Mol Gen Genet (1987) 207:349-354 © Springer-Verlag 1987 The xylS gene positive regulator of TOL plasmid pWWO: Identification, sequence analysis and overproduction leading to constitutive expression of meta cleavage operon Nicolas Mermod, Juan L. Ramos, Amos Bairoch, and Kenneth N. Timmis* Department of Medical Biochemistry, University of Geneva, CH-1211 Gen~ve 4, Switzerland Summary. The Pseudomonas putida TOL plasmid pWWO carries an operon that specifies a meta-cleavage pathway for the catabolism of benzoate and toluates whose tran- scription is positively regulated by the xylS gene product. Stimulation of transcription of the operon is thought to result from activation of this protein by pathway substrates/ effectors. In the present study, overexpression of the xylS gene has led to identification of the regulator as a 33 kDa protein. Overexpression of xylS also resulted in partially constitutive, i.e. effector-independent expression of the meta-cleavage operon. Determination of the polynucleotide sequence of the xylS gene revealed amino acid sequence homology with several DNA binding proteins, particularly with the araC products of Escherichia coli and Salmonella typhimurium and with the nifA and ntrC products of Kleb- siella pneumoniae. Homologous sequences were mainly lo- cated in an ~-helix-turn-c~-helix domain of the polypeptide. Interestingly, amino acid sequence homology was also found with sigma factors of E. coli (ntrA and htpR prod- ucts) and Bacillus subtilis (spolIG and phage SPOI Gp34 products) and other RNA polymerase core-interacting pro- teins, such as the E. coli nusA product. Key words: Pseudomonas - TOL plasmid - Positive regula- tion - DNA binding proteins - Sigma factors Introduction Pseudomonasputida TOL plasmid pWWO encodes enzymes required for the catabolism of toluene and substituted deriv- atives of toluene (Kunz and Chapman 1981; Worsey and Williams 1975). The genes coding for these enzymes are grouped into two operons : the upper pathway operon speci- fies oxidation of toluene, m- and p-xylenes to the corre- sponding carboxylic acids, benzoate or toluates, while the recta-cleavage pathway operon specifies further oxidation of benzoate and toluates to Krebs cycle precursors (Fig. 1). Transcription of the two operons is determined by positive- ly regulated promoters (Inouye et al. 1984a, 1984b; Mer- rood et al. 1984). Transcription of the meta-cleavage path- way operon is activated by the xylS gene product in combi- nation with benzoate or toluates, whereas that of the upper pathway operon is induced by the xylR gene product in combination with toluene or xylene or their alcohol deriva- tives (Fig. 1). Interestingly, upper pathway substrate/effec- tors such as xylene, in combination with xy/R gene product, Offprint requests to ." K.N. Timmis activate expression of the meta-cleavage pathway operon (Franklin etal. 1981, 1983; Inouye et al. 1981, 1983). This activation also involves the xyIS product but does not re- quire meta-cleavage pathway substrates/effectors. In terms of its genetics and biochemistry, the TOL path- way is the most extensively characterized catabolic pathway of a soil microorganism, and it represents an important model system for other aromatic degradation pathways (e.g., Mermod et al. 1986b). Nevertheless, the mechanism of action of the xylR and xylS gene products, and their interaction in the xylR-mediated induction of transcription of the meta operon, are poorly understood. As an initial step towards elucidation of these activities, we have overex- pressed the xylS gene and thereby identified its product. We have determined its amino acid sequence through se- quencing of its gene, and have analysed salient features of this sequence by comparison with other proteins that stimulate transcription. Materials and methods Bacterial strains and general methods. Strains and plasmids are listed in Table 1 and were handled as reported pre- viously (Mermod et al. 1986c). Standard protocols were used for DNA manipulations and sequencing, and for as- saying catechol 2,3-dioxygenase (Mermod et al. 1984; Har- ayama et al. 1986b). Mutagenesis of pKT800 with TnlO00 (Guyer 1978), and in vivo labelling with 3SS-methionine of plasmid-specified proteins in maxicells (Sancar et al. 1979) was performed as described previously. DNA and protein sequence analysis. Computer assisted se- quence analysis was performed using a program package (PC/GENE) developed by one of us (A.B.). The EMBL nucleotide sequence data library and the Swiss-Prot Protein Sequence Data Bank were scanned for sequence relatedness, using the method of Needleman and Wunsch (1970) as im- plemented by Dayhoff (1978). Secondary structure predic- tions were computed using conformational parameters of Chou and Fasman (1978). Results and Discussion xylS gene localization and product identification The 1.6 kb BamHI fragment of pNM185 containing the xylS gene was inserted into the lambda PL promoter expres- sion vector pLV85 in the orientation that results in tran-