Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204.

摘要: Abstract A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. Tn5 transposon mutagenesis by means of suicide donor plasmid pLG221 yielded mutant derivatives defective in metabolism. These were characterized identification products which they accumulated when grown presence assay enzyme activities cell extracts. Based on results obtained, following metabolic pathway is proposed: isopropylbenzene----2,3-dihydro -2,3-dihydroxyisopropylbenzene----3-isopropylcatechol----2 -hydroxy-6-oxo-7-methylocta-2,4-dienoate----isobutyrate + 2-oxopent-4-enoate----amphibolic intermediates. Plasmid DNA isolated from RE204 restriction cleavage analysis. Isopropylbenzene-negative isolates carried a insert within 15-kilobase region 105-kilobase pRE4. fragments pRE4 carrying genes encoding catabolic enzymes cloned Escherichia coli with various vectors; clones identified (i) selection for Tn5-encoded kanamycin resistance case plasmids, (ii) screening dioxygenase-catalyzed oxidation indole indigo, (iii) use Tn5-carrying fragment, derived pRE4::Tn5 plasmid, probe wild-type fragments. subsequently used generate insertion map