MECHANISM INVOLVED IN THE METABOLISM OF NITROPHENYL- CARBOXYLIC ACID COMPOUNDS BY MICROORGANISMS'

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1 MECHANISM INVOLVED IN THE METABOLISM OF NITROPHENYL- CARBOXYLIC ACID COMPOUNDS BY MICROORGANISMS' YANG-HSIEN KE,2 LYNN L. GEE, AND NORMAN N. DURHAM Department of Bacteriology, Oklahoma State University, Stillwater, Oklahoma The stepwise reduction of nitrate has been studied rather extensively during the past years and metabolic schemes have been postulated from the data obtained from growth, inhibition, and respiration experiments. The reduction of nitrate to nitrite is generally accepted although the pathway below nitrite is still rather poorly defined. Hydroxylamine has long been postulated as an intermediate in the scheme of reduction of nitrite to ammonia and this proposal has received support from a number of workers. Taniguchi and co-workers (1953) demonstrated that cellfree extracts of Bacillus pumilis reduced nitrate, nitrite, and hydroxylamine to ammonia when reduced methylene blue was present as an electron donor. Silver and McElroy (1954) have postulated a tentative scheme for nitrate reduction in Neurospora crassa and discussed the possibility that free hydroxylamine existed as an intermediate in the pathway of nitrite reduction. These workers also carried out experiments designed to study the reduction of m-dinitrobenzene in support of the postulation that nitrate metabolism might involve incorporating the inorganic nitrogen into an organic molecule before reduction to the amino level as proposed by de la Haba (1950). Related studies have shown that certain nitrogen containing carboxylic acid compounds may readily be metabolized by microorganisms (Durham, 1958). In an attempt to clarify some of the questions that have been raised concerning hydroxplamine as an intermediate in nitrate and nitrite assimilation we have undertaken studies involving the metabolism of nitrophenyl-carboxylic acid com- I This work was supported in part by contract no. AT(11-1)-71 with the Atomic Energy Commission, Division of Biology and Medicine, and in part by Oklahoma Agricultural Experiment Station, Project No Present address: Department of Bacteriology, Iowa State College, Ames, Iowa. Received for publication October 10, 1958 pounds. These compounds were proposed as models in an attempt to eliminate the toxic effect as well as to increase the stability of the hydroxylamine group. This information should also elucidate how the carbon skeleton of these carboxylic acids is metabolized. The results obtained from studies conducted with o-nitrobenzoic acid are presented in this paper. MATERIALS AND METHODS A bacterium capable of growing on a chemically defined medium with o-nitrobenzoic acid as the sole source of organic carbon and nitrogen was isolated from the soil by enrichment technique. This organism was cultured and tentatively identified as a member of the genus Flavobacterium. Stock cultures were maintained on defined medium containing o-nitrobenzoic acid as the sole source of energy. The chemically defined medium used throughout the study had the following basal composition: NaCl, 0.2 g; KH2PO4, 0.32 g; K2HPO4, 0.42 g; and 0.1 ml of a mineral salt solution in 100 ml of distilled water. o-nitrobenzoic acid was incorporated into the medium as a sole source of organic carbon and nitrogen at a final concentration of 0.1 per cent. Simultaneous adaptation (Stanier, 1947) was the principal method of investigation. The described medium, containing a single compound as the source of carbon and energy, was also used for producing cell suspensions with desired enzymatic patterns for manometric studies. The specific compound that served as a source of carbon and energy was added in a final concentration of 0.1 to 0.2 per cent and the ph adjusted to 7.2. Cells "unadapted" to the carboxylic acid compounds were grown on this medium with 0.1 per cent asparagine replacing the o-nitrobenzoic acid and 0.1 per cent NH4Cl was added during some cultivations to serve as an additional nitrogen supply. The enzymatically adapted cell suspensions used in manometric 593

2 594 KE, GEE, AND DURHAM7[VOL. 77 studies were prepared by harvesting the growth from agar plate cultures 18 to 20 hr old, washing twice, and resuspending in 0.01 M phosphate buffer of ph 7.2. Because of the limited supply of o-nitrosobenzoic acid and o-hydroxylamine benzoic acid, specific induction was achieved by a previously described method (Stanier and Tsuchida, 1949) in which the initially unadapted (asparaginegrown) cell suspension was exposed to a small amount of the compound in question. rather than growing the cells on the compound. The activation of these resting cell suspensions was conducted in Warburg flasks with double side arms. One side arm contained the inducer and the other side arm contained the test substrate. After the addition of the inducer, the course of inducible enzyme formation was followed by measuring oxygen uptake until the compound was completely metabolized, as judged by a return of oxygen consumption to the endogenous respiratory rate. The second compound was then added from the other side arm. All respirometer experiments were performed in the Warburg apparatus (Umbreit et al., 1957) at a temperature of 30 C with air as the gas phase. Each flask contained 2.0 ml of the cell suspension in the main chamber, 0.2 ml of 20 per cent KOH in the center well, and 4,umoles of substrate in the side arm. When o-nitrosobenzoic acid was used as a substrate, experiments were also conducted in which approximately 0.5 mg of the compound were placed in the side arm of the flask. These experiments were designed to confirm the results obtained with previous experiments since o-nitrosobenzoic acid appears to be somewhat unstable in aqueous solution. The o-nitrosobenzoic acid used in these studies was obtained from Bios Laboratories. o-hydroxylamine benzoic acid was supplied through the courtesy of our Department of Biochemistry. Other chemicals were obtained commercially. RESULTS Utilization of the test substrates by inducible enzymes. Since a prerequisite for applying the theory of simultaneous adaptation in tracing metabolic pathways is that the intermediates in doubt must be metabolized by inducible enzymes, experiments were conducted to study the mechanism employed by this organism for attacking a number of different compounds which LJ CZ, Q ṁ z 35io - 30)O - ANTHRANILIC ACID I x IC 5 o-hydroxylamine 0' / BENZOIC ACID--,,/ / o- NITROBENZOIC / /o-nitrobenzyl- ACID ' ALCOHOL 50 A n~~~~,._.-s-h"doreido0 'vo Figure 1. The oxidation of o-nitrobenzoic acid, o-hydroxylamine benzoic acid, anthranilic acid, and o-nitrobenzyl alcohol by cells of Flavobacterium after growth on asparagine. might possibly serve as intermediates. Results from these experiments indicated that this organism formed inducible enzymes in response to o-nitrobenzoic acid, o-hydroxylamine benzoic acid, anthranilic acid, salicylic acid, protocatechuic acid, benzoic acid, catechol, and o-nitrobenzyl alcohol as indicated by marked lags in oxygen uptake when the asparagine grown cells were exposed to the compounds in question. Figure 1 shows the results obtained when anthranilic acid, o-hydroxylamine benzoic acid, o-nitrobenzoic acid, and o-nitrobenzyl alcohol were used as substrates. Compounds such as o-nitrophenol, o-aminophenol, o-nitrobenzaldehyde, 2,4-dihydroxybenzoic acid, 2,4-dinitrophenol, aniline, and nitroso-phenyl-hydroxylamine were not attacked by this microorganism as evidenced by the lack of oxygen consumption. Evidence for the intermediate roles of o-nitrosobenzoic acid and o-hydroxylamine benzoic acid. To elucidate the metabolic pathway by which o-nitrobenzoic acid is metabolized, the organism was grown on this compound as a sole source of carbon and nitrogen and the cells studied with regard to their ability to metabolize suspected

1959] METABOLISM OF NITROBENZOIC ACIDS 595 u, 0 250 o o- HYDROXYLAMINE 200 - BENZOIC ACID :150 100 -NITROS$BENZ0IC ACID j o-nitenzoic ACID 5050kv/ - ~~~E14DOGE14S - 0 30 60 90 120 150 Figure 2.

3 1959] METABOLISM OF NITROBENZOIC ACIDS 595 u, o o- HYDROXYLAMINE BENZOIC ACID : NITROS$BENZ0IC ACID j o-nitenzoic ACID 5050kv/ - ~~~E14DOGE14S Figure 2. The oxidation of o-nitrobenzoic acid, o-nitrosobenzoic acid, o-hydroxylamine benzoic acid, and anthranilic acid by cells of Flavobacterium grown on o-nitrobenzoic acid. intermediates. Figure 2 shows the data obtained in this experimentation. These results indicate that o-nitrobenzoic acid, o-nitrosobenzoic acid, and o-hydroxylamine benzoic acid are metabolized immediately by o-nitrobenzoic acid-grown cells. Since these cells apparently are simultaneously adapted to o-nitrosobenzoic acid and o-hydroxylamine benzoic acid, this suggests that these compounds are possible intermediates in the metabolic pathway of o-nitrobenzoic acid. Anthranilic acid, salicylic acid, protocatechuic acid, benzoic acid, catechol, o-nitrobenzyl alcohol, and o-nitrosophenol were not attacked immediately by the o-nitrobenzoic acid-grown cells, thus indicating that these compounds apparently do not act as principal intermediates in the dissimilation of o-nitrobenzoic acid. The finding that anthranilic acid, the corresponding amino compound, does not appear to be an intermediate in the dissimilative pathway of o-nitrobenzoic acid was a very interesting observation since ammonia has been implicated as an intermediate in the metabolism of nitrate and nitrite. To substantiate the foregoing findings additional experimentation was conducted. Influence of ultraviolet irradiation on the metalolism of test substrates by o-nitrobenzoic acid-grown cells. Since ultraviolet irradiation inhibits the induction of enzyme biosynthesis in various organisms without affecting the activity of preexisting enzymes, experiments were conducted to study the adaptive utilization of the compounds in question by comparing ultraviolet irradiated and nonirradiated o-nitrobenzoic acid-grown cells. Cells grown on o-nitrobenzoic acid were suspended in an 0.01 M phosphate buffer solution of ph 7.2, placed in a flat bottom petri dish, and exposed to a 15 w General Electric Germicidal Lamp for 4 min. The distance from the lamp to the cells was 25 cm. After treatment, the irradiated cells and the nonirradiated controls were added to Warburg flasks and oxygen consumption was measured as previously described. Results from these experiments are given in figure 3. These data show that the biosynthesis of inducible enzymes to attack anthranilic acid and protocatechuic acid are completely suppressed by ultraviolet irradiation under the described conditions, while the preexisting enzymes that attack o-nitrobenzoic acid, o-nitrosobenzoic acid, and o-hydroxylamine benzoic acid were not affected. The intermediary roles of o-nitrosobenzoic acid and o-hydroxyl- 250 NON-IRRADIATED CELLS IRRADIATED CELLS =200 OBA B SBA ilso~~~~~~~~~b 100 R C.)~~~~~TM IN 050 ANTHB IUE 0~ ~ ~ ~~~~ ~~'7CANT Figure S. The oxidation of various substrates by ultraviolet irradiated and nonirradiated cells of Flavobacterium after growth on o-nitrobenzoic acid. END, endogenous; OBA, o-nitrobenzoic acid; SBA, o-nitrosobenzoic acid; HBA, o-hydroxylamine benzoic acid; ANT, anthranilic acid; and PRO, protocatechuic acid.

4 596 KE, GEE, AND DURHAM [VOL. 77 amine benzoic acid, and the nonintermediary roles of anthranilic acid and protocatechuic acid in the dissimilation of o-nitrobenzoic acid are further verified by these results. Enzymatic activity of o-nitrosobenzoic acid and o-hydroxylamine benzoic acid-adapted cells. In order to elucidate the intermediary position of o-nitrosobenzoic acid and o-hydroxylamine benzoic acid in the metabolic scheme, cells enzymatically adapted to these compounds were studied under the same conditions as those employed in the initial experiments. Representative results of these experiments are shown in figures 4 and 5. These data indicate that cells previously exposed to o-nitrosobenzoic acid immediately attack o-hydroxylamine benzoic acid (figure 4) as indicated by the absence of a lag period but are not simultaneously adapted to o-nitrobenzoic acid. Thus indicating an intermediary role for o-hydroxylamine benzoic acid but not for o-nitrobenzoic acid in the metabolism of the nitroso- derivative. Cells previously exposed to o-hydroxylamine benzoic acid utilize only that substrate and are not simultaneously adapted to o-nitrosobenzoic acid, anthranilic acid, or o-nitrobenzoic acid (figure 5). The results indicate that these compounds apparently do not serve as intermediates in the assimilation of Ul, C co- C., -20C i- 15C31. I00O 501 o-hydroxylamine BENZOIC ACID o-nitrosobenzoic/a--- - AGID-\0/ /, / - //8 o-nitrobenzoic /,' /r ~~~ACID // X _NDOGENO/ ~~~~~x, Figure 4. The oxidation of o-nitrobenzoic acid, o-nitrosobenzoic acid, and o-hydroxylamine benzoic acid by cells of Flavobacterium previously exposed to o-nitrosobenzoic acid. / cr -J 0 300h o-nitrosobenzoic ACID j C) z 0 2C 10 5 o- NITROBENZOIC ACID-yA o-hydroxylamine oo_o / BENZOIC ACID a ~ ~~~~~ Ix ANTHRANILIC io/0///, ACID ~ / X.EADOGENOUS C The oxidation of o-nitrobenzoic acid, Figure 5. o-nitrosobenzoic acid, o-hydroxylamine benzoic acid, and anthranilic acid by cells of Flavobacterium adapted to o-hydroxylamine benzoic acid. o-hydroxylamine benzoic acid and must therefore precede the hydroxylamine derivative in the metabolic scheme. Since control experiments indicated that o-nitrosobenzoic acid might be unstable in aqueous solution, confirmation of the results with both the aqueous solution and crystals were carried out as described above. Enzymatic activity of anthranilic acid-grown cells. Although the results do not indicate that anthranilic acid serves as an intermediate in the metabolism of either o-nitrobenzoic acid, o-nitrosobenzoic acid, or o-hydroxylamine benzoic acid, preliminary experiments were conducted to determine the enzymatic activity of anthranilic acid-grown cells. The data indicate that anthranilic acid-grown cells were not enzymaticallv adapted to o-nitrobenzoic acid, o-nitrosobenzoic acid, or o-hydroxylamine benzoic acid but were capable of metabolizing these compounds after a lag period indicating the formation of inducible enzymes. However, these cells were capable of oxidizing salicylic acid immediately, suggesting that the organism was simultaneously adapted to salicylate and implicating this compound as an intermediate in the metabolism of anthranilic acid. Although no further studies were conducte(d

1959] METABOLISM OF NITROBENZOIC ACIDS 597 at the time it appears that anthranilic acid is probably metabolized via a pathway similar to the para-configuration compounds since p-aminobenzoic acid

5 1959] METABOLISM OF NITROBENZOIC ACIDS 597 at the time it appears that anthranilic acid is probably metabolized via a pathway similar to the para-configuration compounds since p-aminobenzoic acid reportedly is assimilated through the corresponding hydroxy containing acid, p-hydroxybenzoic acid (Durham, 1956). DISCUSSION The data obtained in these studies indicate that o-nitrobenzoic acid, a nitrophenyl-carboxylic acid, may be readily metabolized by certain microorganisms as a source of carbon and nitrogen for aerobic growth. The organism capable of utilizing this compound has been identified as belonging to the genus Flavobacterium. Other studies (Durham, 1958) have shown that the corresponding carboxylic acid with the paraconfiguration may be assimilated by Pseudomonas fluorescens. Simultaneous adaptation was explored as a mechanism by which the metabolic pathway for the utilization of o-nitrobenzoic acid might be followed. The asparagine-grown cells demonstrated inducible enzyme formation for o-nitrobenzoic acid, o-hydroxylamine benzoic acid, anthranilic acid, salicylic acid, protocatechuic acid, benzoic acid, catechol, and o-nitrobenzyl alcohol indicating that the organism may readily attack a number of different aromatic compounds. Evidence that o-hydroxylamine and o-nitrosobenzoic acids fulfilled roles as intermediates was accumulated when the o-nitrobenzoic acid-grown cells metabolized these compounds without a lag period. These results indicate that the cells were simultaneously adapted to o-hydroxylamine and o-nitrosobenzoic acids and suggests that these compounds occupy an intermediary role in the dissimilation of o-nitrobenzoic acid. Additional studies revealed that the o-nitrobenzoic acidgrown cells were not simultaneously adapted to anthranilic acid, salicylic acid, protocatechuic acid, benzoic acid, catechol, o-nitrobenzyl alcohol, or o-nitrosophenol. These data were interpreted as meaning that these compounds apparently do not occupy an intermediary position in the metabolism of o-nitrobenzoic acid. Interpretation of the results indicate that the conclusions may be formulated as shown in the sequence of scheme 1. NO2 COOH NO COOH Scheme 1 cellular materials These studies suggest that o-nitrobenzoic acid apparently is metabolized via a different pathway than the corresponding para-nitrophenylcarboxylic acid. Durham (1958) reported that p-nitrobenzoic acid was metabolized through the intermediate p-aminobenzoic acid since P. fluorescens cells cultured on the former compound were simultaneously adapted to p-aminobenzoic acid. p-aminobenzoic acid may be metabolized further with p-hydroxybenzoic acid, protocatechuic acid, and,b-ketoadipic acid as intermediates (Durham, 1956). Interpretation of these data indicate that the schemes for the assimilation of the ortho- and para-nitrophenyl-carboxylic acids may be similar in some stages but apparently involve different intermediates as the compounds are further metabolized. The o-nitrobenzoic acid compound proceeds through o-nitrosobenzoic acid and o-hydroxylamine benzoic acid. By analogy, these data could be related to nitrate assimilation and would support the general idea that hydroxylamine serves as an intermediate in the pathway of nitrate reduction. Yamashina et al. (1954) reported that cell-free enzyme preparations from a halotolerant bacterium catalyzed the reduction of o-nitrosobenzoic acid and o-hydroxylamine benzoic acid to anthranilic acid by leucomethylene blue or via a dehydrogenase system in the presence of an electron carrier. Our results do not indicate that anthranilic acid serves as an amino containing intermediate during the metabolism of o-nitrobenzoic acid. Studies with ultraviolet irradiated cells substantiated this finding. This discrepancy might be the result of the method employed in the experimentation (simultaneous

6 598 KE, GEE, AND DURHAM [VOL. 77 adaptation). However, similar procedures have demonstrated that p-aminobenzoic acid served as an intermediate in the assimilation of p-nitrobenzoic acid (Durham, 1958). In general this would tend to emphasize the tremendous capabilities that microorganisms possess regarding different mechanisms of substrate metabolism. SUMMARY Studies involving the metabolism of nitrophenyl-carboxylic acids by microorganisms were undertaken in an attempt to clarify the biochemical mechanism of nitrate reduction and elucidate the metabolism of the carbon skeleton involved. Enrichment techniques were employed to isolate an organism capable of utilizing o-nitrobenzoic acid as a sole source of organic carbon and nitrogen for aerobic growth. This organism was identified as belonging to the genus Flavobacterium and the enzyme responsible for the assimilation of o-nitrobenzoic acid is inductive in nature. Cells grown on o-nitrobenzoic acid showed immediate oxygen uptake on o-nitrosobenzoic acid and o-hydroxylamine benzoic acid, indicating that these compounds appear to be intermediates in the metabolic scheme of o-nitrobenzoic acid. Cells previously adapted to o-nitrosobenzoic acid showed simultaneous adaptation to o-hydroxylamine benzoic acid but not to o-nitrobenzoic acid or anthranilic acid, whereas cells exposed to o-hydroxylamine benzoic acid were not adapted to o-nitrobenzoic acid, o-nitrosobenzoic acid, or anthranilic acid. These results suggest that o-nitrosobenzoic acid and o-hydroxylamine benzoic acid serve as intermediates in the metabolism of o-nitrobenzoic acid and the sequence of appearance is in this order. Additional compounds were studied but were eliminated as possible intermediates since they were either metabolized after a period of adaptation or showed no increase in oxvgen uptake when compared with endogenous controls. The significance of these results with regard to the biochemical mechanism of nitrate reduction is discussed. REFERENCES DE LA HABA, G Studies oni the mechanism of nitrate assimilation in Neurospora. Science, 112, DURHAM, N. N lbacterial oxidation of p-aminobenzoic acid by Pseudomonas fluorescens. J. Bacteriol., 72, DURHAM, N. N Studies on the metabolism of p-nitrobenzoic acid. Can. J. Microbiol., 4, SILVER, W. S. AND MCELROY, W. 1) Enzyme studies on nitrate and nitrite mutants of Neurospora. Arch. Biochem. Biophys., 51, STANIER, R. Y Simultaneous adaptation: A new technique for the study of metabolic pathways. J. Bacteriol., 54, STANIER, R. Y. AND TSUCHIDA, M Adaptive enzyme patterns in the bacterial oxidation of tryptophane. J. Bacteriol., 58, TANIGUCHI, S., MITSUI, H., TOYODA, J., YAMADA, T., AND EGAMI, F The successive reduction from nitrate to ammonia by cell-free bacterial enzyme systems. J. Biochem. (Tokyo), 40, UMBREIT, W. W., BURRIs, R. H., AND STAUFFER, J. F Manometric techniques. Burgess Publishing Co., Minneapolis. YAMASHINA, I., SHIKATA, S., AND EGAMI, F Enzymic reduction of aromatic nitro, nitroso, and hydroxylamino compounds. Bull. Chem. Soc. Japan, 27,

decarboxylation. Further work with the enzyme systems involved has shown

decarboxylation. Further work with the enzyme systems involved has shown THE BACTERIAL OXIDATION OF AROMATIC COMPOUNDS IV. STITDIES ON THE MECHANISM OF ENZYMATC DEGRADATION OF PROTOCATECHuiC ACID' R. Y. STANIER Department of Bacteriology, University of California, Berkeley,