J Mol Evol (1994) 38:328-335 Journal of Molecular Evolution © Springer-Verlag New York Inc, 1994 Codon Usage Patterns Suggest Independent Evolution of Two Catabolic Operons on Toluene-Degradative Plasmid TOL pWW0 of Pseudomonas putida Shigeaki Harayama Department of Medical Biochemistry, University Medical Center, University of Geneva 1, rue Michel-Servet, 1211 Geneva 4, Switzerland Received: 25 April 1993 / Revised: 1 June 1993 Abstract. TOL plasmid pWW0 of Pseudomonas puti- da encodes a set of enzymes responsible for the degra- dation of toluene. The structural genes for these cato- bolic enzymes are clustered into two operons--namely, the xyICMAB and xylXYZLTEGFJQKIH operons. We examined the codon usage patterns of these catabolic genes by measuring the codon-usage distances between pairs of these catabolic genes. The codon-usage dis- tance, d, between gene 1 and gene 2 was defined as d = [~(pj _ qj)2] 1/2, where pj and qj are the frequencies of the j-th codon in gene 1 and 2, respectively, j being any one of the 64 possible codons. We found that the genes in the same operon exhibit similar codon-usage patterns while genes in the different operons exhibit different codon bias. This observation suggests that genes in the same operon have coevolved, and that the ancestors of the xyICMAB and xylXYZLTEGFJQKIH operons evolved in different organisms. Key words: TOL plasmid -- Pseudomonas putida -- Biodegradation -- Molecular evolution -- Codon usage -- Evolution of metabolic pathway Introduction TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes responsible for the degradation of Correspondence to: S. Harayama. Present address: Marine Biotech- nology Institute, Kamaishi Laboratory, 3-75-1 Heita, Kamaishi City, Iwate 026, Japan toluene. This substrate is initially oxidized by upper- pathway enzymes encoded by the xylCMAB genes to benzoate, which is further transformed to catechol by two enzymatic steps encoded by the xylXYZL genes. Catechol thus formed is then transformed to Krebs-cy- cle intermediates via a meta-cleavage route encoded by the xylTEGFJQKIH genes (Fig. 1). The xylCMAB genes for the upper-pathway enzymes are clustered in one operon (Harayama et al. 1989b), whereas the genes responsible for the degradation of benzoate, xylXYZLTEGFJQKIH, are encoded in another operon (Harayama and Rekik 1990). The degradation of naph- thalene and phenol also occurs via the meta-cleavage route, and the structural genes for the enzymes of the meta-cleavage route are also clustered in naphthalene and phenol-degrading organisms (Yen and Gunsalus 1982; Singler et al. 1992). The comparison of the orders and nucleotide sequences of the meta-cleavage route genes for the toluene, naphthalene, and phenol- catabolic pathways has indicated that an ancestral meta-cleavage route gene cluster equivalent to the xylTEGFJQKIH sequences has spread horizontally and been integrated into contemporary catabolic operons (Singler et al. 1992; Harayama et al. 1987; Ghosal et al. 1987; You et al. 1991; Harayama et al. 1991a; Bartil- son and Shingler 1989; Nordlund and Singler 1990; Nakai et al. 1983b; Horn et al. 1991). There are nu- merous bacteria which can use benzoate as a sole source of carbon and energy. In Acinetobacter calcoaceticus, for example, benzoate is transformed to catechol as in the TOL-plasmid-encoded pathway in P. putida, but catechol thus formed is not metabolized via meta-cleav-