Benzoic Acid). the Production of This Compound by Cultures Growing On

THE BACTERIAL OXIDATION OF AROMATIC COMPOUNDS I. ADApnvE PArERNS wITH RESPECT TO POLYPHENOLIC COMPOUNDS1 B. P. SLEEPER AND R. Y. STANIER Department of Bacteriology, University of California, Berkeley, California Received for publication October 24, 1949 Studies by the technique of simultaneous adaptation (Stanier, 1947, 1948) have shown that there are at least four initially distinct pathways for the oxidation of simple aromatic compounds by Pseudomonas fluorescens. The four primary substrates on these pathways are phenylacetic acid, mandelic acid, phenol, and p-hydroxybenzoic acid, mandelic acid being dissimilated via phenyl- glyoxylic acid, benzaldehyde, and benzoic acid. This work left open the questions of how the benzene ring itself is attacked and whether the initially distinct oxidations of the four primary substrates merge at some point or points in common intermediates. Recently we have begun to reinvestigate these problems; the present paper is concerned with the nature of the initial attack on the aromatic nucleus. In a previous paper (Stanier, 1948) the problem of ring oxidation was analyzed with reference to benzoic acid, and it was concluded that the available data ruled out one of the most obvious possibilities-namely, attack by the successive introduction of phenolic groups on the ring. Such an attack should yield a monohydroxy derivative as the first intermediate. However, of the four possible monohydroxy derivatives of benzoic acid, three (phenol and o- and m-hydroxybenzoic acids) had been shown to be unoxidizable by many strains of P. fluorescens that could readily oxidize benzoic acid. The fourth possible derivative, p-hydroxybenzoic acid, had been eliminated as an intermediate by virtue of the fact that benzoate-grown cells showed an adaptive lag in the oxidation of p-hydroxybenzoate. For these reasons, it did not appear worth while to explore further the possibilities of an oxidation via phenolic intermediates. Fresh light was thrown on the problem by the work of Evans (1947), who obtained strong evidence for the occurrence of polyphenolic intermediates in the oxidation of aromatic compounds by another aerobic bacterium, Vibrio 01. By making periodic qualitative tests on cultures of this organism growing at the expense of aromatic substrates, Evans was able to detect, and subsequently to isolate, several presumed intermediates. Cultures grown on m- and p-hydroxybenzoic acid gave rise to the formation of protocatechuic acid (3,4-dihydroxybenzoic acid). The production of this compound by cultures growing on benzoic acid was also reported, but has subsequently been shown not to occur (Evans, personal communication). Cultures grown on phenol produced catechol. In view of this work, it seemed desirable to re-examine the possibility that 1This work was done in part under a grant-in-aid from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council. 117 118 B. P. SLEEPER AND R. Y. STANIER [VOL. 59 phenolic intermediates might occur in the oxidation of aromatic substrates by P. fluorescens. We have, accordingly, investigated the adaptive patterns of this organism with respect to catechol, protocatechuic acid, and other related polyphenolic compounds. MATERIALS AND METHODS The methods of cultivation and of testing adaptive behavior have been described in previous papers (Stanier, 1947, 1948). For most of the experiments reported, P. fluorescens A.3.12 has been used. This strain is, however, incapable of attacking phenol, and a phenol-utilizing strain (A.3.8) was used instead to study the adaptive patterns of phenol-grown cells. For the most part, specific adaptation was achieved as in our earlier work by growing celLs on a medium containing a specific aromatic compound as the sole source of energy. This procedure proved impractical in the case of catechol, which decomposes slowly in solution, with the formation of products that inhibit bacterial growth. Catechol-adapted cells were obtained by conducting the adaptation in Warburg vessels, as described by Stanier and Tsuchida (1949). RESULTS Evidence for the intermediate roles of catechol and protocatechuic acid. Like all other aromatic substrates that we have tested, both catechol and protocatechuic acid are adaptively attacked by P. fluorescens, as evidenced by the marked lags in oxygen uptake that occur with these substrates after growth on yeast agar or asparagine agar. The oxidation of these compounds and of mandelic acid (included for comparison) by asparagine-grown cells is shown in figure 1. CelLs grown on mandelic acid, benzoic acid, and phenol show the same adaptive patterns with respect to catechol and protocatechuic acid (figures 2, 3, and 4). In each case, there is complete simultaneous adaptation to catechol, which suggests that this substance is a common intermediate in the oxidations of phenol and of the members of the mandelate-benzoate reaction chain. With protocatechuic acid, on the other hand, there is always a slight adaptive lag. This lag is always much shorter than the lag with protocatechuic acid shown by cells unadapted to any aromatic compound; as a rule there is measurable oxygen uptake in the first 5 minutes after substrate addition, and the maximum rate is established within 15 to 20 minutes. This behavior suggests that growth on the above-mentioned substrates brings about a partial activation of the enzymes acting upon protocatechuic acid; however, the existence of a lag, even if very brief, implies that protocatechuic acid cannot actually play a major intermediate role in the oxidation of those substrates. The role of protocatechuic acid as a major intermediate in these oxidations could be clearly eliminated if it were possible to establish with certainty that the initial level of activity against protocatechuic acid in the cells is extremely low. A very low initial level of activity is suggested by the shape of the curves for oxygen uptake with protocatechuic acid, but under the experimental conditions used adaptation occurs so rapidly that accurate measurement of the (A) w 1~-x 0 0 a-

x 0

0 40 80 120 TIME, MINUTES Figure 1. The oxidation of catechol, protocatechuic acid, and mandelic acid (2 micromoles of each) by P. fluorescens grown on asparagine.

30______MANDELATE- 3ROWN

250 ___i; _A

2604 0~~~

uW15C

0 1000 4 0.

0 ~~20 40 60 TIME, MINUTES Figure S. The oxidation of mandelic acid, catechol, and protocatechuic acid (2 micromoles of each) by P. fluorescens grown on mandelate. 119 120 B. P. SLEEPER AND B. Y. STANIER [VOL. 59 initial rate is impossible. Attempts were made to determine the initial level of activity by treating the cells with sodium azide and 2,4-dinitrophenol, which are known to inhibit adaptation in other microorganisms (Monod, 1944; Spiegel- man, 1946). Unfortunately, these inhibitors do not act selectively against either adaptation or assimilation by P. fluore8cens. Subsequently, we obtained indirect evidence for a low level of activity against protocatechuic acid in mandelate-, benzoate-, and phenol-adapted cells from the study of dried cell preparations (Sleeper, Tsuchida, and Stanier, 1950). Such dried cells contain only 2 to 3 per cent of the activity against protocatechuic acid that is found in cells whose

o 10 20 30 TIME, MINUTES Figure 3. The oxidation of benzoic acid, catechol, and protocatechuic acid (2 micromoles of each) by P. fluorescens grown on benzoate. prior conditions of cultivation have resulted in complete adaptation to this substrate. The adaptive patterns with respect to catechol and protocatechuic acid shown by cells grown on p-hydroxybenzoate are the reverse of those shown by cells grown on mandelate, benzoate, or phenol. There is complete simultaneous adaptation to protocatechuic acid, but a marked adaptive lag with catechol (figure 5). This suggests that protocatechuic acid is an intermediate in the oxidation of p-hydroxybenzoic acid but catechol is not, and bears out the earlier conclusion (Stanier, 1947) that p-hydroxybenzoic acid is attacked by a pathway distinct from that of the mandelate-benzoate reaction chain. Were p- 0

0 r20.

0 20 40 60 TIME, MINUTES ,Figure 4. The oxidation of phenol, catechol, and protocatechuic acid (2 micromoles of each) by P. ,fuore8cen. grown on phenol.

250 P- HYDROXYBE1 ZOATE- GROWN

200 (A cr. w 1.- I 0 ir 0:150 ______5 L. W

IL9 m 100 W z w x 0 50 /

O 0 20 40 60 TIME, MINUTES Figure 5. The oxidation of p-hydroxybenzoic acid, catechol, and protocatechuic acid (2 micromoles of each) by P. fluorescens grown on p-hydroxybenzoate. 1. 121~~~~ENDOGENOUS1 122 B. P. SLEEPER AND R. Y. STANIER [voL. 59 hydroxybenzoic acid an intermediate in the oxidation of benzoic acid, as has been several times suggested (Bernheim, 1945; Evans, 1947; Evans, Parr, and Evans, 1949), the adaptive patterns with respect to catechol and protocatechuic acid should be the same in benzoate- and p-hydroxybenzoate-adapted cells. Cells grown on protocatechuate show adaptive lags with both catechol and p-hydroxybenzoic acid (figure 6), whereas cells adapted to catechol show;adaptive lags with both protocatechuic and benzoic acids (figure 7).

25C PROTOCATEC UATE-GROWN

z 0

0 50

0 20 40 Go TIME, MINUTES Figure 6. The oxidation of protocatechuic acid, p-hydroxybenzoic acid,land cateohol (2 micromoles of each) by P. .fuorescens grown on protocatechuate. Experiments with other polyphenolic compounds. In addition to protocatechuic acid and catechol, the following polyphenolic compounds were also tested as possible intermediates: 2,3-dihydroxybenzoic acid, 2,3,4-trihydroxybenzoic (pyrogallol carboxylic) acid, 3,4,5-trihydroxybenzoic (gallic) acid,' pyrogallol, phloroglucinol, and hydroxyhydroquinone. For these experiments both benzoate- and p-hydroxybenzoate-adapted cells were used. In each case, the rates of oxygen uptake with the listed compounds were compared to the rate of oxygen uptake with the substrate to which the cells had been adapted. The results, which will not be described in detail, showed that none of the listed compounds with the dubious exception of hydroxyhydroquinone can play a major intermediate role in the dissimilation of either benzoic or p-hydroxybenzoic acid. Phloroglucinol and 2,3-dihydroxybenzoic acid never gave rise to an oxygen up- 1950] BACTERIAL OXIDATION OF AROMATIC COMPOUNDS 123 take in excess of the autorespiratory one. With pyrogallol carboxylic acid, gallic acid, and pyrogallol the oxygen consumption was slightly greater than the autorespiratory oxygen consumption, but the rates were clearly far too low,for any

TIME, MINUTES Figure 7. The oxidation of catechol, protocatechuic acid, and bensoic acid (2 micromoles of each) by cells of P. /luorescens previously exposed to catechol. TABLE 1 Oxygen consumption by benzoate-adapted cells with various polyphenolic compound.l (S micromoles of each) OXYaGEN CONSUMION, MICROUMTLS SUBST"RAT 30mn 60 mn

None...... 17 30 Benzoic acid ...... 230 259 2,3-dihydroxybenzoic acid...... 13 29 2,3,4-trihydroxybenzoic acid...... 22 46 3,4,5-trihydroxybenzoic acid...... 16 42 Phloroglucinol...... 10 22 Pyrogallol...... 22 41 of these three compounds to serve as intermediates. Typical data from an experiment with benzoate-grown cells are shown in table 1. Hydroxyhydroquinone proved readily oxidizable but the evidence indicates that it is probably not an intermediate in the oxidation of benzoate and p- 124 B. P. SLEEPER AND R. Y. STANER [VOL. 59 hydroxybenzoate. As shown in figure 8, the rate of oxidation of hydroxyhydroquinone by benzoate-grown cells is considerably lower than the rates of benzoate and catechol oxidation. Furthermore, the total oxygen uptake with hydroxyhydroquinone is less than half of that with a molar equivalent of catechol; it should be much greater on the assumption of a sequential relationship between the two, as the following calculation shows: the oxygen uptake per micromole of catechol is approximately 100 microliters, or 4.5 micromoles. Since hydroxyhydroquinone contains one more oxygen atom than catechol, it should give rise (ass ng that the oxidation of catechol were to proceed entirely via

300 BENZOATE - GROWN

250 vI

(n w 200 -J 0

Id U

.4 I.- I-ia z 100 0

0 ______0 20 40 60 TIME, MINUTES Figure 8. The oxidation of benzoic acid, catechol, and hydroxyhydroquinone (2 micromoles of each) by P. fluorescens grown on benzoate. hydroxyhydroquinone) to an oxygen uptake of approximately 4 micromoles, or 90 microliters, per micromole; i.e., approximately twice the oxygen uptake actually observed. This suggests that the oxidation of hydroxyhydroquinone takes place by a mechanism not normally involved in the oxidation of catechol. The addition of hydroxyhydroquinone to a cell suspension results in the immediate appearance of a pink color, which deepens in the course of half an hour to a reddish brown; similar colored substances are never produced by the action of P. fluorescens on other oxidizable aromatic substrates. When cells grown on asparagine were tested for their action on hydroxyhydroquinone, an immediate rapid oxygen uptake resulted, accompanied by the afore- 19501 BACTERIAL OXIDATION OF AROMATIC COMPOUNDS 125 mentioned color changes. This suggested that asparagine-grown cells possessed the necessary enzymes for an attack on hydroxyhydroquinone; however, in view of the fact that every aromatic substrate previously tested with P. fluorescens had been found to undergo attack in a strictly adaptive manner, such behavior seemed anomalous. It occurred to us that in view of the extreme instability of hydroxyhydroquinone, the decomposition observed might be, at least in part, nonenzymatic in nature. Such proved to be the case; a suspension of boiled, asparagine-grown cells oxidized hydroxyhydroquinone at the same rate as did living cells and with identical color changes. The total oxygen uptake with hydroxyhydroquinone by asparagine-grown cells (living or killed) is somewhat lower than that by benzoate-grown cells, which suggests that in the latter case a certain amount of enzymatic degradation may be superimposed on the chemical decomposition. However, in view of the data as a whole, we consider that hydroxyhydroquinone is not a major intermediate, even though the evidence is not as straightforwaxd as that presented for the other compounds discused in this section. DISCUSSION The observation that cells adapted to mandelic acid, benzoic acid, or phenol are simultaneously adapted to catechol suggests that catechol is a common intermediate in the oxidation of all three substrates. Similar evidence indicates that protocatechuic acid is an intermediate in the oxidation of p-hydroxybenzoic acid. Since mandelate-, benzoate-, and phenol-adapted cells are unadapted to protocatechuic acid, whereas p-hydroxybenzoate-adapted cells are unadapted to catechol, it is clear that the pathway through catechol is distinct from that through protocatechuic acid and that these oxidative pathways, if they merge at all, must do so at a later point. The conclusions may be formulated in the following sequences: CHOHCOOH COOH OH 1. 0 , ( ?OH

COOH COOH

K~,2 KsOH OH OH The negative outcome of the experiments with 2,3,4- and 3,4,5-trihydroxybenzoic acids, phloroglucinol, pyrogallol, and hydroxyhydroquinone eliminates 126 B. P. SLEEPER AND R. Y. STANIER [VOL. 59 most of the possible triphenolic substances that might be anticipated as later intermediates on the two pathways; the nature of the next step in ring oxidation is therefore unknown. The problem of how benzoic acid is initially attacked has now become more obscure than ever. As mentioned in the introduction, earlier evidence showed conclusively that none of the four possible monohydroxy derivatives functioned as an intermediate; yet the present findings, which are fully corroborated by recent work of Evans et at. (personal communication), strongly suggest that the diphenolic substance catechol is an intermediate. One is thus led to the hypoth- esis that some mechanism exists for the simultaneous introduction of two hydroxy groups into the benzoic acid molecule. Even this formulation is not adequate to explain the available data. A simple substitution of two adjacent hydroxy groups in the benzoic acid molecule should yield either 2,3- or 3,4- dihydroxybenzoic acid, but as shown in the present paper the former compound is unutilizable and the latter is ruled out by virtue of the lack of adaptation to it in cells adapted to benzoic acid. Since every possible aromatic intermediate between benzoic acid and catechol has now been tested and found wanting, one is forced to conclude either (a) that the transformation occurs as a single complex step reaction or (b) that the aromatic character of the ring is lost and then subsequently restored. Of these, the second possibility appears more plausible. ACKNOWLEDGMENTS We take this opportunity to express our thanks to Dr. J. Cason for supplying us with hydroxyhydroquinone and 2,3-dihydroxybenzoic acid, and to Dr. W. C. Evans for supplying us with pyrogallol carboxylic acid. SUMMARY Cells of Pseudomonas fluorescens grown on mandelate, benzoate, or phenol show complete simultaneous adaptation to catechol, but not to protocatechuic acid. Cells of P. fluorescens grown on p-hydroxybenzoate show complete simultaneous adaptation to protocatechuic acid, but not to catechol. These facts suggest that catechol is a common intermediate in the oxidation of the members of the mandelate-benzoate reaction chain and of phenol, and that protocatechuic acid is an intermediate in the oxidation of p-hydroxybenzoic acid. Other polyphenolic compounds were tested as possible intermediates with negative results. REFERENCES BERNHEIM, F. 1945 The oxidation of benzoic acid and related substances by certain mycobacteria. J. Biol. Chem., 143, 383-389. EvANS, R. A., PARR, W. H., AND EvANs, W. C. 1949 The bacterial oxidation of aromatic compounds. Biochem. J., 44, Proc. Biochem. Soc., viii. EvANS, W. C. 1947 Oxidation of phenol and benzoic acid by some soil bacteria. Bio- chem. J., 41, 373-382. 1950] BACTERIAL OXIDATION OF AROMATIC COMPOUNDS 127

MONOD, J. 1944 Inhibition de l'adaptation enzymatique chez B. coli en prdsence de 2,4-dinitrophdnol. Ann. inst. Pasteur, 70, 381-384. SLEEPER, B. P., TsUCHIDA, M., AND STANIER, R. Y. 1950 The bacterial oxidation of aromatic compounds. II. The preparation of enzymatically active dried cells, and the influence thereon of prior patterns of adaptation. J. Bact., 59, 129-133. SPIEGELMAN, S. 1946 Inhibition of enzyme formation. Federation Proc., 5, 100. STANIER, R. Y. 1947 Simultaneous adaptation: a new technique for the study of meta- bolic pathways. J. Bact., 54, 339-348. STANIER, R. Y. 1948 The oxidation of aromatic compounds by fluorescent pseudo- monads. J. Bact., 55, 477-494. STANIER, R. Y., AND TSUCHIDA, M. 1949 Adaptive enzymatic patterns in the bacterial oxidation of tryptophan. J. Bact., 58, 45-60.