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

Abstract

A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. TnS transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism. These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts. Based on the results obtained, the 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 was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis. Isopropylbenzene-negative isolates carried a TnS insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4. DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors; clones were identified by (i) selection for TnS-encoded kanamycin resistance in the case of TnS mutant plasmids, (ii) screening for isopropylbenzene dioxygenase-catalyzed oxidation of indole to indigo, and (iii) use of a TnS-carrying restriction fragment, derived from a pRE4::TnS mutant plasmid, as a probe for clones carrying wild-type restriction fragments. These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4.The monoalkylbenzenes isopropylbenzene (cumene), ethylbenzene, and toluene are important industrially as synthetic intermediates and solvents (32). As a consequence of their widespread use and subsequent introduction into the environment, the latter two compounds were included on the U.S. Environmental Protection Agency's list of priority pollutants (39).The bacterial metabolism of toluene has been shown to occur by two different pathways. One, exemplified by that encoded by the well-studied TOL plasmid (54), is initiated by a multistep oxidation of the methyl group to give benzoate (40); the other, which is apparently chromosome encoded (26), is initiated by a dioxygenase-catalyzed oxidation of the aromatic ring to give 2,3-dihydro-2,3-dihydroxytoluene which is subsequently dehydrogenated to give 3-methylcatechol (28,29). Similarly, ethylbenzene has been shown to be primarily metabolized through 2,3-dihydro-2,3-dihydroxyethylbenzene to 3-ethylcatechol (27). A general pathway for the complete metabolism of these and other monoalkylbenzenes in which the side chain is not oxidized before ring cleavage has been described (Fig. 1), and evidence for this is summarized in two recent reviews (31, 47). Some evidence has been presented for the metabolism of isopropylbenzene through the ring-cleavage product IV depicted in Fig. 1 (29) and for the metabolism of this compound to isobutyrate, pyruvate, and ace...

Cloning of genes involved in carbazole degradation of Pseudomonas sp. strain CA10: nucleotide sequences of genes and characterization of meta-cleavage enzymes and hydrolase

“…In this study, we observed that E. coli harboring pUCA1 forms indigo on 2ϫ YT plates. This phenomenon was reported in the case of naphthalene dioxygenase (14) and cumene dioxygenase (2,12), suggesting that the pUCA1 insert contains genes encoding CARDO. Further sequence analysis of the upstream and downstream regions of carBC will be done to obtain information on the genes encoding CARDO.…”

Biodegradation of Aromatic Compounds by Escherichia coli

“…13) should be taken into consideration when cloning and expressing heterologous aromatic catabolic clusters in this enterobacterium. In this sense, several reports on the cloning and expression of aromatic ring-hydroxylating dioxygenases in E. coli claim that equivalent enzymes from the host could explain the unexpected activities observed when some of the subunits of the cloned dioxygenases were lacking in the recombinant bacteria (82,167,186,305). The described PP-dioxygenase activity of E. coli (see above) could explain some of these unexpected findings.…”

Bacterial metabolism of naphthalene: construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions

“…Barnsley also partially purified from cell extracts an enzyme (isomerase) (Fig. 1, enzyme D trans-4-(3-Hydroxy-2-thianaphthenyl)-2-oxo-but-3-enoate and trans-4-(3-hydroxy-2-benzofuranyl)-2-oxobut-3-enoate were produced by biotransformation of dibenzothiophene and dibenzofuran, respectively, by P. aeruginosa PAO1(pRE654) (11), while trans-4-(3-hydroxy-2-thienyl)-2-oxobut-3-enoate was a product of biotransformation of benzothiophene by P. putida RE204 (13,15 (26) followed by centrifugation to equilibrium through a cesium chloride-ethidium bromide gradient. Plasmid DNA was isolated from E. coli by the method of Holmes and Quigley (29) when recombinants were screened and by the procedure of Clewell and Helinski (6) for largescale preparations.…”