Cloning and characterization of genes encoding toluene monooxygenases from Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 is able to use numerous substituted benzenes as its sole carbon source. Previous studies have shown that the catabolic pathways for substituted benzene degradation in strain JS150 involve an initial dioxygenation of the aromatic ring to yield a cis-dihydrodiol intermediate, this intermediate then undergoes a dehydrogenation to form the corresponding substituted catechol. The present study shows that this strain also carries genes encoding enzymes for the monooxygenation of substituted benzenes. Two gene clusters were cloned from strain JS150 which allowed Pseudomonas aeruginosa to transform phenol, benzene, toluene, and chlorobenzene to the corresponding catechols. One of the gene clusters, contained on recombinant plasmid pRO2015, was obtained from the chromosome of strain JS150. Pseudomonas aeruginosa (pRO2015) initiated toluene degradation via a 4-monooxygenation of the aromatic ring to yield para-cresol, this compound was subsequently transformed to 4-methylcatechol. The second gene cluster, contained on recombinant plasmid pRO2016, was obtained from one of the plasmids carried by strain JS150. Pseudomonas aeruginosa (pRO2016) initiated toluene degradation by monooxygenation at either positions 2- or 4- of the aromatic ring to yield ortho- and para-cresol, which were subsequently transformed to 3- and 4-methylcatechol, respectively. Oxygen ($\sp{18}$O$\sb2$) incorporation experiments followed by gas chromatography-mass spectrometry of products resulting from toluene oxidation confirmed that toluene hydroxylation was catalyzed through authentic monooxygenase reactions. Molecular characterization of recombinant plasmid, pRO2016 showed that genes encoding two different monooxygenases for the hydroxylation of benzene nuclei had been cloned. One was designated toluene/benzene-4-monooxygenase and the second, toluene/benzene-2-monooxygenase. The expression of the genes encoding the monooxygenases was coordinately regulated by a trans-acting regulatory protein subsequently designated TbmR. Analysis of the deduced amino acid sequences indicated that toluene/benzene-2-monooxygenase was similar to the multicomponent phenol hydroxylases from various Pseudomonas putida strains. Analysis of the partial deduced amino acid sequence of toluene/benzene-4-monooxygenase suggested that it was closely related to the multicomponent toluene-3- and toluene-4-monooxygenases from Burkholderia pickettii PKO1 and Pseudomonas mendocina KR1, respectively.PhDMicrobiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104797/1/9610153.pdfDescription of 9610153.pdf : Restricted to UM users only