and the phenotypes of P. putida harboring the wild-type and xylN mutant TOL plasmids were characterized. The growth of P. putida harboring the wild-type TOL plasmid was inhibited by a high concentration of m-xylene, while that of P. putida harboring the xylN mutant TOL plasmid was not. The apparent Ks value for the oxidation of m-xylene in intact cells of the xylN mutant was fourfold higher than that of the wild-type strain, although the TOL catabolic enzyme activities in cell extracts from the two strains were almost identical. We therefore presume that the xylN gene product is a porin involved in the transport of m-xylene and its analogues across the outer membrane. Western blot analysis confirmed the localization of XylN in the outer membrane.

* Corresponding author. Mailing address: Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan. Phone: (81) 193 26 6544. Fax: (81) 193 26 6592. E-mail: shigeaki.harayama{at}kamaishi.mbio.co.jp.

Journal of Bacteriology, November 2001, p. 6662-6666, Vol. 183, No. 22
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.22.6662-6666.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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