Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 09/11/14 For personal use only.
1496
C A N . J. MICROBIOL. VOL. 23. 1977
coli t o uvrB- and rec- mutants of S. typhimwizitn. It should be pointed out that survival curves showed that TA1950 and TA1535 were equally sensitive t o ultraviolet light, indicating that while the rfa- mutation is able t o suppress the TSY agar sensitivity associated with zrurBit does not suppress ultraviolet light sensitivity. While the physiological mechanisms of the effects reported here are yet t o be determined, it is clear that care should be exercised when enumerating D N A repair-deficient mutants.
1
2
3
4
5
6
7
8
9
1
0
1
1
HOURS
FIG.3. Growth of S. l y p l ~ i t ~ r ~ r r i ~TA1975 rti~ in M-9 supplemented with histidine and TA1535 in M-9 supplemented with histidine and biotin. Plate counts of TA1975 on M-9 plus histidine (A), on TSY (0); plate counts of TA1535 011 M-9 plus histidine and biotin (A), on TSY (@).
medium, supple~nentedwith histidine and biotin, there is no difference between colony counts on TSY and M-9 plates. When each of the five strains were grown in TSY broth, the differences between the M-9 and TSY colony counts were negligible throughout the growth curve. This work has extended the observations of Rosenkranz et 01. (4) with polA- mutants of E.
Acknowledgements This work was supported by grants 2-POIES00597 from the National Institute of Environmental Health Sciences and SMI76-03575 from the National Science Foundation Undergraduate Research Participation Grant, and is contribution No. 3090 froin the Department of Nutrition and Food Science, Massachusetts Institute ofTechnology, Cambridge, MA, U.S.A. 02139. I. ACIES.B. N.. F. D. LEE,and W. E. D u n s - r o ~ 1973. . An improved bacterial test system for the detection and classification of mutagens and c;lscinogens. Proc. Natl. Acad. Sci. U.S.A. 70: 782-786. 2. GOCIEZ, R. F., and A. J. S I N S K E Y1974. . DNA breaks rrt)~ in rifampin-treated S N / I ) I ~ I I C t/ y/ ~p ~h i ~ ~ ~ r ~ r i LT-2 after exposure to nittritionally complex media. Nature. 247: 21 1-212. 3. GOCIEZ, R. F.. A. J . S I N S K E YR., DAVIES, and T. P. L,\suz,\. 1973. Minimal medium recovery of heated S r r l t ~ ~ o t ~typl~irtrrrriri~~i ell~ LT-2. J . Gen. Microbiol. 74: 267-274. 4. R O S E N K ~ ~ \H. NZ S.,, H. S. C A R R and . C. MORGAN. 1971. Unusual growth properties of a bacterial strain lacking DNA polymerase. Biochem. Biophys. Res. Commun. 44: 546-549.
Metabolism of mandelic acid by Neurospora crassa
Accepted June 22, 1977 R A M A K R I S HRAO, N A D. N., and C. S. V A I D Y A N A T H A 1977. N . Metabolism of mandelic acid by Nerrrosparo crrrssn. Can. J. Microbiol. 23: 1496-1499. Preliminary studies on the metabolism of mandelic acid by Nerrrosporn urrssn reveal the operation of a pathway for its degradation which involves benzoyl formic acid, benzaldehyde, benzoic acid, Chydroxybenzoic acid. and protocatechuic acid as the intermediates. This pathway is different from that followed by bacterial systems and is the same as that observed in
Aspergillu.~niger .
NOTES
I497
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 09/11/14 For personal use only.
RAM.AKKISHN-\RAO.D. N . , et C. S. V.AIDY,AW;\-I.H,\V. 1977. Metabolism of rnandelic acid by Nelrrosporcr uccssrr. Can. J. Microbial. 23: 1496-1499. Des e t i ~ d e preliminaires s sur le metabolisme de I'acide mendelique demontrent qu'il existe une voie pour sa dCgl-adation chez Neicrosporrr C I - c r ~ ~ L'acicle ri. ben~oyl-formique,le benzaldehyde, I'acide benzoiqi~e, I'acide 4-hydroxybenzoique et I'acide protocatechuique sont d e s intermediaires metaboliques de cette voie. Cette derniere differe cle celle que I'on observe dans les systemes bacteriens: mais elle est la meme que celle retrouvee chez Aspergillcts tligo.. (Traduit par le journal]
Mandelic acid metabolisni has been extensively studied in Psezr~lolomonasputida and bacterium N.C.I.B. 8250 (1, 2, 3, 6, 10). The pathway involves the intermediary formation of benzoyl formic acid, benzaldehyde, benzoic acid, and catechol. In fungi, however, mandelate metabolism has not been investigated in detail except for a report from this laboratory demonstrating the operation of a protocatechuic acid pathway for the degradation of mandelate by As~)ergillus n i g o (5). In this communication, we report our preliminary studies on the metabolism of this conipound by Neurospora crassu. Neurospora crassn FGSC 352, obtained from the Fungal Genetic Stock Center, Humbaldt State College, Arcata, CA., wasgrown on a modified Vogel's medium (120 ml) (9) containing 0.1% (w/v) DL-mandelic acid in 1-t! conical flasks, for 48 h at 30°C. The same conditions were used for obtaining cells grown on glucose except that mandelic acid was not present in the medium. The mycelial felts thus obtained (5 g) from a still culture were harvested, washed three times with sterile water, and replaced in 50 ml of 0.05 M sodium phosphate buffer pH 7.0 containing 1 mg/ml of the various intermediates. The replacement culti~reswere kept on a rotary shaker (250 rpm) a t 30°C for 6 h and the products formed were analyzed. The cultures were filtered through a filter paper. The medium was acidified to pH 2 with 3 N HC1 and extracted three times with an equal volume of ethylacetate. The pooled organic layer was dried over anhydrous sodium sulfate, concentrated, and subjected to ascending paper chromatography on Whatman No. 1 filter paper using the following solvent systems: (A) formic acid water, 2:98 (v/v); (B) benzene - acetic acid water, 10:7:3 (vjv) (organic phase); (C) isopropanol-ammonia-water, 20: 1:2 (v/v). Phenolic compounds were located by ultraviolet (UV) light and also by spraying diazotized 4-nitroaniline followed by alkali. Benzoic acid was identified with broniophenol blue, and carbonyl compounds as their 2,4-dinitrophenylhydrazones.
The products were identified by comparing their RJ values with those of authentic samples. Cell-free extracts of N , crnssa were prepared as follows: The fi~ngalmat was washed three times with distilled water and weighed. It was then macerated with a n equal weight of glass r extracted powder in a chilled porcelain ~ n o r t a and with 0.025 M sodium phosphate buffer pH 7.0 (3 ml/g mat). The contents were centrifuged at 12 000 x g for 15 min and the supernatant was used as the crude enzyme. Protein was estiniated by Lowry's method (7). Benzoylfor~iiate decarboxylase (EC 4.1.1.7) was assayed by following the disappearance of substrate using 2,4-dinitrophenyl11yd1-azine( 5 ) in a reaction mixture consisting of 0.2 M sodium phosphate buffer p H 6, thiamine pyrophosphate (50 pg), benzoylfor~iiic acid 0.05 pmol, and enzyme protein. Protocatechuic acid dioxygenase (EC 1.13.1 1.3) was assayed by following the disappearance of protocatechuic acid (8) in a reaction niixti~recontaining 0. I pmol of protocatechuic acid, enzyme, and 0.2 M sodii~niphosphate buffer pH 7.5. Catechol dioxygenase (EC 1.13.11.I) activity was measured similarly i~sing catechol instead of protocatechuic acid. One unit of enzyme activity is defined as the amount of enzynie catalyzing the conversion of 1 pmol of substrate into product per minute. Specific activity is expressed as milliunits per milligram protein. All enzyme activities were measured at 30°C. Table 1 shows the I-esults of replacement culture studies. It is evident from the table that the metabolis~nof niandelic acid proceeds via benzoylformic acid, benzoic acid, 4-hydroxybenzoic acid, and protocatechuic acid, Both D- and Lmandelic acids are used in N. crnssa, either by a racelnase or by dehydrogenases specific for the D- and L-enantiomers. Catechol could not be detected in the medium. These results show that N . crnssn follows the same pathway'as that of As/)ergillus tiiger (5). T o elucidate the intermediary nature of benzaldehyde, benzoylforrnate decarboxylase (EC 4.1.1.7) from this organism
CAN. J.
MICROBIOI-. VOL.
23. 1977
TABLE 1. Results of replacement culture studies
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 09/11/14 For personal use only.
Compounds detected in the replacement culture medium Substrate used in the replacement c~llturemedium
Benzoyl formic acid
DL-Mandelic acid D(-) Mandelic acid L ( + ) Mandelic acid Benzoyl formic acid Benzoic acid 4-Hydroxy benzoic acid Protocatechuic acid
Benzoic acid
4-Hydroxy benzoic acid
Protocatechuic acid
+ + + + -
-
-
TABLE2. Specific activity of some of the enzymes of mandelate pathway Specific activity Enzynies
Glucose grown
Mandelate grown
Protocatechuate dioxygenase Benzoyl formate decarboxylase Catechol dioxygenase
0.00
was isolated and soille of the properties studied. catechuate dioxygenase activity (Table 2) while As expected, the product of the enzymatic reac- there was no catechol dioxygenase activity, theretion was found to be benzaldehyde. The enzyme by suggesting that protocatechuic acid and not has been purified froiii the crude extracts by catechol is the intermediate in the nietabolis~nof protamine sulfate treatment, negative adsorption ~nandelicacid by this organism. As there was n o on calcium phosphate gel, and DEAE cellulose accumulation of yellow-coloured products, which chroinatography to about fivefold with an overall are normally formed during extradiol cleavage reactions, it appears that this reaction involves recovery of 70%. Benzoylformate decarboxylase activity ex- an intradiol dioxygenase activity. Based on these hibited a pH optimum of 6 and a temperat~~re results the following pathway is proposed for optimuni of 40°C. Citrate, which is an inhibitor the degradation of mandelic acid by N. crassa. of A. tziger benzoylformate decarboxylase (4), also inhibited the enzyme from N. crassa. SulfDL-Mandelicacid + benzoylformic acid + hydryl reagents s ~ ~ as c h4-chloromercuribenzoate benzaldehyde + benzoic acid + and Hg2+ inhibited the enzyme activity. 4-hydroxybenzoic acid + protocatechuic acid While M n 2 + and Z n 2 + activated the enzyme, + ring-cleaved products. CLI" and F e z + inhibited it. The enzyme from N. crassa was found to be stable for 48 h when The results clearly show that N. crassa follows stored at - 15"C, while that from A. tziger lost a different pathway from that of bacterial systems all activity after 12 h. Substrate analogs such as and is similar t o the pathway followed by Asperphenylpyruvate and phenylacetic acid tested at gillus niger for the degradation of mandelic aicd. 1 mM concentration had no effect on the enzyme Further purification, study of the properties, and activity. comparison of these enzymes from N. crassa and The cell-free extracts possessed powerful proto- A. niger are in progress.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 09/11/14 For personal use only.
NOTES
I . GUNSALUS,I. C., C. F. GUNSALUS.and R. Y. S T A N I E R1953. . T h e enzymatic conversion of mandelic acid. I . Gross fractionation of the system into soluble and particulate components. J. Bacteriol. 66: 538-542. 2. GUNSALUS, C. F., R. Y. SI-ANIER,and I. C. G U N SALUS,1953. The enzymatic conversion of mandelic acid. 11. Properties of particulate fraction. J. Bacteriol. 66: 543-547. 3. H E G E M A NG. , D. 1966. Synthesis of the enzymes of the mandelate pathway by Pselrc1omo11cr.sprrri(l(r. I. Synthesis of the enzymes by the wild type. J. Bacteriol. 91: 1140-1 154. 4. J A M A L U D D IM N . 1971. Metabolism of mandelic acid by Aspergillus niger. Ph.D. thesis. Indian Institute of Science, Bangalore. 5. ~ M A L U D D IM., N , P. V. SUBB-\RAO. and C. S . V A I D Y A N - \ T H A N . 1970. Involvement of protoc21techuate pathway in the metabolism of mandelic acid by Aspergilllrs ,tiger. J . Bacteriol. 101: 786-793.
1499
6. K E N N E D Y ,S. I. T . , and C. A. FEWSON. 1968. Metabolism of mandelate and related compounds by Bacterium NCIB 8250. J . Gen. Microbiol. 53: 259-273. 7. L O W R Y0 , . H . , N . J. ROSEBROUGH, A . L . F A K R and , R. J. RANDALL.1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193: 265-275. 8. N A I R , P. M., and C. S. VAIDYANAI-H-\N. 1964. A colorimetric method for determination of pyrocatechol and related substances. Anal. Biochem. 7: 315321. 9. P A D M A N A B AGN. ., and P. S . SAKMA.1965. Studieson iron metabolism in Nerrrosporcr crcrsscr. Archiv. Biochem. Biophys. 11: 147-152. 10. S T A N I E RR., Y. 1948. T h e oxidation of aromatic compounds by fluorescent Pseudomonas. J. Bacteriol. 45: 477-494.
1496
C A N . J. MICROBIOL. VOL. 23. 1977
coli t o uvrB- and rec- mutants of S. typhimwizitn. It should be pointed out that survival curves showed that TA1950 and TA1535 were equally sensitive t o ultraviolet light, indicating that while the rfa- mutation is able t o suppress the TSY agar sensitivity associated with zrurBit does not suppress ultraviolet light sensitivity. While the physiological mechanisms of the effects reported here are yet t o be determined, it is clear that care should be exercised when enumerating D N A repair-deficient mutants.
1
2
3
4
5
6
7
8
9
1
0
1
1
HOURS
FIG.3. Growth of S. l y p l ~ i t ~ r ~ r r i ~TA1975 rti~ in M-9 supplemented with histidine and TA1535 in M-9 supplemented with histidine and biotin. Plate counts of TA1975 on M-9 plus histidine (A), on TSY (0); plate counts of TA1535 011 M-9 plus histidine and biotin (A), on TSY (@).
medium, supple~nentedwith histidine and biotin, there is no difference between colony counts on TSY and M-9 plates. When each of the five strains were grown in TSY broth, the differences between the M-9 and TSY colony counts were negligible throughout the growth curve. This work has extended the observations of Rosenkranz et 01. (4) with polA- mutants of E.
Acknowledgements This work was supported by grants 2-POIES00597 from the National Institute of Environmental Health Sciences and SMI76-03575 from the National Science Foundation Undergraduate Research Participation Grant, and is contribution No. 3090 froin the Department of Nutrition and Food Science, Massachusetts Institute ofTechnology, Cambridge, MA, U.S.A. 02139. I. ACIES.B. N.. F. D. LEE,and W. E. D u n s - r o ~ 1973. . An improved bacterial test system for the detection and classification of mutagens and c;lscinogens. Proc. Natl. Acad. Sci. U.S.A. 70: 782-786. 2. GOCIEZ, R. F., and A. J. S I N S K E Y1974. . DNA breaks rrt)~ in rifampin-treated S N / I ) I ~ I I C t/ y/ ~p ~h i ~ ~ ~ r ~ r i LT-2 after exposure to nittritionally complex media. Nature. 247: 21 1-212. 3. GOCIEZ, R. F.. A. J . S I N S K E YR., DAVIES, and T. P. L,\suz,\. 1973. Minimal medium recovery of heated S r r l t ~ ~ o t ~typl~irtrrrriri~~i ell~ LT-2. J . Gen. Microbiol. 74: 267-274. 4. R O S E N K ~ ~ \H. NZ S.,, H. S. C A R R and . C. MORGAN. 1971. Unusual growth properties of a bacterial strain lacking DNA polymerase. Biochem. Biophys. Res. Commun. 44: 546-549.
Metabolism of mandelic acid by Neurospora crassa
Accepted June 22, 1977 R A M A K R I S HRAO, N A D. N., and C. S. V A I D Y A N A T H A 1977. N . Metabolism of mandelic acid by Nerrrosparo crrrssn. Can. J. Microbiol. 23: 1496-1499. Preliminary studies on the metabolism of mandelic acid by Nerrrosporn urrssn reveal the operation of a pathway for its degradation which involves benzoyl formic acid, benzaldehyde, benzoic acid, Chydroxybenzoic acid. and protocatechuic acid as the intermediates. This pathway is different from that followed by bacterial systems and is the same as that observed in
Aspergillu.~niger .
NOTES
I497
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 09/11/14 For personal use only.
RAM.AKKISHN-\RAO.D. N . , et C. S. V.AIDY,AW;\-I.H,\V. 1977. Metabolism of rnandelic acid by Nelrrosporcr uccssrr. Can. J. Microbial. 23: 1496-1499. Des e t i ~ d e preliminaires s sur le metabolisme de I'acide mendelique demontrent qu'il existe une voie pour sa dCgl-adation chez Neicrosporrr C I - c r ~ ~ L'acicle ri. ben~oyl-formique,le benzaldehyde, I'acide benzoiqi~e, I'acide 4-hydroxybenzoique et I'acide protocatechuique sont d e s intermediaires metaboliques de cette voie. Cette derniere differe cle celle que I'on observe dans les systemes bacteriens: mais elle est la meme que celle retrouvee chez Aspergillcts tligo.. (Traduit par le journal]
Mandelic acid metabolisni has been extensively studied in Psezr~lolomonasputida and bacterium N.C.I.B. 8250 (1, 2, 3, 6, 10). The pathway involves the intermediary formation of benzoyl formic acid, benzaldehyde, benzoic acid, and catechol. In fungi, however, mandelate metabolism has not been investigated in detail except for a report from this laboratory demonstrating the operation of a protocatechuic acid pathway for the degradation of mandelate by As~)ergillus n i g o (5). In this communication, we report our preliminary studies on the metabolism of this conipound by Neurospora crassu. Neurospora crassn FGSC 352, obtained from the Fungal Genetic Stock Center, Humbaldt State College, Arcata, CA., wasgrown on a modified Vogel's medium (120 ml) (9) containing 0.1% (w/v) DL-mandelic acid in 1-t! conical flasks, for 48 h at 30°C. The same conditions were used for obtaining cells grown on glucose except that mandelic acid was not present in the medium. The mycelial felts thus obtained (5 g) from a still culture were harvested, washed three times with sterile water, and replaced in 50 ml of 0.05 M sodium phosphate buffer pH 7.0 containing 1 mg/ml of the various intermediates. The replacement culti~reswere kept on a rotary shaker (250 rpm) a t 30°C for 6 h and the products formed were analyzed. The cultures were filtered through a filter paper. The medium was acidified to pH 2 with 3 N HC1 and extracted three times with an equal volume of ethylacetate. The pooled organic layer was dried over anhydrous sodium sulfate, concentrated, and subjected to ascending paper chromatography on Whatman No. 1 filter paper using the following solvent systems: (A) formic acid water, 2:98 (v/v); (B) benzene - acetic acid water, 10:7:3 (vjv) (organic phase); (C) isopropanol-ammonia-water, 20: 1:2 (v/v). Phenolic compounds were located by ultraviolet (UV) light and also by spraying diazotized 4-nitroaniline followed by alkali. Benzoic acid was identified with broniophenol blue, and carbonyl compounds as their 2,4-dinitrophenylhydrazones.
The products were identified by comparing their RJ values with those of authentic samples. Cell-free extracts of N , crnssa were prepared as follows: The fi~ngalmat was washed three times with distilled water and weighed. It was then macerated with a n equal weight of glass r extracted powder in a chilled porcelain ~ n o r t a and with 0.025 M sodium phosphate buffer pH 7.0 (3 ml/g mat). The contents were centrifuged at 12 000 x g for 15 min and the supernatant was used as the crude enzyme. Protein was estiniated by Lowry's method (7). Benzoylfor~iiate decarboxylase (EC 4.1.1.7) was assayed by following the disappearance of substrate using 2,4-dinitrophenyl11yd1-azine( 5 ) in a reaction mixture consisting of 0.2 M sodium phosphate buffer p H 6, thiamine pyrophosphate (50 pg), benzoylfor~iiic acid 0.05 pmol, and enzyme protein. Protocatechuic acid dioxygenase (EC 1.13.1 1.3) was assayed by following the disappearance of protocatechuic acid (8) in a reaction niixti~recontaining 0. I pmol of protocatechuic acid, enzyme, and 0.2 M sodii~niphosphate buffer pH 7.5. Catechol dioxygenase (EC 1.13.11.I) activity was measured similarly i~sing catechol instead of protocatechuic acid. One unit of enzyme activity is defined as the amount of enzynie catalyzing the conversion of 1 pmol of substrate into product per minute. Specific activity is expressed as milliunits per milligram protein. All enzyme activities were measured at 30°C. Table 1 shows the I-esults of replacement culture studies. It is evident from the table that the metabolis~nof niandelic acid proceeds via benzoylformic acid, benzoic acid, 4-hydroxybenzoic acid, and protocatechuic acid, Both D- and Lmandelic acids are used in N. crnssa, either by a racelnase or by dehydrogenases specific for the D- and L-enantiomers. Catechol could not be detected in the medium. These results show that N . crnssn follows the same pathway'as that of As/)ergillus tiiger (5). T o elucidate the intermediary nature of benzaldehyde, benzoylforrnate decarboxylase (EC 4.1.1.7) from this organism
CAN. J.
MICROBIOI-. VOL.
23. 1977
TABLE 1. Results of replacement culture studies
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 09/11/14 For personal use only.
Compounds detected in the replacement culture medium Substrate used in the replacement c~llturemedium
Benzoyl formic acid
DL-Mandelic acid D(-) Mandelic acid L ( + ) Mandelic acid Benzoyl formic acid Benzoic acid 4-Hydroxy benzoic acid Protocatechuic acid
Benzoic acid
4-Hydroxy benzoic acid
Protocatechuic acid
+ + + + -
-
-
TABLE2. Specific activity of some of the enzymes of mandelate pathway Specific activity Enzynies
Glucose grown
Mandelate grown
Protocatechuate dioxygenase Benzoyl formate decarboxylase Catechol dioxygenase
0.00
was isolated and soille of the properties studied. catechuate dioxygenase activity (Table 2) while As expected, the product of the enzymatic reac- there was no catechol dioxygenase activity, theretion was found to be benzaldehyde. The enzyme by suggesting that protocatechuic acid and not has been purified froiii the crude extracts by catechol is the intermediate in the nietabolis~nof protamine sulfate treatment, negative adsorption ~nandelicacid by this organism. As there was n o on calcium phosphate gel, and DEAE cellulose accumulation of yellow-coloured products, which chroinatography to about fivefold with an overall are normally formed during extradiol cleavage reactions, it appears that this reaction involves recovery of 70%. Benzoylformate decarboxylase activity ex- an intradiol dioxygenase activity. Based on these hibited a pH optimum of 6 and a temperat~~re results the following pathway is proposed for optimuni of 40°C. Citrate, which is an inhibitor the degradation of mandelic acid by N. crassa. of A. tziger benzoylformate decarboxylase (4), also inhibited the enzyme from N. crassa. SulfDL-Mandelicacid + benzoylformic acid + hydryl reagents s ~ ~ as c h4-chloromercuribenzoate benzaldehyde + benzoic acid + and Hg2+ inhibited the enzyme activity. 4-hydroxybenzoic acid + protocatechuic acid While M n 2 + and Z n 2 + activated the enzyme, + ring-cleaved products. CLI" and F e z + inhibited it. The enzyme from N. crassa was found to be stable for 48 h when The results clearly show that N. crassa follows stored at - 15"C, while that from A. tziger lost a different pathway from that of bacterial systems all activity after 12 h. Substrate analogs such as and is similar t o the pathway followed by Asperphenylpyruvate and phenylacetic acid tested at gillus niger for the degradation of mandelic aicd. 1 mM concentration had no effect on the enzyme Further purification, study of the properties, and activity. comparison of these enzymes from N. crassa and The cell-free extracts possessed powerful proto- A. niger are in progress.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 09/11/14 For personal use only.
NOTES
I . GUNSALUS,I. C., C. F. GUNSALUS.and R. Y. S T A N I E R1953. . T h e enzymatic conversion of mandelic acid. I . Gross fractionation of the system into soluble and particulate components. J. Bacteriol. 66: 538-542. 2. GUNSALUS, C. F., R. Y. SI-ANIER,and I. C. G U N SALUS,1953. The enzymatic conversion of mandelic acid. 11. Properties of particulate fraction. J. Bacteriol. 66: 543-547. 3. H E G E M A NG. , D. 1966. Synthesis of the enzymes of the mandelate pathway by Pselrc1omo11cr.sprrri(l(r. I. Synthesis of the enzymes by the wild type. J. Bacteriol. 91: 1140-1 154. 4. J A M A L U D D IM N . 1971. Metabolism of mandelic acid by Aspergillus niger. Ph.D. thesis. Indian Institute of Science, Bangalore. 5. ~ M A L U D D IM., N , P. V. SUBB-\RAO. and C. S . V A I D Y A N - \ T H A N . 1970. Involvement of protoc21techuate pathway in the metabolism of mandelic acid by Aspergilllrs ,tiger. J . Bacteriol. 101: 786-793.
1499
6. K E N N E D Y ,S. I. T . , and C. A. FEWSON. 1968. Metabolism of mandelate and related compounds by Bacterium NCIB 8250. J . Gen. Microbiol. 53: 259-273. 7. L O W R Y0 , . H . , N . J. ROSEBROUGH, A . L . F A K R and , R. J. RANDALL.1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193: 265-275. 8. N A I R , P. M., and C. S. VAIDYANAI-H-\N. 1964. A colorimetric method for determination of pyrocatechol and related substances. Anal. Biochem. 7: 315321. 9. P A D M A N A B AGN. ., and P. S . SAKMA.1965. Studieson iron metabolism in Nerrrosporcr crcrsscr. Archiv. Biochem. Biophys. 11: 147-152. 10. S T A N I E RR., Y. 1948. T h e oxidation of aromatic compounds by fluorescent Pseudomonas. J. Bacteriol. 45: 477-494.
