xyIUW, Two Genes at the Start of the upper Pathway Operon of TOL Plasmid Pwwo, Appear to Play no Essential Part in Determining its Catabolic Phenotype

Abstract

The upper pathway operon of the toluene catabolic pathway of TOL plasmid pWW0 was shown to carry two open reading frames between the start of transcription and xylC (encoding benzaldehyde dehydrogenase), the first previously reported gene of the operon. These were designated xyIUW: xyIU encoded a protein of 131 amino acid residues (Mr 14244) which bore no relationship with any protein in the databases, and xyIW encoded a protein of 348 residues (Mr 36992) which was strongly homologous to other long-chain Zn-containing alcohol dehydrogenases. Extracts of Escherichia coli carrying xyIUW in expression vector pTrc99A contained a novel protein corresponding to XyIW, but no NAD+ -dependent dehydrogenase activity against benzyl alcohol, mandelate or benzylamine. A mini-Tn5 transposon carrying the meta pathway operon was constructed and from it two strains of Pseudomonas putida were constructed with the normally plasmid-encoded catabolic operons integrated into the chromosome. Three derivatives of plasmid pKNG101 containing modified xyIUW genes were constructed, two of which had frameshifts in xyIU and xyIW, respectively, and a third with a deletion from the 3′ end of xyIU into the 5′ end of xyIW. The wild-type genes of the two Pseudomonas strains were substituted by the mutant alleles by reverse genetics. The ability of the constructed mutant strains to utilize the aromatic substrates of the TOL pathway was not significantly affected.