Eur. J. Biochem. 28, 301-310 (1972)

The Metabolism of Benzoate and Methylbenzoates via the meta-Cleavage Pathway by Pseudomonas arviZZa mt- 2

Keith MURRAY, Clive J. DUGGLEBY, Jose M. SALA-TREPAT, and Peter A. WILLIAMS Department of Biochemistry and Soil Science, University College of North Wales, Bangor

(Received February 16fApril20, 1972)

1. Pseudomonas arvilla mt-2 grows a t the expense of benzoate, m-toluate (3-methylbenzoate) and p-toluate (4-methylbenzoate), but not o-toluate (2-methylbenzoate). Under various condi- tions compounds with spectra characteristic of muconic semialdehydes can be made to accumu- late in the media. These spectra indicate that benzoate is metabolised through catechol and 2-hy- droxymuconic semialdehyde (2-hydroxy-6-oxohexa-2,4-dienoate), m-toluate through 3-methyl- catechol and 2-hydroxy-6-oxohepta-2,4-dienoate and p-toluate through 4-methylcatechol and 2-hydroxy-5-methylmuconic semialdehyde (2-hydroxy-5-methyl-6-oxohexa-2,4-dienoate).

2. Freshly harvested cells grown on all three substrates only consume oxygen a t a significant rate when presented with the growth substrates themselves and catechol and the methylcate- chols, but not with salicylate, protocatechuate or phenol or the cresols.

3. Extracts of cells grown on these substrates contain high induced levels of the suite of meta cleavage enzymes, including both the hydrolytic branch and the 4-oxalocrotonate branch.

4. The substrate specificity of the early enzymes of the pathway suggests that only one non- specific enzyme expresses each activity in cell-free extracts and that it is nonspecifically induced during growth on all three carbon sources.

5. It is suggested that the metabolic role of the hydrolytic branch of the pathway is the assi- milation of 3-methylcatechol and its precursor, and that of the 4-oxalocrotonate branch is to assimilate catechol and 4-methylcatechol and their precursors.

6. o-Toluate is not metabolised because it is neither a substrate for the benzoate oxidase system nor an inducer of any of the meta pathway enzymes.

7 . It appears that benzoate and m- and p-toluates are the substrate inducers of the meta pathway enzymes in this organism. The ortho pathway is induced on incubating cells with cate- chol and the first inducer appears to be &,cis-muconate or possibly catechol itself.

The coexistence of two enzymes able to degrade the meta cleavage product of catechol, 2-hydroxy- muconic semialdehyde, was first demonstrated in cell-free extracts of both benzoate-grown Azotobacter species [l] and a naphthalene-grown Pseudomonas strain NCIB 9816 [2]. Sala-Trepat and Evans [3] elucidated the two resulting pathways, which after diverging a t 2-hydroxymuconic semialdehyde con- verge again a t 2-oxopent-4-enoate ; they concluded that in Azotobacter only the 4-oxalocrotonate branch, involving as the first step the NADf-dependent dehydrogenation of 2- hydroxymuconic semialdehyde, was of any metabolic significance since the 2-hy- droxymuconic semialdehyde hydrolase was present

Enzymes. Catechol 1,2-oxygenase or catechol: oxygen 1,2-oxidoreductase (EC 1.13.1.1); catechol 2,3-oxygenase or catecho1:oxygen 2,3-oxidoredoctase (EC 1.13.1.2); NAD nuc- leosidase or NAD glycohydrolase (EC 3.2.2.6).

21 Eur. J. Blochem., Vol.28

in only very low uninducible levels. In NCIB 9816 all the enzymes of the 4-oxalocrotonate branch are present [4] and again it has been proposed that cate- chol is metabolised mainly by this pathway, since although the hydrolase is induced by growth on naphthalene, the level of induction is much less than that of the 2-hydroxymuconic semialdehyde dehydro- genase. As a result of work on these two organisms, a programme was initiated to investigate whether the two branches coexist in other organisms capable of rneta cleavage, and to study their respective metabolic roles.

In Pseudomonas putida NCIB 10015, the organism in which the hydrolytic pathway was first described [5,6], enzymes of both branches are present and are induced to high levels by growth on phenol or the cresols (methylphenols) [7,8]. From a study of the specificity of the ring-fission product-metabolising

302 Benzoate and Methylbenzoate Metabolism by Pseudomonas arvilla Em. J. Biochem.

1 . Benzoate 2. m-Toluate (3-methylbenzoate) (R' = CH3,R2=H)

3 . p - Toluate (4- methylbenzoate) (R' = H ,R2=CH3) ? P O *

Benzoate J oxidase I

Catechol 2 , 3 - 'to. oxygenase -. __

1 . Catechol 2.3-Methylcatechol 3.4-Methylcatechol

1 .2 - Hydroxymuconic semialdehyde G (2 - Hydroxy - 6 - oxohexa - 2,4 - dienoate ) 2. 2 -Hydroxy -6-oxohepta- 2,4 -dienoate 3 . 2 - Hydroxy - 5 - methylmuconic semialdehyde (:-~ydroxy - 5 -methyl COOH 1.4-Oxa~ocrotonate (m)

COOH (2-hydroxy -hexa -2 ,4 -d iene -186-d ioa te ) 3. 2-Methyl -4-oxalocrotonate (enol)

- 2,4 -diene - 1,6 -dioate)

1 . 2 - Oxopent - 4 - enoate 2 . 2 - Oxopent - 4 - enoate 3 . 2 - Oxohex - 4 - enoate

( 2 - oxohex - 4 -ere- t ,6 - dioate)

( 5 - methyl - 2-oxohex - 4 - ene - 1,6 -dioate) 3. 2-Methyl - 4-oxalocrotonate (&)

2 - Oxopent - 4 - enoate hydratase -___

J.

''OH 1 . 4- hydrLxy - 2-oxovalerate 2 4 - hydroxy - 2-oxovalerate 3 4- hydroxy - 2 - oxohexanoate

4- Hydroxy -2-oxovalerate HO c R2 __

aldolase 0 R2CH2CH0

CH3;o COOH

Fig. 1. Proposed pathway for metabolism of benzoate and m- and p-toluates by P. arvilla. Metabolites of benzoate are denoted (l), those of m-toluate ( 2 ) and those of p-toluate (3)

enzymes it is proposed that in this organism the 4-oxalocrotonate branch is used for the metabolism of catechol and 4-methylcatechol and their precursors (in this case phenol and p-cresol) and the hydrolytic branch for the metabolism of 3-methylcatechol and its precursors (0- and m-cresol) [S, 91.

This paper further extends the study to Pseudo- monas arvilla mt-2 which is a representative of the species Pseudomonas putida, biotype A [lo], typical in all nutritional and cultural characteristics except that it metabolises benzoate via the meta pathway [Ill. Some aspects of the meta cleavage pathway in this organism have been reported by Feist and Hege- man [Ill. The results presented here show the co- existence of both branches of the meta cleavage pathway in cells grown on benzoate and m- and p-toluates (methylbenzoates) (Fig. I ) ; the metabolic

role of the two branches appears to be the same as in P. putida NCIB 10015.

EXPERIMENTAL PROCEDURE MATERIALS

Microorganisms Pseudomonas arvilla mt-2 was a gift from Dr.

G. D. Hegeman (Department of Bacteriology and Immunology, University of California, Berkeley) and Pseudomonas putida KBI (NCIB 10521), a gift from Dr M. Knight of this department.

Chemicals The preparation of 4-oxalocrotonate and 4-hy-

droxy-2-oxovalerate has been described [3]. cis,cis-

V01.28, N0.3,1972 K. MURRAY, C. J. DUGGLEBY, J. M. SALA-TREPAT, and P. A. WILLIAMS 303

Muconic acid was a gift from Dr M. Robert-Gero (Laboratoire d' Enzymologie, C.N .R.S . , Gif-sur- Yvette,France). All other chemicals and reagents were from commercial sources.

METHODS

Maintenance and Culture of Microorganisms Pseudomonas arvilla mt-2 was maintained on

nutrient agar slopes. Liquid media contained 1 .O g/1

5 mg/l FeSO, * 7H20, 0.5 g/l nitrilotriacetic acid, 1 ml/l of stock salts solution [I21 and carbon source as indicated; the pH was adjusted to 7.0 with 5M NaOH before sterilisation. Carbon sources were added before sterilisation except for salicyclic and the methylsalicylic acids, which were neutralised with NaOH and sterilised separately by filtration. Growth in all cultures was initiated by addition of a loo/, inoculum of succinate-grown cells.

For oxygen-uptake experiments cells were grown in 50ml medium in conical flasks maintained a t 30 "C in a rotary shaker. The cells were sedimented on a bench centrifuge, washed twice with 100mM phosphate buffer, pH 7.5, and resuspended in the same buffer to an absorbance a t 610 nm of 0.40-0.45 ready for immediate use.

Large scale batch cultures for preparation of cell-free extracts were grown under forced aeration in I01 Pyrex bottles in a water bath a t 30 "C. Cells were harvested when still growing exponentially in an air-driven Sharples Supercentrifuge a t room tem- perature. The freshly harvested cells were washed twice with 100 mM phosphate buffer, pH 7.5, and if not required for immediate use, stored a t -25 "C.

Pseudomonas putida KBl was maintained on agar slopes containing 0.50/, succinate. Cultures were grown in liquid medium containing 0.5 g/1 (NH,),SO,, 10 g/l KH,PO,, 1.0 g/1 nitrilotriacetic acid, 5 g/1 sodium succinate and 1 ml/l stock salts solution [12], which was adjusted to pH 7.0 with 5 M NaOH. 10 1 batch cultures were grown and cells harvested, washed and stored as described for Pseudo- moms arvilla.

(NHJ2SO4, 5.0 g/l KHZPO,, 0.1 g/1 MgSO4 * 7HZ0,

Preparation of Cell-Free Extracts of Pseudomonas arvilla

2 g wet weight of cells were suspended in I0 ml 100 mM phosphate buffer, pH 7.5, containing loo/, acetone, and broken by sonicating at 0 "C for 5 min in an M.S.E. IOOW ultrasonic disintegrator. Cell debris was removed by centrifuging a t 90000 x g for 60 min.

Preparation of NAD Glycohydrolase (NADase) 4 g wet weight of cells of Pseudomonas putida

KB1 were suspended in 10 ml 100 mM phosphate 21'

buffer, pH 7.5, and broken by sonicating a t 0 "C for 5 min in an M.S.E. ultrasonic disintegrator. Cell debris was removed by centrifuging a t I15000 x g for 90 min. The cell-free extract was heated on a boiling-water bath for 5-10 min, and the protein precipitate removed by centrifiiging at 65 000 x g for 15 min. The preparation was assayed for NADase activity by the KCN method [13], and was stored at -25 "C until required.

Preparation of the Ring- Fission Products of the Catechols

These compounds were prepared in solution by the action of heat-treated cell-free extracts of Pseudo- monas putida NCIB 10015 on the corresponding catechols, as describe3 previously [S].

Enzyme Assays All the spectrophotometric assays were carried

out a t ambient temperatures in a Unicam SPSOO recording spectrophotometer. In performing a series of assays of the meta pathway enzymes in a partic- ular cell-free extract, the activities of catechol 2,3-oxygenase end the ring-fission product-metabol- ising enzymes were measured immediately after preparation of the extract and the 4-oxalocrotonate tautomerase, the 4-oxalocrotonate decarboxylase and the 4-hydroxy-2-oxovalerate aldolase activities measured on the following day. The extract was kept between 0 "C and 4 "C until all these assays were completed.

The procedures for assaying and definition of the units of activity of catechol2,3-oxygenase, 2-hydroxy- muconic semialdehyde hydrolase, 2-hydroxymuconic semialdehyde dehydrogenase, 4-oxalocrotonate tauto- merase and 4-hydroxy-2-oxovalerate aldolase were as previously described [3,8].

4-Oxalocrotonate decarboxylase was assayed as previously described [3], but with a modified reaction mixture containing 5-10 pl cell-free extract, 60 p1 of an ethanolic solution of 4-oxalocrotonate, 0.1 ml 100 mM MgSO, and I00 mM phosphate buffer, pH 7.5 to 3 ml. The unit of activity remains as before.

Catecholl,2-oxygenase was assayed by measuring the increase in absorbance a t 260 nm due to formation of cis,&-muconate in a reaction mixture containing 10-50 p1 cell-free extract, 0.2 ml 500 mM EDTA, 0.2 ml 1 mM catechol and 100 mM phosphate buffer, pH 7.5, to 3.0ml. The blank cell contained all except substrate. The unit of activity is defined as the amount of enzyme required to produce 1 pmol of &,cis-muconate in 1 min, equivalent to an increase in absorbance of 5.63.

cis&-Muconate lactonising enzyme was assayed by measuring the decrease in absorbance a t 260 nm due to the disappearance of substrate in a reaction mixture containing 20-50 pl cell-free extracts,

304 Benzoate and Methylbenzoate Metabolism by Pseudomonas arzdla Eur. J. Biochem.

0.3 ml 1 mM &,&muconate, 50 pl 100 mM MnC1, and 100 mM Tris-HC1 buffer, pH 8.0, to 3.0 ml. The blank cell contained all except substrate. The unit of activity is defined as that amount of enzyme which will destroy 1 pmo1 &&-muconate in 1 min, equi- valent to a decrease in absorbance of 5.75.

Protein Determination The concentration of protein in cell-free extracts

was measured by the biuret method [14].

RESULTS The Metabolism of Benzoate and m- and p-Toluates

Pseudomonas arvilla was able to grow both solid and in liquid media on only benzoate and m- and p-toluates of the compounds tested as sole carbon sources; no growth was observed upon salicylate, 3-methylsalicylate, 4-methylsalicylate or 5-methyl- salicylate or upon o-toluate a t 1 mM, 2.5 mM or 5 mM.

Liquid cultures grown on benzoate and m- and p-toluates showed transient yellow colours charac- teristic of 2-hydroxymuconic semialdehyde and its analogues formed as a result of meta cleavage. Freshly harvested cells grown upon any of the three acids also produced the characteristic yellow colour after a few minutes incubation with catecho1 or either of the methylcatechols.

In order to ascertain which of the ring-fission products was formed during growth on each sub- strate, media from cultures from which the cells had been removed by centrifugation were examined for the characteristic ring-fission product spectra formed on making alkaline; only in benzoate-grown cultures did sufficient accumulate to produce a reasonable spectrum (Fig. 2A). In the case of the tolu- ates, freshly harvested and washed cells grown on each of the acids were resuspended in media contain- ing the growth substrate but under conditions far from optimal for growth. The ring-fission products from the toluates accumulated best in those cultures maintained a t acid pH values (Fig.2B, C ) . The ring- fission product produced from m-toluate by m-tolu- ate-grown cells is identical under both acid and alka- line conditions with that of authentic 2-hydroxy-6- oxohepta-2,4-dienoate, the ring-fission product of 3-methylcatechol prepared from 3-methylcatechol by heat-treated extracts of P. putida NCIB 10015 (Fig.2B). In the case of the accumulated products from benzoate and p-toluate, the identification was not as definite, since on increasing the pH to 12, the lmsx of the spectrum produced in each case was about 2 n m below that of 2-hydroxymuconic semi- aldehyde and 2-hydroxy-5-methylmuconic semi- aldehyde, respectively, and also showed a distinct broadening on the low wavelength side (Fig.ZA, C).

3 3 0 350

I

"

- 330 350 400 451

Wavelength ( n r n )

Fig. 2. Spectra of culture supernatants of P. arvilla cells grown on benzoate and the toluutes, compared with spectra of authentic meta ring-fission. products of mtechol and the methyleatechols. (A) Supernatant from 10 1 culture of benzoate-grown cells (I), made alkaline to pH 12, compared authentic ring-fission products of catechol (11), 4-methylcatechol(III) and 3-methyl catechol (IV), all at pH 12. (B) Supernatant from 3-h incuba- tion of m-toluate-grown cells in medium at pH 5.5 containing 5 mM m-toluate, at p H 2 (I) and at pH 12 (11), compared with authentic ring-fission product of 3-methylcatechol a t p H 2 (111) and pH 12 (IV). (C) Supernatant from 3-h incuba- tion of p-toluate-grown cells in medium at pH 5.7 containing 5 mM p-toluate, at pH 12 (I) compared with authentic ring- fission product of 4-methylcatechol a t p H 12 (11). (D) Fresh ring-fission product of 4-methylcatechol, adjusted to pH 12 (I) and after standing in aqueous solution overnight before adjusting to pH 12 (11). Authentic ring-fission products were prepared as described in Experimental Methods. The p H was adjusted to 12 or 2 by addition of a few drops of

5 M NaOH or 5 M HCI

Part of this may be due to the presence of other me- tabolites absorbing a t low wavelengths, but may also be due to some chemical alteration taking place during the incubation since a similar but not identical effect could be produced either by incubating fresh ring-fis- sion products with medium from succinate-grown cells from which the cells had been removed by centrifuga- tion or merely on standing (Fig.2D). Since it is well authenticated that benzoate is metabolised via 2-hy- droxymuconic semialdehyde [ll], and the spectrum of the yellow compound formed in the culture super- natant shows the characteristic spectral changes of 2-hydroxymuconic semialdehyde except for the broadening of the spectrum on the low wavelength side and the slight lowering of the value of lmax a t high pH, it is not unreasonable to assume that the accumulated compound is mainly 2-hydroxymuconic

K. MURRAY, C. J. DUOOLEBY, J. M. SALA-TREPAT, and P. A. WILLIAMS 305 V01.28, N0.3,1972

Table 1. Rates of oxygen uptake by freshly harvested cells of Pseudomonas arvilla

Rates of oxygen uptake were measured with a Clark oxygen electrode. In each case 1.8 ml of a suspension of cells, freshly harvested, washed and resuspended to an absorbance value at 610 nm of 0.40-0.45 in 100 mM phosphate buffer, pH 7.5, were added to 0.2 ml water (to measure endogenous uptake) or to 0.2 ml 10 mM assay substrate. Endogenous uptake was subtracted from uptake in the presence of substrate. Rates of uptake <27 yl/h per mg dry weight cells, an average value for endogenous uptake, are considered insignificant. n.d.

= not determined

Growth substrate

Benzoate m-Toluate p-Toluate ( 6 mM) (5 mM) (5 mM)

Assay substrate

~1 x h-' x mg-1 Experiment I Benzoate 139 281 93 m-Toluate 95 241 107 p-Toluate 40 192 100 o-Toluate 37 25 26 Catechol 394 65 1 242 3-Methylcatechol 224 530 305 4-Methylcatechol 487 719 514 Salicylate 0 11 30 Protocatechuate n.d. 12 0 Experiment I1 Catechol 338 196 342 Phenol 2.3 3.4 2.3 0-Cresol 0 0 2.3 m-Cresol 0 6.8 15 p-Cresol 0 2.6 2.3

Table 2. Specific activities of enzymes in cell-free extracts of Pseudomonas arvilla

Assays were carried out as described in Experimental Procedure. n.d. = not determined

Growth substrate

Activity Succinate Benzoate m-Toiuate p-Toluate (11mM) (10mM) (5mM) (5mM)

meta pathway Catechol 2,3-oxy-

genase 2-Hydroxymuconic

semialdehyde hydrolase

2-Hydroxymuconic semialdehyde dehy drogenase

4-Oxalocrotonate tautomerase

4-Oxalocrotonate decarboxylase

4-Hydroxy-2-0x0- valerate aldolase

ortho pathway Catechol l,2-0xy-

genase &,cis-Muconate

lactonising enzyme

228

1.3

3.3

250

16

2.6

2.7

2.6

mU/mg protein

7250 4160

27 20

163 90

50500 59300

910 320

49 19

0 n.d.

21 n.d.

4 020

16

80

36 700

360

18

n.d.

n.d.

semialdehyde. It then follows that the identical effect shown with the yellow compound formed from p-toluate is the meta cleavage product of 4-methylcatechol. In fact if p-toluate is metabolised by the meta pathway in a straightforward manner, 4-methylcatechol is the only methylcatechol that could be formed. Where there is doubt is in the metabolism of m-toluate which could be converted to either 3-methylcatechol or 4-methylcatechol ; in this case only does the identification of the ring- fission product from its spectrum appear definitive with no complicating features.

Freshly harvested cells grown on benzoate, m- and p-toluates showed significant oxygen uptake when incubated with catechol, 3- and 4-methyl- catechol, benzoate and m- and p-toluates, but negligible uptake with o-toluate, salicylate, proto- catechuate or phenol or the cresols (Table I). The relative rates of uptake by cells grown on the different substrates stimulated by the catechols is similar, but cells grown on benzoate show a higher relative uptake with benzoate than do cells grown on either of the toluates.

Assays of cell-free extracts of cells grown on ben- zoate and m- and p-toluates show the presence of high levels of catechol 2,3-oxygenase, Z-hydroxy- muconic semialdehyde hydrolase, the enzymes of

the 4oxalocrotonate branch and 4-hydroxy-2-0x0- valerate aldolase (Table 2). All these enzymes are strongly induced by grown on the aromatic sub- strates, although, as is well authenticated for Pseudo- m0na.s arvilla, the constitutive level of catechol 2,3-oxygenase in succinate-grown cells is considerable [Ill. No catechol 1,2-oxygenase could be detected in benzoate-grown cells, although the level of the other enzyme of the ortho pathway assayed, &,&muconate lactonising enzyme, did appear to be about eight times higher than in succinate-grown cells.

The specificity of the catechol 2,3-oxygenase activities in extracts of cells grown on the different substrates is very similar (Table 3), the relative rates of oxidation of catechol, 4-methylcatechol and 3-me- thylcatechol being 100 : 80 : 60 respectively ; by com- parison the rates of oxygen utilisation by whole cells on the catechols shows much greater variation (Table I). However, from the enzyme assays it seems very likely that P . arvilla contains only one catechol 2,3-oxygenase, present in fairly high levels in succin- ate-grown cells and induced about 20- to 40-fold by growth on any of the substrates.

The same situation also appears to be true for both the ring-fission product-metabolising enzymes (Table 4) : the 2-hydroxymuconic semialdehyde hydrolase acts upon the ring-fission product of 3-me- thylcatechol a t a much greater rate than those of catechol or 4-methylcatechol, whereas the converse

306 Benzoate and Methylbenzoate Metabolism by €?seudomonas arvilla Eur. J. Biochem.

is true of the 2-hydroxymuconic semialdehyde de- hydrogenase, which oxidises the two semialdehydes a t similar rates, but is unable to act upon the ketone group of 2-hydroxy-6-oxohepta-2,4-dienoate.

The Induction of the meta-Pathway Enzymes We were unable to repeat the observation of

Feist and Hegeman [I I] that salicylate appeared to be a nonmetabolisable inducer of the meta cleavage

Table 3. Specificity of catechol2,d-oxygenase activity in cell-free extracts of Pseudomonas arvilla

The assay was carried out as described in Experimental methods with the substitution, where appropriate, of the corresponding substrate for catechol. The increase in absorb- ances were measured at 375, 388 or 382 nm, the character- istic I,,, values for the ring-fission product 5 of catechol, 3-methylcatechol and 4-methylcatechol, respectively, and the specific activities calculated assuming conversion of 0.1 pmol substrate in the 3 ml reaction mixture produced increases in

absorbance of 1.2, 0.5 and 1.05, respcctively

Specific Relativc activity activity Assay substrate Growth

substrate

Succinate 11 mM

Benzoate 10 mM

m-Toluate 5 mM

p-Toluate 5 mM

mU/mg protein ' lo Catechol 228 100

3-Methylcatechol 140 61 4-Methylcatechol 185 81

Catechol 7250 100 3-Methylcatechol 4100 57 4-Methylcatechol 5610 77

Catechol 4160 100 3-Methylcatechol 2560 62 4-Methylcatechol 3570 86

Catechol 4020 100 %-Methylcatecho1 2730 63 4-Methylcatechol 3400 85

enzymes. Neither did freshly harvested cells grown upon succinate in the presence of salicylate or either of three methylsalicylates take up oxygen a t signi- ficant rates with the benzoates or the catechols (Table 5), nor did cell-free extracts of cells grown on the same media contain levels of meta pathway en- zymes greater than those in succinate-grown cells (Table 6), rather the levels were lower. Growth on succinate plus benzoate under identical conditions produced cells which both oxidised the catechols a t significant rates (although the rates of oxidation of the benzoates were very low) and also contained levels of the meta pathway enzymes, except for the 4-hy- droxy-2-oxovalerate aldolase, which were about twice those in succinate-grown cells. The discrepancy be- tween our results and those of Feist and Hegeman regarding the role of salicylate may be due to a great- er degree of repression brought about by the 11 mM succinate used by us as aliphatic substrate compared with the 20mM acetate used by them. Other attempts to find a nonmetabolisable inducer using 2-, 3- and 4-chlorobenzoates were unsuccessful.

We did find that P. arvilla was able to grow on catechol as sole carbon source on agar plates contain- ing 2 mM catechol, and a reduced level of salts in the medium (FeSO, - 7H,O reduced to 2 mg/l and the stock salt solution to 0.5 ml/l) ; this also disagrees with an observation of Feist and Hegeman [11]. Rather than grow P. arvilla on catechol in liquid medium, which presents some difficulties due to its toxicity and ease of oxidation, we carried out an experiment similar to one used by Johnson and Stanier with Alcaligenes eutrophus [15]. P. arvilla was grown upon 10 1 11 mM succinate until the end of the logarithmic phase, when the cells were harvest- ed and washed. They were then divided into two

Table 4. Specificity of ring-fission-?Yfoduct-metabolis~~ enzymes in cell-free extract of Pseudomonas arvilla The assays were performed as described in Experimental methods with the substitution where appropriate of the corresponding substrate. The rates of decrease of absorbance at 375,388 and 382 nm were followed spectrophotometrically and the activities

calculated using the absorbance values given in Table 3 2-Hydroxymuconic semialdehyde 2-Hydroxymuconic semialdehyde

hydrolase dehydrogenase Assay substrate Growth

substrate Specific Relative Specific Relative activity activity activity activity

Succinate 11 mM

Benzoate 10 mM

m-Toluate 5mM

p-Toluate 5 mM

2-Hydroxymuconic semialdehyde 2-Hydroxy-6-oxohepta-2,4-dienoate 2-Hydroxy-5-methylmuconic semialdehyde 2-Hydroxymuconic semialdehyde 2-Hydroxy-6-oxohepta-2,4-dienoate 2-Hydroxy-5-methylmuconic semialdehyde 2-Hydroxymuconic semialdehyde 2-Hydroxy-6-oxohepta-2,4-dienoate 2-Hydroxy-5-methylmuconic semialdehyde 2-Hydroxymuconic semialdehyde 2-Hydroxy-6-oxohepta-2,4-dienoate 2-Hydroxy-5-methylmuconic semialdehyde

mU/mg '/a

1.3 6 23 100

1.0 4 27 3

840 100 31 4 20 4

470 100 16 3 16 4

420 100 17 4

mU/mg O l O

3.3 100 0 0 4.5 140

163 100 0 0

189 115 80 100 0 0

113 140 90 100 0 0

104 115

VO1.28, N0.3,1972 K. MURRAY, C . J. DUCGLEBY, J. Y. SALA-TREPAT, and P. A. WILLIAMS 307

Table 5. Rates of oxygen uptake by freshly harvested cells of Pseudomonas arvilla grown on various media Experimental method as described in Table 1. n.d. = not determined

Oxygen uptake with growth substrates

Assay substrate Benzoate mDf Salicylate mM 3-Methylsalicylate 4-Methylsalicylate 5-Methylsalicylate o-Toluate + succinate 11 mM + succinate 11 mM 5 mM + snccinate 5 mM + succinate 5 mM + succinate 5 mM + succinate

11 mM 11 mM 11 mM 11 mill ~ _____ ~ ~ _ _ _ _ _ _ _ _ _ _

~1 x h-' x mg-'

Benzoate 30 9.4 23 18 1.4 n.d. o-Toluate 32 3.1 n.d. 8 n.d. n.d. m-Toluate 17 14 n.d. 13 n.d. n.d. p-Toluate 18 0 n.d. 0 n.d. n.d. Cat e c h o 1 106 7.8 30 8 0 6.6 4-Methylcatechol 90 11 n.d. 11 n.d. 8.2

Salicylate n.d. 9.4 10 11 2.8 n.d. Protocatechuate 23 3.1 n.d. 5 11.5 n.d.

3-Methylcatechol 47 17 n.d. 13 n.d. 0

Table 6. specific activities of meta-cleavage enzymes in cell-free extracts of Pseudomonas arvilla grown on various media Assay as described in Experimental Procedure

Specific activity in growth media

Benzoate (5 mM) Salicylate (5 mM) o-Toluate (5 mM) Suctlinatoa Activity + Succiuate (11 mM) + Succinate (11 mM) + Succiuate (11 mM) (11 mM)

mU/mg protein

Catechol 2,a-oxygenase 500 54 51 228 2-Hydroxymuconic semialdehyde

2-Hydroxymuconic semialdehyde de-

4-Oxalocrotonate tautomerase 2070 260 780 250 4-Oxalocrotonate decarboxylase 23 2.4 0 16

hydrolase 2.5 0.4 0.6 1.3

hydrogenase 6.4 1.4 0.5 3.3

4-Hydroxy-2-oxovalerate aldolase 0.7 6 5 2.5

a Results taken from Table 2.

2 4 6 8 llJII

2 4 6 8 Time (h)

Fig. 3. Chunges in concentration of substrate and 2-hydroxy- muconic semialdehyde in medium during incubation of succin- ate-grown cells of Pseudomonas arvilla with ( A ) benzoate and ( B ) catechol. Concentration of benzoate (O), catechol (o), 2-hydroxymuconic semialdehyde (0) . The details of the ex- periment are described inthetext. Concentrations of benzoate,

catechol and 2-hydroxymuconic semialdehyde were estimated by sampling the medium, removing the cells by centrifuga- tion and measuring the absorbances a t 271,275 and 375 nm, respectively, using values for the absorbance of 1 mM solu- tions at the respective wavelengths of 0.54,2.4 and 43.2. The arrows denote the times of addition of substrate (1, 2, 4, 5),

start of aeration (3) and harvesting of cells (6)

308 Benzoate and Methylbenzoate Metabolism by Pseudomonas arvilla Em. J. Biochem.

Table 7. Specific activities of enzymes in cell-free extracts of succinate-grown cells of Pseudomonas arvilla after incabation

with catechol or benzoate Succinate-grown cells were resuspended in mineral salts medium to an absorbance value at 610 nm of 0.34, and either benzoate or catro'lol were added in four equal increments over the course of 6 h to a final concentration of 5 mM. Aera- tion was gradually increased during the course of the experi- ment. Cells were harvested 8 h after the start of the experi- ment and cell-extract prepared and assayed as described in

Experimental Procedure. n.d. = not determined

Specific activities

Activity Extracts of Extracts of catechol-treated benzoate-t,reated

cells cells

mU/mg protein meta pathway Catechol2,3-oxygenase 0 2 580 2-Hydroxymuoonic

8-Hydroxymuconic

4-Oxalocrotonate

4- Oxalocrotonate

4-Hydroxy-2-oxovalerate

ortho pathway

cic,cis-Muconate

semialdehyde hydrolase 0.17 6.7

semialdehyde dehydro- genase 0.08 31

tautomerase n.d. 47 500

decarboxylase n.d. 160

aldolase n.d. 4.8

Catechol 1,Z-oxygenase 270 0

lactonising enzyme 190 43

portions and each was resuspended in mineral salts medium a t pH 7.0 without carbon source, to an absorbance a t 610 nm of 0.34. To these two suspcn- sions benzoate and catechol, respectively, were added in four equal increments over the course of 6 h to a final concentration of 5 mM. The cultures were only stirred for the first 3.5 h, but subsequently were aerat- ed at an increasing rate until, for the last 2 h, they were aerated very vigorously. During the experi- ment samples were taken, the cells removed by centri- fugation and the concentration of catechol, benzoate and 2-hydroxymuconic semialdehyde determined from their characteristic absorbances (Fig. 3). After 8 h, during which time there was little detectable increase in cell density, the cells were harvested and washed. Enzyme assays of the cell-free extracts are shown in Table 7. The cells incubated with benzoate contained high levels of the meta pathway enzymes, about 20-30°/, of those in the fully induced ben- zoate-grown cells ( e f . Table 2) , no detectable catechol 1,2-oxygenase and a level of &,cis-muconate lactonis- ing enzyme somewhat elevated above the constitu- tive value, but of the same order as found in benzoate- grown cells. However, extracts of cells incubated with catechol had no detectable catechol 2,3-oxygenase activity even after heating t o 55 "C for 10 min to

destroy the heat-labile catechol 1,2-oxygenase-activ- ity, and the levels of the 2-hydroxymuconic semialde- hyde metabolising enzymes appear to be considerably repressed. However, the levels of the ortho pathway enzymes measured, catecholl,2-oxygenase and cis,& muconate lactonising enzyme are considerably induc- ed. This occurred in spite of the fact that the concen- tration of 2-hydroxymuconic semialdehyde rose to 0.12 mM in the medium during incubation, presum- ably through the action of the high constitutive levels of catechol 2,3-oxygenase present in succinate- grown cells, and furthermore appeared to be metab- olised during the last 3 h of the incubation.

The results of this experiment indicate very strongly that benzoate is the substrate inducer of a t least the first enzymes of the meta pathway. Neither our results nor those of Feist and Hegeman [ill exclude the possibility that the later enzymes, in particular the 4-hydroxy-2-oxovalerate aldolase, are induced by products further down the pathway.

DISCUSSION Few reports of bacterial metabolism of toluates

have been made. Omori and Yamada [16] isolated p-toluate from the medium of p-xylene-grown cells of Pseudomonas aeruginosa, and proposed its further metabolism via p-cresol, p-hydroxybenzoate and protocatechuate. They have also isolated m-toluate from cultures of the same organism growing on m-xylene [17]. An alternative scheme was proposed by Davis, Hossler and Stone [18] for the metabolism of p-xylene by a Pseudomonas species via p-toluic acid, 4-methylcatechol and 2-hydroxy-5-methyl- muconic semialdehyde.

The results presented here for Pseudomonas arvilla mt-2 are consistent with the pathway shown in Fig. 1 for the metabolism of benzoate and m- and p-toluates. Some of the scheme however has not been verified directly. As in our experiments with Pseudomonas putida NCIB 10015 [8] we have not demonstrated the participation of 2-oxopent-4-enoate or its methyl analogue, nor have we been able to assay for 2-0x0- pent-4-enoate hydratase. However, in other organisms using the meta pathway 2-oxopent-4-enoate has been shown to be the product of both2-hydroxymuco- nic semialdehyde hydrolase [3,6] and of 4-oxalo- crotonate decarboxylase [3], and 2-oxopent-4-enoate hydratase has been purified to homogeneity [19]. Nor have we demonstrated the participation of the ulti- mate products of the semialdehyde formed from 4-methylcatechol, nor the ability of the enzymes present to degrade these products. Although 2-me- thyl-4-oxalocrotonate has not yet been chemically identified, Dagley and his co-workers have shown that 2-oxohex-4-enoate and 4-hydroxy-2-oxohex-4- enoate are subsequently formed during meta cleavage of 4-methylcatechol [6,20]. We feel, therefore, that

Vo1.28, No.3, 1972 K. MURRAY, C. J. DUQQLEBY, J. M. SALA-TREPAT, and P. A. WILLIAMS 309

the scheme presented is consistent with the informa- tion a t present available.

The specificities of the ring-fission product- metabolising enzymes show that 3-methylcatechol and its precursor m-toluate can only be metabolised through the hydrolytic branch of the pathway, and suggest very strongly that catechol and 4-methyl- catechol and their precursors benzoate and p-toluate are metabolised, if not exclusively, then mainly by the 4-oxalocrotonate branch. This is not unequivocal- ly proved by these results since the metabolite flux through the 4-oxalocrotonate branch will depend on the NAD* levels in the intact cell and also on the specificity of the tautomerase and the decarboxylase towards Z-rnethyl-4-oxalocrotonate, which we have not determined. However, such a scheme is in accord- ance with our findings that in two organisms, Azoto- bacter [3] and Alcaligenes eutrophus (unpublished observations of Murray and Williams) which possess only the 4-oxalocrotonate branch, the only mono- phenolic compounds metabolised by the meta path- way are phenol and p-cresol, which are converted to catechol and 4-methylcatechol, respectively. The role of the two branches appears to be analogous there- fore to that shown in Pseudomonas putida NCIB 10015 for the metabolism of phenol and the cresols [S,9].

The similarity between the specificities of the catechol 2,3-oxygenase, 2-hydroxymuconic semi- aldehyde hydrolase and 2-hydroxymuconic semi- aldehyde dehydrogenase activities in extracts of cells grown upon either succinate or any of the aro- matic substrates suggests that for each of these activ- ities there is only one enzyme, which is present in the succinate-grown cells a t low constitutive levels and which is induced by growth on either aromatic substrate. In the case of the benzoate oxidase system which cannot be assayed in cell-free extracts the situation is less clear but a t least the results are not inconsistent with there being only one such system present in cells grown on either benzoate or the toluates.

Benzoate (or possibly 3,5-cyclohexadiene- 1,Z-diol- 1-carboxylate [21]) appears to be the inducer of a t least benzoate oxidase, catechol 2,3-oxygenase and the 2-hydroxymuconic-semialdehyde-metabolising enzymes. This is consistent with the induction “from the top” by nonmetabolisable inducers of those meta pathway enzyme assayed by Feist and Hegeman 1111.1. Since benzoate appears to be an inducer it is not unreasonable to propose that in m- and p-toluate- grown cells the induction of this same set of enzymes is also by the growth substrate. As with Pseudomonas putida NCIB 10015 [8,22] and Pseudomonm aerugi- nosa T1 [14], the meta pathway enzymes are both nonspecific in their action and also nonspecifically induced, thus giving the cell a considerable range of metabolic potential with a limited amount of genetic material.

During the incubation with catechol both catechol 1,2-0xygenase and &,cis-muconate lactonising en- zyme are induced, and presumably the other enzymes of the ortho pathway. The most likely explanation is that the inducer of the meta pathway enzymes is not present, and therefore, even though there is considerable breakdown of the catechol to 2-hydroxy- muconic semialdehyde due to the high constitutive levels of catechol 2,3-oxygenase, the meta pathway cannot be utilised for metabolism. There are however low constitutive levels of catechol 1,2-oxygenase in succinate-grown cells and these will complete for the catechol and convert some to cis,&-muconate, which is the only compound so far shown to induce catechol 1,Z-oxygenase and hence is the primary inducer of the ortho pathway [24--271. Our results do not exclude catechol itself as the inducer of cate- chol 1,2-0xygenase in Pseudomonm arvilla, but we consider this possibility remote both because of the apparent universality of cis,&-muconate as the inducer in other organisms and because of the direct participation of catechol in benzoate metabolism. It has been our experience, however, that it is ex- tremely difficult to accumulate catechol in benzoate- grown cultures of Pseudomonus arvilla even after a number of serial subcultures in iron- free media, because of the very high levels of catechol 2,3-oxy- genase, and it may be that in exponentially growing cultures the level of catechol never rises sufficiently high to cause induction of the ortho enzymes.

The relationship between the induction of ortho and meta pathways therefore appears to be the same as in Pseudomonus putidu NCIB 10015, where phenol and the cresols being inducers “from the top” of the meta pathway enzymes are assimilated by this pathway, but substrates which are converted to catechol and which are not inducers of the meta pathway, as benzoate in the case of Pseudomonas putida NCIB 10015, are assimilated by the ortho pathway [ZZ]. These experiments confirm the obser- vations in other organisms [8,22,23] that the meta- bolic function of the meta pathway is the nonspecific assimilation of catechol and methylcatechols and their precursors, whereas the ortho pathways appear capable of only metabolising catechol and its pre- cursors.

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K. Murray, C. J. Duggleby, and P. A. Williams Department of Biochemistry and Soil Science, University College of North WaIes, Memorial Buildings, Bangor, Cearnarvonshire, Great Britain.

J. M. Sala-Trepat's present address: Laboratoire d'Enzymologie, C.N.R.S., F-91 Gif-sur-Yvette, France