JOURNAL OF BACTERIOLOGY, Sept 1976, p. 1098-1107 Copyright ©D 1976 American Society for Microbiology
Vol. 127, No. 3 Printed in U.S.A.
Catechol Oxygenases of Pseudomonas putida Mutant Strains R. C. BAYLY* AND D. I. McKENZIE Department of Microbiology, Monash University Medical School, Commercial Road, Prahran, Victoria, Australia
Received for publication 7 June 1976
Investigation of a mutant strain of Pseudomonas putida NCIB 10015, strain PsU-E1, showed that it had lost the ability to produce catechol 1,2-oxygenase after growth with catechol. Additional mutants of both wild-type and mutant strain PsU-E1 have been isolated that grow on catechol, but not on benzoate, yet still form a catechol 1,2-oxygenase when exposed to benzoate. These findings indicate that either there are separately induced catechol 1,2-oxygenase enzymes, or that there are two separate inducers for the one catechol 1,2-oxygenase enzyme. Comparisons of the physical properties of the catechol 1,2-oxygenases formed in response to the two different inducers show no significant differences, so it is more probable that the two proteins are the product of the same gene. Sufficient enzymes of the ortho-fission pathway are induced in the wild-type strain by the initial substrate benzoate (or an early intermediate) to commit that substrate to metabolism by ortho fission exclusively. A mechanism exists that permits metabolism of catechol by meta fission if the ortho-fission enzymes are unable to prevent its intracellular accumulation. The numerous reports on the induction and regulation of the ortho-fission pathway for dissimilation of aromatic compounds has been recently reviewed (20). There have been fewer observations reported on these aspects of the meta-fission pathway. Farr and Cain (6) reported that catechol induced a catechol 2,3-oxygenase (EC 1.13.11.2) in a strain of Pseudomonas aeruginosa and that other metabolites of the meta-fission pathway elicited catechol 1,2oxygenase (EC 1.13.11.1). A study with P. putida (7) showed that the phenolic precursors of catechol and mono-methylcatechols induced all the enzymes of the meta-fission pathway and that growth on benzoate or catechol elicited the enzymes of only the ortho-fission pathway. Using a different strain of P. aeruginosa from that used by Farr and Cain (6), Ribbons (18) reported that catechol induced catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde hydrolase, and phenol hydroxylase. Although it has been accepted generally that the ortho- and meta-fission pathways do not operate concurrently, Trecanni et al. (21) found both catechol 1,2- and 2,3-oxygenase in cells of P. desmolyticum grown on either naphthalene, benzoate, salicylate, or 5-methylsalicylate. The results of Ribbons (18) also suggested that benzoate may be degraded by both pathways operating concurrently. A recent report (13) on P. arvilla mt-2 has shown that cells that contain high levels of meta-fission enzymes after
growth on benzoate also contain low levels of ortho-fission enzymes. Bayly (Ph.D. thesis, Monash Univ., Victoria, Australia 1969) reported that catechol induced both catechol 1,2and 2,3-oxygenase in P. putida NCIB 10015 and that the physiological state of the cells before exposure to catechol appeared to affect the ratio of the specific activities of the two oxygenases. Murray and Williams (12) have reported that, for the same strain of P. putida, catechol induced enzymes of the meta-fission pathway, as well as being converted to cis,cis-muconate by a constitutive basal level of catechol 1,2oxygenase, by this means initiating induction of the ortho-fission pathway. In view of the above, it was decided to investigate further the mechanism that controls initiation of the ortho-fission route. The present report describes some properties of mutant strains of P. putida NCIB 10015 in which the inductive responses to aromatic compounds differ from that in the wild-type strain. The results are used to explain why, in this strain, the ortho-fission pathway of benzoate and the metafission pathway of phenol are divergent from the common intermediate, catechol. MATERIALS AND METHODS Organism and methods of cultivation. The wildtype strain used was P. putida NCIB 10015, strain U, of Dagley and Gibson (4), which will be referred to as strain PsU-O. P. putida strain A.3.12 was ob-
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CATECHOL OXYGENASES OF P. PUTIDA MUTANTS
VOL. 127, 1976
tained from P. J. Chapman. The maintenance and growth of the cultures were as described by Bayly and Wigmore (2). The basal medium used was the mineral salts base of Hegeman (9) with appropriate additions of sources of carbon. Acetate, fumarate, and succinate were used as carbon sources for growth at the concentrations stated in the text. Generation times were determined in basal medium that contained either phenol (4 mM), benzoate (4 mM), or acetate (20 mM). When phenol, benzoate, or catechol were used as inducers they were added to a final concentration of 2.5 mM. For induction experiments the cells were grown in basal medium with acetate (20 mM) as carbon source until the culture was in the early exponential phase (absorbance of 0.1 to 0.15, measured at 440 nm in a Bausch and Lomb Spectronic 20) at which time the inducer was added. The period of induction was 2 h, during which time absorbance increased to 0.6 to 0.7. For experiments investigating the time course of induction of enzymes, the cells were harvested at intervals of up to 8 h after the addition of each aromatic compound. Preparation of cell extracts. After induction the cells were harvested and cell extracts were prepared by sonic oscillation as described by Dagley and Gibson (4). Detection of catechol 2,3-oxygenase activity in colonies of cells grown on solid media. The presence of catechol 2,3-oxygenase (2,3ox) activity was detected by spraying colonies grown on solid basal medium plus an appropriate carbon source with an aqueous solution of 4-methylcatechol (10 mM). If 2,3ox is present the colonies turn yellow owing to the formation of 2-hydroxy-5-methylmuconic semialdehyde (2). Mutagenesis. Mutant strains were obtained by treatment of strain PsU-O with ethylmethane sulphonate as described by Bayly and Wigmore (2). Selection of mutant strains. (i) Mutants with
1099
detectable catechol 2,3-oxygenase activity after growth at the expense of benzoate. Mutant strains were obtained by incubation of mutagenized cells for 48 h on basal medium plus benzoate (2.5 mM). Colonies that formed 2,3ox were selected. (ii) Mutant strains unable to convert benzoate to catechol. Mutant strains unable to convert benzoate to catechol were selected from strains PsU-O and PsU-E1 by the penicillin/cycloserine method of Ornston et al. (16) using the modifications described by Wigmore et al. (23). The selective carbon source was catechol (2.5 mM) and the contra-selective carbon source was benzoate (2.5 mM). The mutagenized cells were grown on fumarate before first exposure to the contra-selective carbon source. The frequency of appearance of the mutant types was
Vol. 127, No. 3 Printed in U.S.A.
Catechol Oxygenases of Pseudomonas putida Mutant Strains R. C. BAYLY* AND D. I. McKENZIE Department of Microbiology, Monash University Medical School, Commercial Road, Prahran, Victoria, Australia
Received for publication 7 June 1976
Investigation of a mutant strain of Pseudomonas putida NCIB 10015, strain PsU-E1, showed that it had lost the ability to produce catechol 1,2-oxygenase after growth with catechol. Additional mutants of both wild-type and mutant strain PsU-E1 have been isolated that grow on catechol, but not on benzoate, yet still form a catechol 1,2-oxygenase when exposed to benzoate. These findings indicate that either there are separately induced catechol 1,2-oxygenase enzymes, or that there are two separate inducers for the one catechol 1,2-oxygenase enzyme. Comparisons of the physical properties of the catechol 1,2-oxygenases formed in response to the two different inducers show no significant differences, so it is more probable that the two proteins are the product of the same gene. Sufficient enzymes of the ortho-fission pathway are induced in the wild-type strain by the initial substrate benzoate (or an early intermediate) to commit that substrate to metabolism by ortho fission exclusively. A mechanism exists that permits metabolism of catechol by meta fission if the ortho-fission enzymes are unable to prevent its intracellular accumulation. The numerous reports on the induction and regulation of the ortho-fission pathway for dissimilation of aromatic compounds has been recently reviewed (20). There have been fewer observations reported on these aspects of the meta-fission pathway. Farr and Cain (6) reported that catechol induced a catechol 2,3-oxygenase (EC 1.13.11.2) in a strain of Pseudomonas aeruginosa and that other metabolites of the meta-fission pathway elicited catechol 1,2oxygenase (EC 1.13.11.1). A study with P. putida (7) showed that the phenolic precursors of catechol and mono-methylcatechols induced all the enzymes of the meta-fission pathway and that growth on benzoate or catechol elicited the enzymes of only the ortho-fission pathway. Using a different strain of P. aeruginosa from that used by Farr and Cain (6), Ribbons (18) reported that catechol induced catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde hydrolase, and phenol hydroxylase. Although it has been accepted generally that the ortho- and meta-fission pathways do not operate concurrently, Trecanni et al. (21) found both catechol 1,2- and 2,3-oxygenase in cells of P. desmolyticum grown on either naphthalene, benzoate, salicylate, or 5-methylsalicylate. The results of Ribbons (18) also suggested that benzoate may be degraded by both pathways operating concurrently. A recent report (13) on P. arvilla mt-2 has shown that cells that contain high levels of meta-fission enzymes after
growth on benzoate also contain low levels of ortho-fission enzymes. Bayly (Ph.D. thesis, Monash Univ., Victoria, Australia 1969) reported that catechol induced both catechol 1,2and 2,3-oxygenase in P. putida NCIB 10015 and that the physiological state of the cells before exposure to catechol appeared to affect the ratio of the specific activities of the two oxygenases. Murray and Williams (12) have reported that, for the same strain of P. putida, catechol induced enzymes of the meta-fission pathway, as well as being converted to cis,cis-muconate by a constitutive basal level of catechol 1,2oxygenase, by this means initiating induction of the ortho-fission pathway. In view of the above, it was decided to investigate further the mechanism that controls initiation of the ortho-fission route. The present report describes some properties of mutant strains of P. putida NCIB 10015 in which the inductive responses to aromatic compounds differ from that in the wild-type strain. The results are used to explain why, in this strain, the ortho-fission pathway of benzoate and the metafission pathway of phenol are divergent from the common intermediate, catechol. MATERIALS AND METHODS Organism and methods of cultivation. The wildtype strain used was P. putida NCIB 10015, strain U, of Dagley and Gibson (4), which will be referred to as strain PsU-O. P. putida strain A.3.12 was ob-
1098
CATECHOL OXYGENASES OF P. PUTIDA MUTANTS
VOL. 127, 1976
tained from P. J. Chapman. The maintenance and growth of the cultures were as described by Bayly and Wigmore (2). The basal medium used was the mineral salts base of Hegeman (9) with appropriate additions of sources of carbon. Acetate, fumarate, and succinate were used as carbon sources for growth at the concentrations stated in the text. Generation times were determined in basal medium that contained either phenol (4 mM), benzoate (4 mM), or acetate (20 mM). When phenol, benzoate, or catechol were used as inducers they were added to a final concentration of 2.5 mM. For induction experiments the cells were grown in basal medium with acetate (20 mM) as carbon source until the culture was in the early exponential phase (absorbance of 0.1 to 0.15, measured at 440 nm in a Bausch and Lomb Spectronic 20) at which time the inducer was added. The period of induction was 2 h, during which time absorbance increased to 0.6 to 0.7. For experiments investigating the time course of induction of enzymes, the cells were harvested at intervals of up to 8 h after the addition of each aromatic compound. Preparation of cell extracts. After induction the cells were harvested and cell extracts were prepared by sonic oscillation as described by Dagley and Gibson (4). Detection of catechol 2,3-oxygenase activity in colonies of cells grown on solid media. The presence of catechol 2,3-oxygenase (2,3ox) activity was detected by spraying colonies grown on solid basal medium plus an appropriate carbon source with an aqueous solution of 4-methylcatechol (10 mM). If 2,3ox is present the colonies turn yellow owing to the formation of 2-hydroxy-5-methylmuconic semialdehyde (2). Mutagenesis. Mutant strains were obtained by treatment of strain PsU-O with ethylmethane sulphonate as described by Bayly and Wigmore (2). Selection of mutant strains. (i) Mutants with
1099
detectable catechol 2,3-oxygenase activity after growth at the expense of benzoate. Mutant strains were obtained by incubation of mutagenized cells for 48 h on basal medium plus benzoate (2.5 mM). Colonies that formed 2,3ox were selected. (ii) Mutant strains unable to convert benzoate to catechol. Mutant strains unable to convert benzoate to catechol were selected from strains PsU-O and PsU-E1 by the penicillin/cycloserine method of Ornston et al. (16) using the modifications described by Wigmore et al. (23). The selective carbon source was catechol (2.5 mM) and the contra-selective carbon source was benzoate (2.5 mM). The mutagenized cells were grown on fumarate before first exposure to the contra-selective carbon source. The frequency of appearance of the mutant types was
