JOURNAL OF BACTERIOLOGY, Dec. 1984, p. 1003-1009 Vol. 160, No. 30021-9193/84/121003-07$02.00/0Copyright © 1984, American Society for Microbiology

Isolation and Characterization of Pseudomonas putida PpF1Mutants 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 cycleintermediates. The initial reaction was catalyzed by a multicomponent enzyme, toluene dioxygenase, whichoxidized toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol). Theenzyme consisted of three protein components: NADH-ferredoxint.0 oxidoreductase (reductaset01), ferredox-into,, and a terminal oxygenase which is an iron-sulfur protein (ISPt.1). Mutants blocked in each of thesecomponents were isolated after mutagenesis with nitrosoguanidine. Mutants occurred as colony morphologyvariants when grown in the presence of toluene on indicator plates containing agar, mineral salts, agrowth-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 lightblue with a dark blue center, and strains that lacked ISPto, gave dark blue colonies. Blue color differences inthe mutant colonies were due to variations in the extent ofNBT reduction. Strains lacking all three componentsappeared white. Toluene dioxygenase mutants were characterized by assaying toluene dioxygenase activity incrude cell extracts which were complemented with purified preparations of each protein component. Between40 and 60% of the putative mutants selected from the NBT-TTC indicator plates were unable to grow withtoluene as the sole source of carbon and energy. This method should prove extremely useful in isolating mutantsin other multicomponent oxygenase enzyme systems.

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

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

The formation of cis-toluene dihydrodiol by P. putidaPpF1 is catalyzed by a multicomponent enzyme system thathas been termed toluene dioxygenase (42). The individualprotein components have been identified as NADH-fer-redoxin,01 oxidoreductase (reductaseto, [34]), an iron-sulfurferredoxin (ferredoxinto, [18, 42]), and an iron-sulfur protein(ISPto, [18, 33]). The proposed organization of the toluenedioxygenase enzyme system is shown in Fig. 2. In thiscommunication, the gene designations todA, todB, and todChave 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 multi-component enzyme systems have not been studied in detail.Such investigations have been hindered by the lack of asuitable screening procedure for isolating mutants in theindividual components of these multicomponent enzymes.We now describe the use of two different tetrazolium redoxdyes to facilitate the screening and subsequent isolation ofmutants in each component of toluene dioxygenase. Furtherbiochemical characterization of each mutant strain was ac-complished by complementation of crude cell extracts withpurified preparations of each protein component. The resultsobtained suggest that tetrazolium dyes may also be used toscreen for mutant strains that are deficient in the individualprotein components of other multicomponent oxygenases.(A partial summary of these results was presented at the

81st Annual Meeting of the American Society for Microbiol-ogy [Abstr. Annu. Meet. Am. Soc. Microbiol. 1981, K39, p.144].)

MATERIALS AND METHODS

Organism and growth conditions. P. putida PpF1 is theorganism previously described by Gibson et al. (17). Cells

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1004 FINETTE, SUBRAMANIAN, AND GIBSON

OH3

A

CH20H

Benzyl Alcohol

Dihydrodiol p-iresol

CH3 COOH

N OH OOHHOH H

Catechol 3-Methyl Cotechol OH

Protocotechuote

Ring Fsion

FIG. 1. Pathways used for the oxidation of toluene to ring fissionsubstrates 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 asdescribed previously (7). Mutants defective in toluene di-oxygenase activity were detected on a mineral salts indicatormedium that contained the following: agar, 2%; arginine,0.02%; Nitro Blue tetrazolium (NBT), 20.4 mg/liter; and2,3,5-triphenyl-2H-tetrazolium chloride (TTC), 25 mg/liter.

Isolation of mutants defective in the toluene dioxygenaseenzyme system. P. putida PpF1 was grown to stationaryphase in 5.0 ml of L broth. The cells were washed twice with0.1 M sodium citrate buffer (pH 5.5) and resuspended in 10.0ml of the same buffer containing 1.0 mg of N-methyl-N'-nitro-N-nitrosoguanidine (NTG). After standing at room

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

vapor phase to each plate, and cells were allowed to grow for48 to 72 h at 30°C. This procedure resulted in many differentcolony morphology variants. Small colonies that were white,white with light blue centers, light blue with dark bluecenters, and dark blue were isolated, purified, and tested forgrowth on glucose and toluene. Presumptive toluene-nega-tive strains were subjected to biochemical complementationanalyses.

Complementation analyses. P. putida PpF1 and toluene-negative mutant strains were grown in 500 ml of mineral saltsmedium containing 0.1% arginine for 18 to 24 h. Toluene wassupplied in the vapor phase. Cells were harvested by cen-trifugation, washed twice with 0.05 M potassium phosphatebuffer (pH 7.2), and suspended in 3.0 ml of PEG buffer (42).Cell extracts were prepared from each concentrated cellsuspension by passage through an Aminco French pressurecell at 19,800 lb/in2. The supernatant fluid obtained aftercentrifugation at 100,000 x g for 1 h was used immediatelyfor complementation analyses. Each cell extract was as-sayed for toluene dioxygenase activity in the presence andabsence of purified preparations of the individual compo-nents of the toluene dioxygenase enzyme system. Toluenedioxygenase activity was determined by measuring the rateof formation of cis-[14C]toluene dihydrodiol from [methyl-14C]toluene (42). Purified protein components were added tothe assay mixture at the following concentrations: reduc-tasetol, 20 to 40 ,ug/ml; ferredoxintol, 30 to 50 ,ug/ml; ISPtol, 25to 40 ,ug/ml. The components of toluene dioxygenase wereprepared as described previously (33, 34, 42) and stored at-70°C. The activity of each component was checked beforebeing used for complementation analyses. The protein con-centration in crude cell extracts was determined by themethod of Lowry et al. (27).

Materials. The following materials were obtained from thesources indicated: [methyI-14C]toluene (specific activity, 26.4mCi/mmol), Amersham Searle Corp., Arlington Heights,Ill.; NBT, ampicillin, and D-cycloserine, Sigma ChemicalCo., St. Louis, Mo.; TTC, Eastman Kodak Co., Rochester,N.Y.; toluene, MCB Manufacturing Chemists Inc., Cincin-nati, Ohio.

RESULTSRationale for the isolation of mutants defective in the

toluene dioxygenase enzyme system. To avoid confusion withthe nomenclature used for genes associated with tolerance tocolicin (tol), we have chosen to designate the genes codingfor the enzymes responsible for the degradation of tolueneby P. putida PpF1 with the prefix tod. Electrons weretransferred from NADH to ferredoxin,01 by the flavoproteinreductaset.0 (Fig. 2). Ferredoxint,0 reduced the terminaloxygenase component ISPto, which then catalyzed the incor-poration of molecular oxygen into toluene to form cis-tolu-ene dihydrodiol (16). In addition to participating in thetransfer of electrons to ISP,01, reductaseto, can also transferelectrons from NADH to other electron acceptors, includingthe redox dye NBT. In addition, the total amount of dyereduced is increased in the presence of ferredoxin,01. NBTreduction was dependent on the presence of NADH andreductasetol. Blue color formation was not observed in theabsence of NADH or in the presence of NADH and fer-redoxintol. These observations suggested that NBT could beused in solid media to select for specific mutants defective inthe toluene dioxygenase enzyme system. For example,strains lacking all three components or reductaset,0 shouldappear as small white colonies. Those strains which lackferredoxinto, but which have an active reductaseto, compo-

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P. PUTIDA TOLUENE DIOXYGENASE MUTANTS 1005

CH3

NAD ~(FADH) (red.)TOL (SPTOL$3

OH

NADH+H+ - RdTOL 2FdTL ISPTO 0(FAD) (red.) ~~(oxd.)' 2 Toluene

Fe H3H

NAD ~ RdTo 2Fd ISPO(FADH2)70 (oxd.)TOL (red.rOL O

I Hcis-TolueneDihydrodiol

I. I1.

Molecular WeightSubunitMolecular WeightGeneDesignation

VFerredoxinTOLReductose(ReductaseTO}

46,000

46,000

todA

FerredoxinTOL

15,400

Iron-SulfurProteinTOL

151,000

15,400 52,50020,800

todB todC/\

todC todC2(52,500 MW (20,800 MWa Subunit) /3 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, whereascolonies of ISP,01 mutants should be small and dark blue asa result of NBT reduction by reductase,01 and ferredoxin,01.In addition, we chose to use TTC to distinguish betweenmutant and wild-type colonies. Bochner and Savageau (3)have previously shown that, in the presence of TTC, colo-nies that can fully catabolize a test substrate are red,whereas those unable to do so remain white. When P. putidaPpF1 was grown in the presence of toluene on NBT-TTCplates containing limiting amounts of arginine, all colonieswere large and red. When toluene was omitted from theplates, only small white colonies were observed. Treatmentof the parent organism with NTG gave rise to the predictedcolony morphology variants (Fig. 3).Complementation analyses of mutants. Between 40 and

60% of the putative mutants selected from NBT-TTC platesafter treatment with NTG were unable to grow with tolueneas the sole source of carbon and energy. The majority ofthese mutants were quite stable, with reversion frequenciesranging from 10' to less than 10-11 (Table 1). Many (1 to5%) of the small white colonies selected were arginineauxotrophs. However, those colonies showing NBT reduc-tion when subjected to complementation analysis gave thepredicted genotype (Fig. 3 and Table 1). In these experi-ments, putative mutant strains were grown with arginine inthe presence of toluene. Crude cell extracts from each strainwere assayed for toluene dioxygenase activity. Enzymeassays were then repeated in the presence of purified prep-arations of each component of the toluene dioxygenasesystem. Enzyme activity was not detected in cell extracts ofP. putida PpF1 that were grown in the absence of toluene.The addition of separate purified preparations of reduc-tase,01, ferredoxintol, and ISP,01 to these cell extracts did notresult in toluene dioxygenase activity unless all three pro-teins were present. In contrast, cell extracts prepared fromseveral mutant strains that were grown with arginine in thepresence of toluene could be complemented with purifiedcomponents of the toluene dioxygenase enzyme system.Extracts from strains that gave white colonies with pale blue

centers on the indicator plates (Fig. 3E) regained significantlevels of toluene dioxygenase activity when they weresupplemented with reductasetol. These mutants, PpF12 andPpF102 (Table 1), have been designated as having a todAgenotype. Antibodies prepared against reductase,01 failed tocross-react with cell extracts prepared from these two strains.In some instances, TOL- colorless colonies that failed toreduce NBT were also shown to have a todA genotype.However, the majority of the colorless small colonies of thetype shown in Fig. 3C gave extracts that could not becomplemented by any double combination of the purifieddioxygenase components. These strains, PpF120, PpF7,PpF211, PpF133, and PpF131 (Table 1), were designated astodABC mutants. Mutant strains that gave light blue colonieswith dark blue centers were typified by PpF3 (Fig. 3D) andPpF126. Extracts from these organisms could be comple-mented with ferredoxinto, (Table 1), and the organisms weredesignated as todB mutants. The same colony morphologywas observed for todBC mutants (Table 1), which requiredboth ferredoxinto, and ISPto0 for toluene dioxygenase activ-ity. Strains such as PpF4 that gave small dark blue colonieson the indicator plates (Fig. 3A) could be complementedwith ISPto0 and were designated as todC mutants. Four ofthese mutants are shown in Table 1.Some of the mutants isolated gave results that were not

easily interpreted by complementation analyses. StrainPpF121 appeared to be unique in that toluene dioxygenaseactivity could be complemented by either ferredoxinto, orISPtol. The reasons for the apparent inhibition of activity inthe 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 theferredoxinto, component of the toluene dioxygenase enzymesystem. Another strain, PpF123, appeared to have a lowlevel or altered activity of the ISPto, component of thetoluene dioxygenase enzyme system.

It is clear that the NBT-TTC indicator plates together withappropriate enzymatic complementation studies have per-mitted the isolation and partial biochemical characterization

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1006 FINETTE, SUBRAMANIAN, AND GIBSON

FIG. 3. Colony morphologies of strain PpFl (wild type) andtoluene dioxygenase mutants grown in the presence of toluenevapors 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 colo-nies were taken with a Zeiss model DRC stereomicroscope, using an80a filter and reflected tungsten light. Magnifications: A, D, and E,x5.25; B and C, x3.375.

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

DISCUSSIONMulticomponent bacterial oxygenases play an essential

role in the aerobic oxidation of hydrocarbons and relatedcompounds. Some examples are benzene (1, 15), toluene(18, 33, 34), naphthalene (12, 13) benzoate (14, 41), andpyrazon (31) dioxygenases and methane (8, 9, 11), octane (4,29) and camphor (26, 35) monooxygenases. In addition,similar enzymes are probably responsible for the oxidationof xylenes and toluates in the catabolic pathway encoded byTOL plasmids (38-40). Little is known about the gene orderand regulation of these enzyme systems. This is mainlybecause of the absence of a suitable screening procedure forthe isolation of mutants defective in individual oxygenasecomponents. Current methods for the isolation of catabolicmutants have focused on certain strains of Pseudomonasspp. and utilize enrichment procedures involving penicillinor D-cycloserine (2, 5, 28). Direct, positive enrichment hasalso been achieved by the use of "suicide substrates" (36,37).The results presented herein demonstrated that a conven-

tional enrichment procedure coupled with the use of theredox dyes NBT and TTC can be used in a selective mannerto identify mutants of P. putida PpF1 that are defective inindividual components of the toluene dioxygenase enzymesystem. In the absence of the redox dyes, all small colonieswould be scored as putative mutants in enzymes involved intoluene degradation. Although the percentage of small colo-nies obtained after enrichment was not determined, it hasbeen our experience that many of these organisms containdefects unrelated to toluene catabolism. Consequently, aconsiderable amount of time and effort is required to screenlarge numbers of mutants for a desired phenotype or geno-type. The advantages of using NBT and TTC are twofold.NBT permits the direct isolation of specific mutants that aredefective in the toluene dioxygenase multicomponent en-zyme system and eliminates the need to test every smallcolony for these properties. In the presence of TTC, a largenumber of small colonies were red. The percentage of suchcolonies was not determined. However every small redcolony tested retained the ability to grow with toluene. Inthe absence of TTC, these colonies would have been con-sidered as putative mutants in the toluene catabolic path-way. Thus, the use of both redox dyes provided a rapid andeffective screening procedure for tod mutants.The principle of the procedure is based on the observation

that the extent of NBT reduction by reductaseto, (todA) isenhanced in the presence of ferredoxinto, (todB). Theseresults seem to hold true for intact cells. Although theprocedure is relatively simple, it is important to note thatconsistent dye reduction is only observed with well-isolatedcolonies. Care must also be taken to avoid nonspecificreduction of NBT. This was achieved by adjusting theconcentrations of NBT and the growth-supporting substrateto levels that only permit NBT reduction in the presence ofthe inducing substrate. Under these conditions, both colonysize and extent of NBT reduction can be used to identifymutant strains. Thus, small white colonies observed afterNTG mutagenesis are putative mutants in structural orregulatory genes involved in toluene degradation. The ra-tionale for selection also predicts that mutants in the genecoding for reductaset., (todA) should also show this pheno-type. However, some of the todA mutants selected were

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P. PUTIDA TOLUENE DIOXYGENASE MUTANTS

small white colonies with pale blue centers (Fig. 3E). Thereason for the slight amount of NBT reduction by thesemutants is not known. However, this property made theidentification of some todA mutants a relatively easy task. Itis important to show that small white colonies are notauxotrophic for the growth-limiting substrate since in thepresent study a significant percentage of such colonies wereidentified as arginine auxotrophs.

Small light blue colonies with dark blue centers (Fig. 3D)were identified as mutants defective in the gene coding forferredoxinto, (todB). The same phenotype was observed withtodBC mutants. Small dark blue colonies (Fig. 3A) wereidentified as mutants in the terminal oxygenase component(ISPtol, todC). These mutants, together with those describedabove, were easily distinguished from the parent strain of P.putida PpF1, which formed large red colonies on the indica-tor medium (Fig. 3B). This was due to the reduction of TTCto 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 tothe tight coupling of electron transport in the intact toluenedioxygenase enzyme system. The inclusion of TTC in theindicator medium was based on the observations of Bochnerand Savageau (3), who showed that TTC reduction onlyoccurs if sufficient levels of a carbon and energy source areavailable. Since nonspecific reduction of TTC can occur, it is

important to determine conditions that allow uninduced andmutant colonies to grow without reducing the redox dye.

Colored colonies unrelated to NBT reduction were alsoobserved after NTG mutagenesis. Small brown colonieswere formed by mutants defective in the enzyme 3-methyl-catechol 2,3-dioxygenase. The color was due to the au-tooxidation of accumulated 3-methylcatechol. Small brightyellow colonies were formed by mutants defective in 2-hydroxy-6-oxo-2,4-heptadienoic acid hydrolyase. At alka-line pH, the substrate for this enzyme had a high extinctioncoefficient at 385 nm, which accounted for the yellow colorobserved. The characterization of these mutants will bereported (manuscript in preparation).The predicted color and colony size of mutants defective

in specific components of the toluene dioxygenase enzymesystem were confirmed by in vitro complementation analy-ses with purified protein components. Representative strainsof the different classes of mutants that were obtained areshown in Table 1. Mutants defective in the structural genesfor reductaseto, (todA), ferredoxint.0 (todB), and ISPt.0 (todC)were isolated. The terminal oxygenase component (ISPtol) oftoluene dioxygenase is an iron-sulfur protein that has an a2P2subunit composition. The molecular weights of the a and Psubunits are 52,500 and 20,800, respectively (33). At thistime, the individual subunits have not been isolated, and wewere unable to assign the a and l subunits to separate genes.

TABLE 1. Biochemical complementation analysis of mutantsToluene dioxygenase activitya

P. putida Reversion Genotypicstrain Rd,01 Fd,.0 ISP,0 RD,01 + RDt., + FD,0 + Control' frequency designation

Ft ISP01, isp,01PpFlC 8 Wild type (TOL+)PpFld -e - - - Wild type (TOL+)PpF12 5 - 6 6 i0-9 todAPpF102 6 8 6 <10-11 todAPpF3 6 - 4 6 - -10 todBPpF126 - 7 7 7 5 x 10-8 todBPpF4 4 4 6 <10-11 todCPpF106 - 3 - 2 4 10-9 todCPpF1O 2 2 4 10-10 todCPpFlll - 6 - 5 8 3 x 10-9 todCPpF24 10 <10-11 todBCPpF25 - 10 <10-11 todBCPpF130 - 7 2 x 10-10 todBCPpF118 - 5 <10-11 todBCPpF22 10 <10-11 todBCPpF27 - 5 10-8 todBCPpF26 - 8 3 x 10-9 todBCPpF31 - 4 <101- todBCPpF120 - - <10-11 todABCPpF7 - <10-11 todABCPpF211 2 x 10-11 todABCPpF133 - <10-1- todABCPpF131 - 10-11 todABCPpF121 4 7 1 2 5 2 x 10-9 todB/CPpF123 2 --< 101 todAB[CrfPpF26a 1 - 6 1 -0-10 todAiB]PpF28 6 6 1 5 2 x 10-7 tod[BJPpF103 3 8 3 6 3 5 3 5 x 10-9 todfB]PpF23 - 3 4 2 x 10-7 todlB]aNanomoles of cis-[14C]toluene dihydrodiol formed per minute. Purified components of the toluene dioxygenase system were added to crude cell extracts as

indicated in the text. Rdto,, Reductaset,1; Fd,01, ferredoxint,l; ISPt,,, iron-sulfur proteint,0; ISPt,1, iron-sulfur protein,..bToluene dioxygenase activity in crude cell extracts of strains in the absence of purified toluene dioxygenase components.Cell extracts prepared from cells grown with arginine in the presence of toluene (induced).d Cell extracts prepared from cells grown with arginine (uninduced).No activity (<1 nmol cis-[14C]toluene dihydrodiol formed per min).

f Brackets indicate low enzyme levels or altered catalytic activities.

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1008 FINETTE, SUBRAMANIAN, AND GIBSON

Bacterial Strain

PpFI12, PpFI02

P,F3, PpFl26

PpF23, PpIF28, PpFl3

PpFl 21 -

Pp F26a -

PpF4, PPFIOPp Fll, Pp Fl06

Pp F27, Pp F24, Pp F25, Pp F22 -

Pp Fl 30, Pp Fl 18, Pp F26, PPF 31

Pp F123 -

Pp F21 1, PpF 7, Pp Fl20Pp Fl 3 1, Pp F l33

CH3

NADHXH+ Rd ToL>..2FdToL ISPL °(AD) NJ (red.)TO loxd) 0 0

todA todB todC 2FeHC H

NADt Rd - <2Fd -/?ISP1\ OH(FADH2)TOL (oxd.)T (red)TOL OH

--- -_ _ q

-- - _-

i ~~-----4

Genotype

tLodA

todB

- tod [B]

tod B/tod C

_ todA[B]

_ tod C

_ tod BC

ttodAB[C]

ttod ABC

I

- Inactive Gene ProductI- --- Low Enzyme Level or Altered Cotolytic Activity

*-_ Mutotion Complemented with Either Gene Product

FIG. 4. Summary of NTG-generated mutations defective in the toluene dioxygenase enzyme system.

Since the components of toluene dioxygenase form acomplex in vivo, the interpretation of in vitro complemen-tation analysis of some mutant strains may be difficult. It isquite possible that a mutation in one component resulting ina structural or conformational change may be complementedby another component of the system. Further biochemicaland genetic analyses should clarify this situation and shedsome light on the interactions and complexing of this multi-component enzyme system.

It should also be noted that some toluene dioxygenasemutants may be a result of double mutations or deletionsgenerated by NTG. This would explain why in certain casesrevertants of mutant strains were not isolated. It is also quitelikely that some revertants were a result of a pseudorever-sion event, especially since the components of toluenedioxygenase form a complex in the cell.

In many Pseudomonas species, the genes coding forenzymes involved in the degradation of aromatic hydro-carbons and related compounds are carried on transmissibleplasmids (20). However, plasmid isolation techniques (19,21, 22, 24, 25) and curing experiments (6, 23, 39) have failedto indicate the presence of an extrachromosomal element inP. putida PpF1. In addition, we were unable to demonstrateconjugal transfer of the todABC genes from a tryptophanauxotroph of the parent strain to any of the mutants isolatedin the present study. Thus, the genes responsible for en-zymes of the dihydrodiol pathway for toluene degradationappear to be located on the chromosome.The availability of a wide variety of mutants defective in

the multicomponent toluene dioxygenase system will facili-tate future studies on the biochemistry and genetic regula-tion of toluene degradation by P. putida PpF1. In addition,the redox dye screening procedure should be applicable toother organisms that contain important multicomponentoxygenases (1, 4, 8, 12, 14, 31, 35, 41).

ACKNOWLEDGMENTS

This investigation was supported by Public Health Service grantGM 29909 from the National Institutes of Health. B.A.F. is aPredoctoral Fellow supported by grant F-440 from the Robert A.Welch Foundation.We thank John La Claire II for his assistance with photographing

bacterial colonies and Ann Rhode and Gregg Whited for theirassistance in preparing the figures. We also thank Catherine Potterfor assistance in preparing the manuscript.

LITERATURE CITED

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2. Bayly, R. C., and G. J. Wigmore. 1973. Metabolism of phenoland cresols by mutants of Pseudomonas putida. J. Bacteriol.113:1112-1120.

3. Bochner, B. R., and M. A. Savageau. 1977. Generalized indica-tor plate for genetic, metabolic, and taxonomic studies withmicroorganisms. Appl. Environ. Microbiol. 33:434-444.

4. Cardini, G., and P. Jurtshuk. 1970. The enzymatic hydroxyla-tion of n-octane by Corynebacterium sp. strain 7E1C. J. Biol.Chem. 245:2789-2796.

5. Carhart, G., and G. D. Hegeman. 1975. Improved method ofselection for mutants of Pseudomonas putida. Appl. Environ.Microbiol. 30:1046-1047.

6. Chakrabarty, A. M. 1972. Genetic basis of the biodegradation ofsalicylate in Pseudomonas. J. Bacteriol. 112:815-823.

7. Claus, D., and N. Walker. 1964. The decomposition of tolueneby soil bacteria. J. Gen. Microbiol. 36:107-122.

8. Colby, J., and H. Dalton. 1978. Resolution of the methanemonooxygenase of Methylococcus capsulatus (Bath) into threecomponents. Biochem. J. 171:461-468.

9. Colby, J., and H. Dalton. 1979. Characterization of the secondprosthetic group of the flavoenzyme NADH-acceptor reductase(component C) of the methane monooxygenase from Methylo-

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Degradation of the benzene nucleus by bacteria. Nature (Lon-don) 202:775-778.

11. Dalton, H. 1980. Oxidation of hydrocarbons by methanemonoxygenase from a variety of microbes. Adv. Appl. Mi-crobiol. 26:71-87.

12. Ensley, B. D., and D. T. Gibson. 1983. Naphthalene dioxygen-ase: Purification and properties of a terminal oxygenase compo-nent. J. Bacteriol. 155:505-511.

13. Ensley, B. D., D. T. Gibson, and A. L. Laborde. 1982. Oxidationof naphthalene by a multicomponent enzyme system fromPseudomonas sp. strain NCIB 9816. J. Bacteriol. 149:948-954.

14. Fujisawa, H., M. Yamaguchi, and T. Yamaguchi. 1976. Evi-dence for participation of NADH-dependent reductase in thereaction of benzoate 1,2-dioxygenase. Adv. Exp. Med. Biol.74:118-126.

15. Geary, P. J., and D. P. E. Dickson. 1981. Mossbauer spectro-scopic studies of the terminal dioxygenase protein of benzenedioxygenase from Pseudomonas putida. Biochem. J. 195:199-203.

16. Gibson, D. T., M. Hensley, H. Yoshioka, and T. J. Mabry. 1970.Formation of (+)-cis-2,3-dihydroxy-1-methyl-cyclohexa-4,6-diene from toluene by Pseudomonas putida. Biochemistry9:1626-1630.

17. Gibson, D. T., J. R. Koch, and R. E. Kallio. 1968. Oxidativedegradation of aromatic hydrocarbons by microorganisms. I.Enzymatic formation of catechol from benzene. Biochemistry7:2653-2662.

18. Gibson, D. T., W.-K. Yeh, T.-N. Liu, and V. Subramanian.1982. Toluene dioxygenase: a multicomponent enzyme systemfrom Pseudomonas putida, p. 51-61. In N. Mitsuhiro et al.(ed.), Oxygenases and oxygen metabolism. Academic Press,Inc., New York.

19. Guerry, P., D. J. LeBlanc, and S. Falkow. 1973. General methodfor the isolation of plasmid deoxyribonucleic acid. J. Bacteriol.116:1064-1066.

20. Haas, D. 1983. Genetic aspects of biodegradation by pseudomon-ads. Experientia 39:1199-1213.

21. Hanson, J. B., and R. H. Olsen. 1978. Isolation of large bacterialplasmids and characterization of P2 incompatibility group plas-mids: pMG1 and pMG5. J. Bacteriol. 135:227-238.

22. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method forthe preparation of bacterial plasmids. Anal. Biochem. 114:193-197.

23. Johnston, J. B., and J. A. Grunau. 1980. Simple method ofstudying drug-induced loss of plasmid DNA (curing), p. 739-741.In J. D. Nelson and C. Grassi (ed.), Current chemotherapy andinfectious disease, vol. I. American Society for Microbiology,Washington, D.C.

24. Kado, C. I., and S. T. Liu. 1981. Rapid procedure for detectionand isolation of large and small plasmids. J. Bacteriol.145:1365-1373.

25. Kahn, M., R. Kolter, C. Thomas, D. Figurski, R. Meyer, E.Regault, and D. R. Helinski. 1979. Plasmid cloning vehiclesderived from plasmids ColEl, F, R6K, and RK2. MethodsEnzymol. 68:268-280.

26. Katagiri, M., B. N. Gauguli, and I. C. Gunsalus. 1968. A solublecytochrome P-450 functional in methylene hydroxylation. J.

Biol. Chem. 243:3543-3546.27. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.

1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

28. Ornston, L. N., M. K. Ornston, and G. Chou. 1969. Isolation ofspontanous mutant strains of Pseudtomonas putida. Biochem.Biophys. Res. Commun. 26:179-184.

29. Peterson, J. A., D. Basu, and M. J. Coon. 1966. Enzymaticoxidation. I. Electron carriers in fatty acid and hydrocarbonhydroxylation. J. Biol. Chem. 241:5162-5164.

30. Rodgers, J. E., and D. T. Gibson. 1977. Purification and prop-erties of cis-toluene dihydrodiol dehydrogenase from Pseudo-monas putida. J. Bacteriol. 130:1117-1124.

31. Sauber, K., C. Frogner, G. Rosenberg, J. Eberspacher, and F.Lingens. 1977. Purification and properties of pyrazon dioxyge-nase from pyrazon-degrading bacteria. Eur. J. Biochem.74:89-97.

32. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966. Theaerobic pseudomonads: a taxonomic study. J. Gen. Microbiol.43:159-271.

33. Subramanian, V., T.-N. Liu, W.K. Yeh, and D. T. Gibson. 1979.Toluene dioxygenase: purification of an iron-sulfur protein byaffinity chromatography. Biochem. Biophys. Res. Commun.91:1131-1139.

34. Subramanian, V., T.-N. Liu, W.-K. Yeh, M. Narro, and D. T.Gibson. 1981. Purification and properties of NADH-ferre-doxint,, reductase. J. Biol. Chem. 256:2723-2730.

35. Tsai, R. L., I. C. Gunsalus, and K. Dus. 1971. Composition andstructure of camphor hydroxylase components. Biochem. Bi-ophys. Res. Commun. 45:1300-1306.

36. Wigmore, G. J., and D. W. Ribbons. 1980. p-Cymene pathwayin Pseudomonas putida: selective enrichment of defective mu-tants using halogenated substrate analogs. J. Bacteriol.143:816-824.

37. Wigmore, G. J., and D. W. Ribbons. 1981. Selective enrichmentof Pseudomonas sp. defective in catabolism after exposure tohalogenated substrates. J. Bacteriol. 146:920-927.

38. Williams, P. A., and K. Murray. 1974. Metabolism of benzoateand the methyl benzoates by Pseudomonas putida (arvilla)mt-2: evidence for the existence of a TOL plasmid. J. Bacteriol.120:416-423.

39. Williams, P. A., and M. J. Worsey. 1976. Ubiquity of plasmidsin coding for toluene and xylene metabolism in soil bacteria:evidence for the existence of new TOL plasmids. J. Bacteriol.125:818-828.

40. Worsey, M. J., and P. A. Williams. 1975. Metabolism of tolueneand xylene by Pseudomonas putida (arvilla) mt-2. Evidence fora new function of the TOL plasmid. J. Bacteriol. 124:7-13.

41. Yamaguchi, M., and H. Fujisawa. 1978. Characterization ofNADH-cytochrome c reductase, a component of benzoate1,2-dioxygenase system from Pseudomonas arvilla C-1. J. Biol.Chem. 253:8848-8853.

42. Yeh, W.-K., D. T. Gibson, and T.-N. Liu. 1977. Toluenedioxygenase: a multicomponent enzyme system. Biochem. Bi-ophys. Res. Commun. 78:401-410.

43. Ziffer, H., D. M. Jerina, D. T. Gibson, and V. M. Kobal. 1973.Absolute stereochemistry of the (+)-cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene produced from toluene by Pseudo-monas putida. J. Am. Chem. Soc. 95:4048-4049.

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