despite their discrete substrate ranges for metabolism. The gene components responsible for substrate specificity between the bph and tod operons were investigated. The large subunit of the terminal dioxygenase (encoded by bphAI and todCl) and the ring meta-cleavage compound hydrolase (bphD and todF) were critical for their discrete metabolic specificities, as shown by the following results. (i) Introduction of todCIC2 (coding for the large and small subunits of the terminal dioxygenase in toluene metabolism) or even only todCl into biphenyl-utilizing P. pseudoalcaligenes KF707 and P. putida KF715 allowed them to grow on toluene-benzene by coupling with the lower benzoate meta-cleavage pathway.Introduction of the bphD gene (coding for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) into tolueneutilizing P. putida F1 permitted growth on biphenyl. (ii) With various bph and tod mutant strains, it was shown that enzyme components of ferredoxin (encoded by bphA3 and todB), ferredoxin reductase (bphA4 and todA), and dihydrodiol dehydrogenase (bphB and todD) were complementary with one another. (iii) Escherichia coli
Engineering of hybrid gene clusters between the toluene metabolic tod operon and the biphenyl metabolic bph operon greatly enhanced the rate of biodegradation of trichloroethylene. Trichloroethylene (TCE) has been recognized as one of the most significant environmental pollutants in soil and groundwater (11). TCE and related compounds have been shown to persist over time in the environment and are suspected to be carcinogenic (7). A recent study showed that TCE could be degraded by aerobic and anaerobic bacteria (1). An aerobic, methane-oxidizing bacterium that was isolated in pure culture degraded TCE by a cometabolic process (8). Nelson and coworkers (9) and Wackett and Gibson (12) showed that toluene dioxygenase plays a role in the degradation of TCE by using mutants of toluene-utilizing Pseudomonas putida Fl. This role was confirmed with Escherichia coli carrying the structural genes (todClC2BA) for toluene dioxygenase (15). Another toluene-degrading species, Pseudomonas mendocina, oxidizes toluene through toluene monooxygenase, which inserts a single atom of oxygen at the para position of toluene to form p-cresol (13). It was demonstrated that E. coli cells carrying a gene cluster coding for multicomponent toluene monooxygenase degraded TCE to carbon dioxide, chloride ion, and water-soluble metabolites. We have cloned from Pseudomonas pseudoalcaligenes KF707 bph genes coding for the catabolism of biphenyl (BP) and polychlorinated biphenyls (BPs) to (chloro)benzoic acids (3). The bphAJA2A3A4 genes, coding for BP dioxygenase (10), are similar to the todClC2BA genes, coding for toluene dioxygenase, in toluene-utilizing P. putida Fl (14), both in gene organization and in nucleotide sequence. The amino acid identities of the large subunit of terminal dioxygenase (BphAl and TodCl), the small subunit of terminal dioxygenase (BphA2 and TodC2), ferredoxin (BphA3 and TodB), and ferredoxin reductase (BphA4 and TodA) are, respectively, 65, 60, 60, and 53% (10). We found previously that E. coli cells expressing BP dioxygenase (composed of BphAlA2A3A4) were totally inactive for toluene, although E. coli cells expressing toluene dioxygenase (composed of TodClC2BA) converted BP to the dihydrodiol (cis-2,3-dihydroxy-1-methylcyclohexa-4,6-diene) (2). During the course of the construction of a hybrid gene cluster involving bphAlA2A3A4 and todClC2BA, it was found that E. coli cells expressing a hybrid gene cluster containing todCl:: * Corresponding author. Mailing address:
The extradiol ring-cleavage dioxygenases derived from seven different Pseudomonas strains were expressed in Escherichia coli and the substrate specificities were investigated for a variety of catecholic compounds. The substrate range of four 2,3-dihydroxybiphenyl dioxygenases from biphenyl-utilizing bacteria, 3-methylcatechol dioxygenase from toluene utilizing Pseudomonas putida F1, 1,2-dihydroxynaphthalene dioxygenase from a NAH7 plasmid, and catechol 2,3-dioxygenase from a TOL plasmid pWW0 were compared. Among the dioxygenases, that from Pseudomonas pseudoalcaligenes KF707 showed a very narrow substrate range. Contrary to this, the dioxygenase from pWW0 showed a relaxed substrate range. The seven extradiol dioxygenases from the various Pseudomonas strains are highly diversified in terms of substrate specificity.
The extradiol ring-cleavage dioxygenases derived from seven different Pseudomonas strains were expressed in Escherichia coli and the substrate specificities were investigated for a variety of catecholic compounds. The substrate range of four 2,3-dihydroxybiphenyl dioxygenases from biphenyl-utilizing bacteria, 3-methylcatechol dioxygenase from toluene utilizing Pseudomonas putida F1, 1,2-dihydroxynaphthalene dioxygenase from a NAH7 plasmid, and catechol 2,3-dioxygenase from a TOL plasmid pWW0 were compared. Among the dioxygenases, that from Pseudomonas pseudoalcaligenes KF707 showed a very narrow substrate range. Contrary to this, the dioxygenase from pWW0 showed a relaxed substrate range. The seven extradiol dioxygenases from the various Pseudomonas strains are highly diversified in terms of substrate specificity.
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