Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product

Abstract

TOL plasmid pWW0 specifies enzymes for the oxidative catabolism of toluene and xylenes. The upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. The synthesis of these enzymes is positively regulated by the product of xylR. Determination of upper pathway enzyme levels in bacteria carrying Tn5 insertion mutant derivatives of plasmid pWW0-161 has shown that the genes for upper pathway enzymes are organized in an operon with the following order: promoter-xylC (benzaldehyde dehydrogenase gene[s])-xylA (xylene oxygenase gene[s])-xylB (benzyl alcohol dehydrogenase gene). Subcloning of the upper pathway genes in a lambda pL promoter-containing vector and analysis of their expression in Escherichia coli K-12 confirmed this order. Two distinct enzymes were found to attack benzyl alcohol, namely, xylene oxygenase and benzyl alcohol dehydrogenase; and their catalytic activities were additive in the conversion of benzyl alcohol to benzaldehyde. The fact that benzyl alcohol is both a product and a substrate of xylene oxygenase indicates that this enzyme has a relaxed substrate specificity.

Primary structure of xylene monooxygenase: similarities to and differences from the alkane hydroxylation system

“…The source of the DNA containing both xylM and xylA was pGSH2836 (5). The methods for DNA * Corresponding author.…”

“…P. putida PaWl carrying the TOL plasmid pWWO and E. coli K12AHlAtrp carrying pGSH2836 have been described previously (5,8).…”

“…Xylene monooxygenase is the first enzyme in this pathway and oxidizes toluene and xylenes to (methyl)benzyl alcohols (26). The enzyme has a broad substrate specificity and also oxidizes (methyl)benzyl alcohols to (methyl)benzaldehydes and indole to indoxyl (5,13). The enzyme consists of two different subunits encoded by xylM and xylA, respectively (8).…”

Identification of a novel composite transposable element, Tn5280, carrying chlorobenzene dioxygenase genes of Pseudomonas sp. strain P51

“…Plasmid pTCB95 was constructed by cloning the 12-kb HindIII pP51 DNA fragment of pTCB60 in pSUP202. This plasmid was then partially digested with BamHI and ligated with a DNA fragment containing the kanamycin resistance (Kmr) gene of Tn903 (17), derived from plasmid pRME1 (19), with BamHI ends. Transformants of E. coli were subsequently analyzed for their plasmid content, and a plasmid carrying the Kmr gene in the BamHI site on the tcbB locus was used (pTCB95R3) (Fig.…”

Posttranscriptional control of puc operon expression of B800-850 light-harvesting complex formation in Rhodobacter sphaeroides

“…A 705-base-pair (bp) StuIApaI restriction endonuclease fragment was removed from pUI601 (21), and the linear plasmid DNA was blunt ended with the T4 DNA polymerase (24). A 1.5-kb HinclI restriction endonuclease fragment containing a Knr cartridge from pRME 1 (16) (35), which serves as a suicide vector in R. sphaeroides (8). The resulting plasmid, pPSUPPST::Kn(AStuI-ApaI), was transformed into E. coli S17-1 and mobilized into R. sphaeroides 2.4.1, and a Knr Tcs double crossover was isolated as previously described (8).…”