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Appl Environ Microbiol, May 1998, p. 1650-1656, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification and Analysis of Genes Involved in Anaerobic Toluene Metabolism by Strain T1: Putative Role of a Glycine Free Radical

Peter W. Coschigano,1,* Thomas S. Wehrman,2,dagger and L. Y. Young2

Department of Biological Sciences and Program in Molecular and Cellular Biology, Ohio University, Athens, Ohio 45701-2979,1 and Biotechnology Center for Agriculture and the Environment, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903-02312

Received 9 December 1997/Accepted 2 March 1998

The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. The tutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.

* Corresponding author. Mailing address: Department of Biological Sciences, Ohio University, Athens, OH 45701-2979. Phone: (740) 593-9488. Fax: (740) 593-0300. E-mail: Coschiga{at}ohiou.edu.

dagger Present address: Department of Molecular Pharmacology, Stanford School of Medicine, Stanford University, Stanford, CA 94305-5332.

Appl Environ Microbiol, May 1998, p. 1650-1656, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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