Isolation and Characterization of Pseudomonas putida PpF1 Mutants ...

JOURNAL OF BACTERIOLOGY, Dec. 1984, p. 1003-1009

Vol. 160, No. 3

0021-9193/84/121003-07$02.00/0 Copyright © 1984, American Society for Microbiology

Isolation and Characterization of Pseudomonas putida PpF1 Mutants Defective in the Toluene Dioxygenase Enzyme System BARRY A. FINETTE, VENKITESWARAN SUBRAMANIAN,t AND DAVID T. GIBSON* Center for Applied Microbiology and Department of Microbiology, The University of Texas at Austin, Austin, Texas 78712 Received 13 June 1984/Accepted 30 August 1984

Pseudomonas putida PpF1 degraded toluene via a dihydrodiol pathway to tricarboxylic acid cycle intermediates. The initial reaction was catalyzed by a multicomponent enzyme, toluene dioxygenase, which oxidized toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol). The enzyme consisted of three protein components: NADH-ferredoxint.0 oxidoreductase (reductaset01), ferredoxinto,, and a terminal oxygenase which is an iron-sulfur protein (ISPt.1). Mutants blocked in each of these components were isolated after mutagenesis with nitrosoguanidine. Mutants occurred as colony morphology variants when grown in the presence of toluene on indicator plates containing agar, mineral salts, a growth-supporting nutrient (arginine), 2,3,5-triphenyltetrazolium chloride (TTC), and Nitro Blue Tetrazolium (NBT). Under these conditions, wild-type colonies appeared large and red as a result of TTC reduction. Colonies of reductaseto0 mutants were white or white with a light blue center, ferredoxinto, strains were light blue with a dark blue center, and strains that lacked ISPto, gave dark blue colonies. Blue color differences in the mutant colonies were due to variations in the extent of NBT reduction. Strains lacking all three components appeared white. Toluene dioxygenase mutants were characterized by assaying toluene dioxygenase activity in crude cell extracts which were complemented with purified preparations of each protein component. Between 40 and 60% of the putative mutants selected from the NBT-TTC indicator plates were unable to grow with toluene as the sole source of carbon and energy. This method should prove extremely useful in isolating mutants in other multicomponent oxygenase enzyme systems.

Toluene can serve as a growth substrate for different Pseudomonas species. However, the metabolic pathway for toluene degradation is not the same in all species that have been examined. For example, Pseudomonas putida mt-2 oxidizes the methyl group of toluene to form benzyl alcohol (40). Subsequent oxidative reactions lead to the formation of catechol, which is the substrate for meta-ring fission of the aromatic nucleus. The genes coding for the enzymes of this pathway are carried on a transmissible plasmid that has been termed TOL (38-40). In contrast, work in our laboratory has revealed two other pathways by which toluene is dissimilated to tricarboxylic acid (TCA) cycle intermediates. Recently, Richardson and Gibson described the initial oxidation of toluene to p-cresol by a strain of Pseudomonas mendocina. These authors suggested that subsequent oxidation leads to protocatechuate, which serves as the substrate for ortho-ring fission of the aromatic nucleus (K. L. Richardson and D. T. Gibson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1984, K54, p. 156). A different strain of P. putida that was isolated in our laboratory initiates the oxidation of toluene by incorporating one molecule of oxygen into the aromatic nucleus to form cis-1(S),2(R)-dihydroxy-3methylcyclohexa-3,5-diene (cis-toluene dihydrodiol [16, 43]). Further oxidation of cis-toluene dihydrodiol leads to the formation of 3-methylcatechol (17, 30), which is then degraded to intermediates of the TCA cycle via the meta-cleavage pathway (10). This organism has now been designated as strain PpF1. The initial reactions used by P. putida PpF1, P. mendocina, and P. putida mt-2 for toluene degradation are shown in Fig. 1.

The formation of cis-toluene dihydrodiol by P. putida PpF1 is catalyzed by a multicomponent enzyme system that has been termed toluene dioxygenase (42). The individual protein components have been identified as NADH-ferredoxin,01 oxidoreductase (reductaseto, [34]), an iron-sulfur ferredoxin (ferredoxinto, [18, 42]), and an iron-sulfur protein (ISPto, [18, 33]). The proposed organization of the toluene dioxygenase enzyme system is shown in Fig. 2. In this communication, the gene designations todA, todB, and todC have been assigned for reductasetol, ferredoxin,01, and ISPto0, respectively. At present, the mechanisms involved in the regulation of gene expression for toluene dioxygenase and similar multicomponent enzyme systems have not been studied in detail. Such investigations have been hindered by the lack of a suitable screening procedure for isolating mutants in the individual components of these multicomponent enzymes. We now describe the use of two different tetrazolium redox dyes to facilitate the screening and subsequent isolation of mutants in each component of toluene dioxygenase. Further biochemical characterization of each mutant strain was accomplished by complementation of crude cell extracts with purified preparations of each protein component. The results obtained suggest that tetrazolium dyes may also be used to screen for mutant strains that are deficient in the individual protein components of other multicomponent oxygenases. (A partial summary of these results was presented at the 81st Annual Meeting of the American Society for Microbiology [Abstr. Annu. Meet. Am. Soc. Microbiol. 1981, K39, p. 144].) MATERIALS AND METHODS and Organism growth conditions. P. putida PpF1 is the organism previously described by Gibson et al. (17). Cells

Corresponding author. t Present address: Dow Chemical Co., Midland, MI 48640. *

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vapor phase to each plate, and cells were allowed to grow for 48 to 72 h at 30°C. This procedure resulted in many different colony morphology variants. Small colonies that were white, white with light blue centers, light blue with dark blue centers, and dark blue were isolated, purified, and tested for growth on glucose and toluene. Presumptive toluene-negative strains were subjected to biochemical complementation

OH3

analyses.

A

CH20H

Benzyl Alcohol Dihydrodiol

CH3 N

OH

^-

p-iresol

COOH

OOH HOH

Catechol

3-Methyl Cotechol

H OH

Protocotechuote

Ring Fsion FIG. 1. Pathways used for the oxidation of toluene to ring fission substrates by: A, P. putida mt-2; B, P. putida PpFl; C, P. mendocina. were grown in L broth or on a mineral salts medium (32) containing 0.2% arginine. Solid media contained 2% agar. Toluene was introduced to cultures in the vapor phase as described previously (7). Mutants defective in toluene dioxygenase activity were detected on a mineral salts indicator medium that contained the following: agar, 2%; arginine, 0.02%; Nitro Blue tetrazolium (NBT), 20.4 mg/liter; and 2,3,5-triphenyl-2H-tetrazolium chloride (TTC), 25 mg/liter. Isolation of mutants defective in the toluene dioxygenase enzyme system. P. putida PpF1 was grown to stationary phase in 5.0 ml of L broth. The cells were washed twice with 0.1 M sodium citrate buffer (pH 5.5) and resuspended in 10.0 ml of the same buffer containing 1.0 mg of N-methyl-N'-

nitro-N-nitrosoguanidine (NTG). After standing at room for 10 min, the cells were washed twice with 0.1 M potassium phosphate buffer (pH 7.0), suspended in mineral salts medium supplemented with 0.1% arginine, and incubated in the presence of toluene for 2 h at 30°C. The cells were then washed with phosphate buffer, resuspended in 5.0 ml of mineral salts medium, and allowed to grow in the presence of toluene for 1 h at 30°C before addition of filter-sterilized ampicillin (200 ,ug/ml) and D-cycloserine (100 ,ug/ml). After 4 h, the cells were washed with phosphate buffer, and 0. 1-ml aliquots (100 to 300 cells) were plated onto NBT-TTC indicator plates. Toluene was supplied in the temperature

Complementation analyses. P. putida PpF1 and toluenenegative mutant strains were grown in 500 ml of mineral salts medium containing 0.1% arginine for 18 to 24 h. Toluene was supplied in the vapor phase. Cells were harvested by centrifugation, washed twice with 0.05 M potassium phosphate buffer (pH 7.2), and suspended in 3.0 ml of PEG buffer (42). Cell extracts were prepared from each concentrated cell suspension by passage through an Aminco French pressure cell at 19,800 lb/in2. The supernatant fluid obtained after centrifugation at 100,000 x g for 1 h was used immediately for complementation analyses. Each cell extract was assayed for toluene dioxygenase activity in the presence and absence of purified preparations of the individual components of the toluene dioxygenase enzyme system. Toluene dioxygenase activity was determined by measuring the rate of formation of cis-[14C]toluene dihydrodiol from [methyl14C]toluene (42). Purified protein components were added to the assay mixture at the following concentrations: reductasetol, 20 to 40 ,ug/ml; ferredoxintol, 30 to 50 ,ug/ml; ISPtol, 25 to 40 ,ug/ml. The components of toluene dioxygenase were prepared as described previously (33, 34, 42) and stored at -70°C. The activity of each component was checked before being used for complementation analyses. The protein concentration in crude cell extracts was determined by the method of Lowry et al. (27). Materials. The following materials were obtained from the sources indicated: [methyI-14C]toluene (specific activity, 26.4 mCi/mmol), Amersham Searle Corp., Arlington Heights, Ill.; NBT, ampicillin, and D-cycloserine, Sigma Chemical Co., St. Louis, Mo.; TTC, Eastman Kodak Co., Rochester, N.Y.; toluene, MCB Manufacturing Chemists Inc., Cincinnati, Ohio. RESULTS Rationale for the isolation of mutants defective in the toluene dioxygenase enzyme system. To avoid confusion with the nomenclature used for genes associated with tolerance to colicin (tol), we have chosen to designate the genes coding for the enzymes responsible for the degradation of toluene by P. putida PpF1 with the prefix tod. Electrons were transferred from NADH to ferredoxin,01 by the flavoprotein reductaset.0 (Fig. 2). Ferredoxint,0 reduced the terminal oxygenase component ISPto, which then catalyzed the incorporation of molecular oxygen into toluene to form cis-toluene dihydrodiol (16). In addition to participating in the transfer of electrons to ISP,01, reductaseto, can also transfer electrons from NADH to other electron acceptors, including the redox dye NBT. In addition, the total amount of dye reduced is increased in the presence of ferredoxin,01. NBT reduction was dependent on the presence of NADH and reductasetol. Blue color formation was not observed in the absence of NADH or in the presence of NADH and ferredoxintol. These observations suggested that NBT could be used in solid media to select for specific mutants defective in the toluene dioxygenase enzyme system. For example, strains lacking all three components or reductaset,0 should appear as small white colonies. Those strains which lack ferredoxinto, but which have an active reductaseto, compo-

P. PUTIDA TOLUENE DIOXYGENASE MUTANTS

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1005

CH3

NADH+H+

RdTOL (FAD) ~(FADH) -

NAD

NAD

~

RdTo

(FADH2)70 I

2Fd (red.)TL

ISPTO ~~(oxd.)'

(SPTOL

(red.)TOL

0 2 Toluene Fe H3H

OH

$3

ISPO O (red.rOL

2Fd

(oxd.)TOL

H

cis-Toluene

VI.

Dihydrodiol

I1.

FerredoxinTOL FerredoxinTOL Iron-Sulfur Reductose

ProteinTOL

(ReductaseTO}

Molecular Weight Subunit Molecular Weight Gene Designation

46,000

15,400

151,000

46,000

15,400

52,500 20,800

todA

todB

todC

/\

todC

todC2

(52,500 MW (20,800 MW /3 Subunit) a Subunit)

FIG. 2. Biochemical organization and gene designations for the multicomponent toluene dioxygenase enzyme system of P. putida PpF1.

nent should appear as small light blue colonies, whereas colonies of ISP,01 mutants should be small and dark blue as a result of NBT reduction by reductase,01 and ferredoxin,01. In addition, we chose to use TTC to distinguish between mutant and wild-type colonies. Bochner and Savageau (3) have previously shown that, in the presence of TTC, colonies that can fully catabolize a test substrate are red, whereas those unable to do so remain white. When P. putida PpF1 was grown in the presence of toluene on NBT-TTC plates containing limiting amounts of arginine, all colonies were large and red. When toluene was omitted from the plates, only small white colonies were observed. Treatment of the parent organism with NTG gave rise to the predicted colony morphology variants (Fig. 3). Complementation analyses of mutants. Between 40 and 60% of the putative mutants selected from NBT-TTC plates after treatment with NTG were unable to grow with toluene as the sole source of carbon and energy. The majority of these mutants were quite stable, with reversion frequencies ranging from 10' to less than 10-11 (Table 1). Many (1 to 5%) of the small white colonies selected were arginine auxotrophs. However, those colonies showing NBT reduction when subjected to complementation analysis gave the predicted genotype (Fig. 3 and Table 1). In these experiments, putative mutant strains were grown with arginine in the presence of toluene. Crude cell extracts from each strain were assayed for toluene dioxygenase activity. Enzyme assays were then repeated in the presence of purified preparations of each component of the toluene dioxygenase system. Enzyme activity was not detected in cell extracts of P. putida PpF1 that were grown in the absence of toluene. The addition of separate purified preparations of reductase,01, ferredoxintol, and ISP,01 to these cell extracts did not result in toluene dioxygenase activity unless all three proteins were present. In contrast, cell extracts prepared from several mutant strains that were grown with arginine in the presence of toluene could be complemented with purified components of the toluene dioxygenase enzyme system. Extracts from strains that gave white colonies with pale blue

centers on the indicator plates (Fig. 3E) regained significant levels of toluene dioxygenase activity when they were supplemented with reductasetol. These mutants, PpF12 and PpF102 (Table 1), have been designated as having a todA genotype. Antibodies prepared against reductase,01 failed to cross-react with cell extracts prepared from these two strains. In some instances, TOL- colorless colonies that failed to reduce NBT were also shown to have a todA genotype. However, the majority of the colorless small colonies of the type shown in Fig. 3C gave extracts that could not be complemented by any double combination of the purified dioxygenase components. These strains, PpF120, PpF7, PpF211, PpF133, and PpF131 (Table 1), were designated as todABC mutants. Mutant strains that gave light blue colonies with dark blue centers were typified by PpF3 (Fig. 3D) and PpF126. Extracts from these organisms could be complemented with ferredoxinto, (Table 1), and the organisms were designated as todB mutants. The same colony morphology was observed for todBC mutants (Table 1), which required both ferredoxinto, and ISPto0 for toluene dioxygenase activity. Strains such as PpF4 that gave small dark blue colonies on the indicator plates (Fig. 3A) could be complemented with ISPto0 and were designated as todC mutants. Four of these mutants are shown in Table 1. Some of the mutants isolated gave results that were not easily interpreted by complementation analyses. Strain PpF121 appeared to be unique in that toluene dioxygenase activity could be complemented by either ferredoxinto, or ISPtol. The reasons for the apparent inhibition of activity in the presence of reductaseto0 have not been determined. Other strains such as PpF26a, PpF28, PpF103, and PpF23, appeared to have low levels or altered activities of the ferredoxinto, component of the toluene dioxygenase enzyme system. Another strain, PpF123, appeared to have a low level or altered activity of the ISPto, component of the toluene dioxygenase enzyme system. It is clear that the NBT-TTC indicator plates together with appropriate enzymatic complementation studies have permitted the isolation and partial biochemical characterization

1006

FINETTE, SUBRAMANIAN, AND GIBSON

J. BACTERIOL.

of a wide variety of mutants defective in the todABC genes of the toluene dioxygenase enzyme system. A summary of the mutants obtained by this procedure is shown in Fig. 4. DISCUSSION ...

FIG. 3. Colony morphologies of strain PpFl (wild type) and toluene dioxygenase mutants grown in the presence of toluene vapors on NBT-TTC indicator plates: A, strain PpF4 todC; B, P. putida PpF1; C, strain PpF120 todABC; D, strain PpF3 todB; E, strain PpF12 todA. Photographs of the mutant and wild-type colonies were taken with a Zeiss model DRC stereomicroscope, using an 80a filter and reflected tungsten light. Magnifications: A, D, and E, x5.25; B and C, x3.375.

Multicomponent bacterial oxygenases play an essential role in the aerobic oxidation of hydrocarbons and related compounds. Some examples are benzene (1, 15), toluene (18, 33, 34), naphthalene (12, 13) benzoate (14, 41), and pyrazon (31) dioxygenases and methane (8, 9, 11), octane (4, 29) and camphor (26, 35) monooxygenases. In addition, similar enzymes are probably responsible for the oxidation of xylenes and toluates in the catabolic pathway encoded by TOL plasmids (38-40). Little is known about the gene order and regulation of these enzyme systems. This is mainly because of the absence of a suitable screening procedure for the isolation of mutants defective in individual oxygenase components. Current methods for the isolation of catabolic mutants have focused on certain strains of Pseudomonas spp. and utilize enrichment procedures involving penicillin or D-cycloserine (2, 5, 28). Direct, positive enrichment has also been achieved by the use of "suicide substrates" (36, 37). The results presented herein demonstrated that a conventional enrichment procedure coupled with the use of the redox dyes NBT and TTC can be used in a selective manner to identify mutants of P. putida PpF1 that are defective in

individual components of the toluene dioxygenase enzyme system. In the absence of the redox dyes, all small colonies would be scored as putative mutants in enzymes involved in toluene degradation. Although the percentage of small colonies obtained after enrichment was not determined, it has been our experience that many of these organisms contain defects unrelated to toluene catabolism. Consequently, a considerable amount of time and effort is required to screen large numbers of mutants for a desired phenotype or genotype. The advantages of using NBT and TTC are twofold. NBT permits the direct isolation of specific mutants that are defective in the toluene dioxygenase multicomponent enzyme system and eliminates the need to test every small colony for these properties. In the presence of TTC, a large number of small colonies were red. The percentage of such colonies was not determined. However every small red colony tested retained the ability to grow with toluene. In the absence of TTC, these colonies would have been considered as putative mutants in the toluene catabolic pathway. Thus, the use of both redox dyes provided a rapid and effective screening procedure for tod mutants. The principle of the procedure is based on the observation that the extent of NBT reduction by reductaseto, (todA) is enhanced in the presence of ferredoxinto, (todB). These results seem to hold true for intact cells. Although the procedure is relatively simple, it is important to note that consistent dye reduction is only observed with well-isolated colonies. Care must also be taken to avoid nonspecific reduction of NBT. This was achieved by adjusting the concentrations of NBT and the growth-supporting substrate to levels that only permit NBT reduction in the presence of the inducing substrate. Under these conditions, both colony size and extent of NBT reduction can be used to identify mutant strains. Thus, small white colonies observed after NTG mutagenesis are putative mutants in structural or regulatory genes involved in toluene degradation. The rationale for selection also predicts that mutants in the gene coding for reductaset., (todA) should also show this phenotype. However, some of the todA mutants selected were

VOL. 160, 1984

P. PUTIDA TOLUENE DIOXYGENASE MUTANTS

small white colonies with pale blue centers (Fig. 3E). The for the slight amount of NBT reduction by these mutants is not known. However, this property made the identification of some todA mutants a relatively easy task. It is important to show that small white colonies are not auxotrophic for the growth-limiting substrate since in the present study a significant percentage of such colonies were identified as arginine auxotrophs. Small light blue colonies with dark blue centers (Fig. 3D) were identified as mutants defective in the gene coding for ferredoxinto, (todB). The same phenotype was observed with todBC mutants. Small dark blue colonies (Fig. 3A) were identified as mutants in the terminal oxygenase component (ISPtol, todC). These mutants, together with those described above, were easily distinguished from the parent strain of P. putida PpF1, which formed large red colonies on the indicator medium (Fig. 3B). This was due to the reduction of TTC to a red formazan in the presence of the growth substrate. Under the described conditions, the parent strain of P. putida PpF1 did not reduce NBT. This was probably due to the tight coupling of electron transport in the intact toluene dioxygenase enzyme system. The inclusion of TTC in the indicator medium was based on the observations of Bochner and Savageau (3), who showed that TTC reduction only occurs if sufficient levels of a carbon and energy source are available. Since nonspecific reduction of TTC can occur, it is

important to determine conditions that allow uninduced and mutant colonies to grow without reducing the redox dye. Colored colonies unrelated to NBT reduction were also observed after NTG mutagenesis. Small brown colonies were formed by mutants defective in the enzyme 3-methylcatechol 2,3-dioxygenase. The color was due to the autooxidation of accumulated 3-methylcatechol. Small bright yellow colonies were formed by mutants defective in 2-

reason

1007

hydroxy-6-oxo-2,4-heptadienoic acid hydrolyase. At alkaline pH, the substrate for this enzyme had a high extinction coefficient at 385 nm, which accounted for the yellow color observed. The characterization of these mutants will be reported (manuscript in preparation). The predicted color and colony size of mutants defective in specific components of the toluene dioxygenase enzyme system were confirmed by in vitro complementation analyses with purified protein components. Representative strains of the different classes of mutants that were obtained are shown in Table 1. Mutants defective in the structural genes for reductaseto, (todA), ferredoxint.0 (todB), and ISPt.0 (todC) were isolated. The terminal oxygenase component (ISPtol) of toluene dioxygenase is an iron-sulfur protein that has an a2P2 subunit composition. The molecular weights of the a and P subunits are 52,500 and 20,800, respectively (33). At this time, the individual subunits have not been isolated, and we were unable to assign the a and l subunits to separate genes.

TABLE 1. Biochemical complementation analysis of mutants Toluene dioxygenase activitya

P. putida strain

PpFlC PpFld PpF12 PpF102 PpF3 PpF126 PpF4 PpF106 PpF1O PpFlll PpF24 PpF25 PpF130 PpF118 PpF22 PpF27 PpF26 PpF31 PpF120 PpF7 PpF211 PpF133 PpF131 PpF121 PpF123 PpF26a PpF28 PpF103 PpF23

Rd,01

Fd,.0

-e 5 6 -

ISP,0

RD,01 +

Ft

6 8 4 7

6 7 -

-

4 3 2 6

-

RDt., +

FD,0 +

ISP01,

isp,01

6 6

-

4 2 2 5

-

Control'

Reversion

Genotypic

frequency

designation

8 i0-9