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Centre of Advanced Study in Botany, University of Madras, Madras 600005, India ..... Acknowledgements G.M. and A.M. thank the University Grants Commission ...

Plant and Soil 69, 163-169 (1982). 0032-079X/82/0692-0163501.05. Ms. 5021 9 1982 Martinus Nijhoff/Dr W. Junk Publishers, The Hague. Printed in the Netherlands.

Degradation of aromatic compounds by Rhizobium spp. G. MUTHUKUMAR, A. ARUNAKUMARI and A. MAHADEVAN Centre of Advanced Study in Botany, University of Madras, Madras 600005, India Received 10 March 1982. Revised September 1982 Key words Biodegradation Catechin p-Hydroxybenzoate Oxidation Rhizobium Salicylate Summary The ability of rhizobia to utilize catechol, protocatechuic acid, salicylic acid, phydroxybenzoic acid and catechin was investigated.The degradation pathway of p-hydroxybenzoate by Rhizobium japonicum, R. phaseoli, R. leguminosarum, R. trifolii and Rhizobium sp. isolated from bean was also studied. R. leguminosarum, R. phaseoli and R. trifolii metabolized p-hydroxybenzoate to protocatechuate which was cleaved by protocatechuate 3,4-dioxygenasevia ortho pathway. R. japonicum degraded p-hydroxybenzoateto catechol which was cleavedby catechol 1,2-dioxygenase. Rhizobium sp., a bean isolate, dissimilatedp-hydroxybenzoate to salicylate.Salicylatewas converted to gentisicacid prior to ring cleavage.The rhizobia converted p-hydroxybenzoateto Rothera positive substance. Catechol and protocatechuic acid were directly cleaved by the species. R. japonicum converted catechin to protocatechuic acid.

Introduction Extensive studies have been c o n d u c t e d o n bacterial d e g r a d a t i o n of phenolic s u b s t a n c e s b o t h in aerobic z a n d a n a e r o b i c 3 e n v i r o n m e n t s . Literature o n the capacity of Rhizobium spp. to cleave a r o m a t i c substances is f r a g m e n t a r y despite their significance in interfering with growth 11. O n l y Hussien et al. 6, 7 did present some d a t a o n the d e g r a d a t i o n of a r o m a t i c substances by Rhizobium spp. in the presence of s o d i u m glutamate. R. japonicum, R. leguminosarum, R. lupini, R.

meliloti, R. trifolii a n d cowpea rhizobia m a d e good growth o n catechol (1), p r o t o c a t e c h u i c acid (2), salicylic acid (3) a n d p - h y d r o x y b e n z o i c acid (4). O n e of us ~1 observed fluctuations in rhizobial p o p u l a t i o n in p e a n u t (Arachis hypogeae) fields treated with a solution of m y r o b a l a n t a n n i n . The p o p u l a t i o n increased 55 days after application. This o b s e r v a t i o n t e m p t e d us to investigate the ability of r h i z o b i a to degrade a few a r o m a t i c substances which c o m m o n l y occur in plants a n d in soils 13.

Materials and methods

R. leguminosarum 110/2011, R.japonicum, R. trifolii 7002 KSJ were kindly supplied by Dr. N. S. Subba Rao, I.A.R.I.,New Delhi. R. trifolii B 13, R. trifolii B 11, R. leguminosarurn B 8, R. phaseoli, Rhizobium sp. from bean and Rhizobium sp. B 16 (groundnut isolate) were obtained from the culture collection of this laboratory. All the strains were maintained on yeast mannitol agar medium15. To study the growth on aromatic substances, synthetic medium6 amended with 1 mM aromatic substance was used. To isolate intermediates, rhizobia were grown in liquid medium and the ceils were separated by centrifuging at 7,000g for 10 min at 4~ Phenols in the spent culture medium were extracted 8. 163

164

M U T H U K U M A R , A R U N A K U M A R I AND MAHADEVAN

For replacement culture studies, cells were washed twice in 0.025 M phosphate buffer and then resuspended in the buffer to get 0 . 1 0 . D . at 600 nm. The test substance was added to get a final concentration of 1 m M and incubated in a shaker at 28~ at 100 rpm for 8 h. Cells were removed by centrifugation and the supernatant was extracted for phenols. For enzyme studies, cells were suspended in 0.025 M phosphate buffer, pH 7.4 to get a concentration of 0.05 O.D. at 600 nm and disrupted by sonication for 3 rain in a ultrasonic disintegrator. The suspension was centrifuged at 10,000 9 for 30 min and the supernatant was used as crude enzyme source. Catechol 1,2-dioxygenase and catechol 2,3-dioxygenase were estimated by Hegeman's procedure 5. The unit of enzyme activity was defined as p-moles of catechol converted to eis, eis-muconic acid at 260 nm per minute per mg of protein. The catechol 2,3-dioxygenase unit was defined as p-moles of catechol converted to eis, cis-muconaldehyde at 375 nm per minute per mg of protein. Protocatechuate 3,4-dioxygenase was assayed by Fujisawa's method 4 The unit of enzyme activity was defined as p-moles of protocatechuate disappearing at 290 nm at 24~ per minute per mg protein. Protein was estimated by Lowry's procedure 10. Rothera's test was conducted to find out the type of ring cleavage 14.

Identification of phenolic intermediates By using Whatman No. 1 chromatographic paper and developing in butanol-acetic acid-water (41-5 v/v, upper phase)8, phenols were detected by spraying with diazotized p-nitroaniline. UV spectra of the substances were scanned in 95% ethanol in a Unicam SP 800 UV spectrophotometer.

Oxidation studies Cells were grown in liquid medium containing p-hydroxybenzoic acid (1 mM) for 3 days at 28~ in a shaker (100 rpm) and after centrifuging the culture at 7,000 g for 10 min, were washed with 0.025 M phosphate buffer to get ca. 3 mg dry weight/ml. The side arm of the manometer contained 2 p-mole of test substance (freshly prepared) and the centre well received 0.2 ml of freshly prepared 20% K O H with a folded filter paper. Respiration was measured according to Muthukumar and Mahadevan 12.

Results All rhizobia except R. leguminosarum utilized catechol and protocatechuic acid. R. leguminosarum 110/2011 exhibited no growth on salicylate whereas others made poor growth (Table 1). p-Hydroxybenzoate was utilized as a carbon source by all the rhizobia except R.japonicum which made poor growth. Catechin (5) supported the growth of all 9 strains of Rhizobium. R. trifolii and R. phaseoli grown on p-hydroxybenzoate rapidly oxidized it and protocatechuate but slowly oxidized catechol and salicylate (Fig. 1). R. leguminosarum cells rapidly oxidized p-hydroxybenzoate and oxygen uptake with protocatechuate at the initial period was more than with catechol. Oxidation of salicylate by the cells was extremely slow (Fig. 1). R. japonicum cells grown on p-hydroxybenzoate rapidly oxidized catechol but slowly oxidized protocatechuic acid and salicylic acid (Fig. 1). Rhizobium sp., a bean isolate, oxidized p-hydroxybenzoate and salicylic acid without any lag. But it oxidized eatechol and protocatechuic acid extremely slowly. R.japonicum, R. leguminosarurn, R. phaseoli, R. trifolii and Rhizobium sp. grown on p-hydroxybenzoic acid gave positive reaction with Rothera's test for keto compounds.

165

D E G R A D A T I O N O F AROMATIC C O M P O U N D S BY R H I Z O B I U M

Isolation of phenols from the spent culture medium of R. leguminosarum, R. phaseoli and R. trifolii and replacement cultures with p-hydroxybenzoate revealed the presence of a substance with Rf value of 0.81 and gave )~max 290 nm. It was identified as protocatechuic acid. p-Hydroxybenzoate spent culture medium and replacement cultures with R.japonicum contained a substance that had an Rfof0.89 with L max 278 nm, which was identified by co-chromatography as catechol. When Rhizobium sp. from bean was grown on p-hydroxybenzoate, the culture medium did not contain any aromatic substance. But cells grown on salicylic acid converted it to a product which exhibited absorbance maximum 330 nm, identified as gentisic acid (6). N o phenolic intermediates were isolated from catechol and protocatechuate media, cultured with rhizobia. Culture filtrate and replacement cultures containing catechin with R.japonicum revealed the presence of a substance which gave Rf of 0.81 with L max 290 nm and was identified as protocatechuic acid. Cells of R. trifolii, R. phaseoli and R. leguminosarum synthesized 0.010, 0.032 and 0.025 units respectively of protocatechuate 3,4-dioxygenase when grown on 1 m M p-hydroxybenzoate, p-Hydroxybenzoate grown R. japonicum cells induced catechol 1,2-dioxygenase (0.012 units) but catechol 2,3-dioxygenase was absent in the cells. Protocatechuate 3,4-dioxygenase was also present which equalled in concentration to the uninduced cells grown on glutamate.

Table 1. Growth* of R h i z o b i u m spp. on aromatic substances Isolate

Salicylic acid

p-Hydroxy benzoic acid

Catechol

Protocatechuic acid

Catechin

R.japonicum

+

+

+

+ +

+ +

R. leguminosarum

-

+ +

-

+

+

+

+ +

+

+

+

R. phaseoli

+

+ +

+

+

+

R. trifolii

+

+ +

+

+ +

+

+

+ +

+ +

+ +

+ +

R. trifolii B 11

+

+ +

+

+ +

+

R h i z o b i u m sp

+

+ +

+

+

+ +

+

+ +

+

+

+

110/2011 R. leouminosarum

B8

7002 KSJ R. trifolii

B313

bean isolate Rhizobium

sp.B 16 * Growth on synthetic medium 6, containing one of the phenols at 1 mM; growth was measured on 3rd day. - = N o growth; + = poor growth; + + = good growth

MUTHUKUMAR, ARUNAKUMARI AND MAHADEVAN

166

x2_

9-

xz

~,~

c~, \ cz.~ ~ v

a:l

eq

~e~dn EO ]r~

Fig. 1. Oxidation of aromatic compounds by Rhizobium spp.

DEGRADATION OF AROMATIC COMPOUNDS BY RHIZOBIUM

167

p-Hydroxybenzoate grown Rhizobium sp. contained both catechol 1,2dioxygenase and protocatechuate 3,4-dioxygenase, 0.009 and 0.007 units, respectively. Discussion

Rhizobia utilized catechol, protocatechuic acid, p-hydroxybenzoate and catechin as sole carbon source. R. phaseoli, R. trifolii and R. leguminosarum converted p-hydroxybenzoate to protocatechuate which was isolated from spent culture medium as well as from replacement cultures. These species produced protocatechuate 3,4-dioxygenase to cleave protocatechuate in an intradiol fashion resulting in the formation of [~-carboxy cis, cis-muconate (7). Ceils grown on p-hydroxybenzoate reacted positively to Rothera's reagent for keto substances. Clearly the metabolism of p-hydroxybenzoate proceeds via ~ketoadipate pathway (Fig. 2). In addition, protocatechuate was oxidized rapidly compared with catechol and salicylate by p-hydroxybenzoate grown cells of R. trifolii, R. leguminosarum and R. phaseoli. Presumably these species degrade p-hydroxybenzoate by a pathway indicated in Fig. 2. Conversion of p-hydroxybenzoate to protocatechuate by R. phaseoli was reported 6. But R. japonicum rapidly oxidized catechol when the cells were previously grown on p-hydroxybenzoate. Failure to detect protocatechuic acid in spent culture medium and in replacement culture also suggested the non-conversion of p-hydroxybenzoate to protocatechuate. Instead, catechol was detected in the spent cultures and the cells induced catechol 1,2-dioxygenase when grown on phydroxybenzoate. Catechol and Rothera positive substance were detected. The conversion of p-hydroxybenzoate to catechol might involve single or multiple step reaction but catechol was cleaved directly by ortho fission to form cis, eismuconic acid (8). Rhizobium sp. exhibited a different pathway of p-hydroxybenzoate degradation, p-Hydroxybenzoate grown cells rapidly oxidized salicylate but slowly oxidized catechol and protocatechuic acid. However, we could not detect salicylic acid in the spent culture medium or in replacement culture. But salicylic acid grown cells converted it to gentisate which might further undergo ring cleavage. R. phaseoli 405 converted salicylate to gentisate 6. Therefore we propose that Rhizobium sp. converted p-hydroxybenzoate to salicylate which is further converted to gentisic acid before ring cleavage (Fig. 2). All the rhizobia readily catabolized catechin. R. japonieum degraded it to protocatechuie acid. This is the first report of degradation of cateehin by a bacterium. Chandra et al. 1 reported the conversion ofcatechin to phloroglucinol carboxylic acid, protocatechuic acid and an unidentified phenol by Aspergillus

flat)us. Admittedly rhizobia are capable of cleaving aromatic substances and utilize

MUTHUKUMAR, ARUNAKUMARI AND MAHADEVAN

168

C00H COqH 9 e/'~

,/~-~0 H

4

~

COOH

H-"~V - O H

5

OH

6

ring cleavage Aromatic ring cleavage ,~ (tntradio[)

$

#'~.co J ~L>/coc-

bocy~-coo" ~./cooU 7

o.-d--.c o oL/coo9 Fig. 2. Degradation pathways of aromatic compounds by Rhizobium spp. 1. catechol a. R. leguminosarum 110/2011 b. R. phaseoli 2. protocatechuic acid 3. salicylic acid c. R. trifolii 7002 KSJ 4. p-OH benzoic acid d. R. japonicum 5. catechin e. Rhizobium sp. bean isolate 6. gentisic acid 7. I}-carboxycis, cis-muconic acid 8. cis, cis-muconic acid 9. 13-ketoadipicacid

them as source of energy. M a n y plants exude substances including phenols. F o r example, germinating seeds of peanut released catechol, p-coumaric acid, gentisic acid, quercetin, rutin, salicylic acid and vanillic acid in the soil 9. These ould not interfere with the multiplication of rhizobial species in the soil and nodulation in p e a n u t roots unless cleaved. This study clearly shows that rhizobia do cleave the substances.

Acknowledgements G.M. and A.M. thank the University Grants Commission for a research grant. A.A.K. acknowledges C.S.I.R. for the award of a Senior Research fellowship.

DEGRADATION OF AROMATIC COMPOUNDS BY RHIZOBIUM

169

References

1

Chandra T, Madhavakrishna W and Nayaudamma Y 1969 Astringency in fruits. I. Microbial degradation of catechin. Can. J. Microbiol. 15, 303-306. 2 Dagley S 1978 Microbial catabolism in the carbon cycle and environmental pollution. Naturwissenschaften 65, 85-95. 3 Evans W C 1977 Biochemistry of the bacterial catabolism of aromatic compounds in anaerobic environments. Nature London 270, 17-22. 4 Fujisawa H 1970 Protocatechuate 3,4-dioxygenase (Pseudomonas). In Methods in Enzymology Vol. 17 Metabolism of amino acids and amines. Eds. H Tabor and C W Tabor. Academic Press, New York. p 526. 5 Hegeman C D 1966 Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. I. Synthesis of enzymes by wild types. J. Bacteriol. 91, 1140-1154. 6 Hussien Y A, Tewfik M S and Hamdi Y A 1974 Degradation of certain aromatic compounds by rhizobia. Soil Biol. Biochem. 6, 377-381. 7 Hussien Y A, Tewfik M S and Hamdi Y A 1975 Metabolism of certain aromatic compounds by rhizobia. Egypt J. Bot. 18, 91-99. 8 Ibrahim R K and Towers G H N 1960 The identification by chromatography of plant phenolic acids. Arch. Biochem. Biophys. 87, 125-128. 9 Kalaichelvan P T 1980 Prohibitins in groundnut (Arachis hypogaea). Ph.D. thesis, University of Madras, Madras. 10 Lowry O H, Rosenberg N J, Farr A L and Randall R J 1951 Protein measurements with Folin phenol reagent. J. Biol. Chem. 193, 265-275. 11 Muthukumar G 1980 Effect of Tannins of Soil Micro-organisms and Crops. Ph.D. Thesis, University of Madras, Madras. 12 Muthukumar G and Mahadevan A 1981 Effect of tannins on soil respiration and glucose oxidation by micro-organisms. Indian J. Exp. Biol. 19, 1083-1085. 13 Rice E L and Pancholy S K 1973 Inhibition of nitrification by climax ecosystems. II. Additional evidence and possible role of tannins. Am. J. Bot. 60, 691-702. 14 Rothera A C H 1908 Note on the sodium nitroprusside reaction for acetone. J. Physiol. 37, 491-494. 15 Vincent J M 1970 A Manual for the Practical Study of Root Nodule Bacteria. International Biological Programme, Blackwell, Oxford, p 3.

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