CLONING, NUCLEOTIDE-SEQUENCE, AND CHARACTERIZATION OF THE GENES ENCODING ENZYMES INVOLVED IN THE DEGRADATION OF CUMENE TO 2-HYDROXY-6-OXO-7-METHYLOCTA-2,4-DIENOIC ACID IN PSEUDOMONAS-FLUORESCENS IP01

A gene cluster encoding cumene (isopropylbenzene)-degrading enzymes wa s cloned in Escherichia coli JM109 from newly isolated Pseudomonas flu orescens strain IP01. E. coli cells containing pIP103 (10.8-kb insert) converted cumene, other monocyclic aromatic hydrocarbons and biphenyl into the meta-cleaved compounds, whereas the cells containing pIP107D (4.8-kb insert) formed the cis-dihydrodiol compounds, and the cells c ontaining pIP107 (5.2-kb insert) formed the catechol derivatives. We p roved that this oxidation system degrades cumene using three enzymes, aromatic-ring dioxygenase, dihydrodiol dehydrogenase, and extradiol di oxygenase. Moreover we determined the nucleotide sequence of a 6,508-b p region encoding aromatic-ring dioxygenase, dihydrodiol dehydrogenase , and extradiol dioxygenase, which subsequently led to identification of six out of seven open reading frames (ORFs) from P. fluorescens IP0 1. The nucleotide and deduced amino acid sequences for ORF1, 2, 4, and 5, designated cumA1A2A3A4, were found to be similar to those for know n multicomponent dioxygenase systems: todC1C2BA from Pseudomonas putid a F1 (51-65% amino acid sequence identity) and bphA1A2A3A4 from Pseudo monas pseudoalcaligenes KF707 (59-78%). Two Cys-His pairs in the large subunit of iron-sulfur proteins were conserved in CumA1, and a simila r Cys-His pair, conserved in the ferredoxin component of other dioxyge nase systems, was found in CumA3. ORF6, designated cumB, was homologou s to the dihydrodiol dehydrogenase gene of the fod (58%) and bph (73%) systems, while ORF7, designated cumC, was homologous to todE (47%) an d bphC (57%) which encode the meta-cleavage enzymes. Analysis of strai n IP01's nucleotide sequence revealed an overall G+C content of 53% fo r the cum gene cluster, less than the general G+C content of the chrom osome of P. fluorescens.