Biochem. J. (1969) 113, 525Printed in Great Britain

The Metabolism of Aromatic Compounds byRhodopseudomonas palustris

A NEW, REDUCTIVE, METHOD OF AROMATIC RING METABOLISM

BY P. L. DUTTON* AND W. C. EVANSDepartment of Biochemi8try and Soil Science, Univer8ity College of North Wales8,

Bangor, Caern8.

(Received 15 January 1969)

1. Rhodop8eudomona8 pal?utri8 grows both aerobically and photosyntheticallyon aromatic acids. p-Hydroxybenzoate and protocatechuate are able to supportaerobic growth; these compounds are metabolized by the protocatechuate 4,5-oxygenase pathway. 2. The photoassimilation of benzoate and hydroxybenzoatesand the effects of air and darkness on the photoassimilation of benzoate aredescribed. 3. Evidence in conflict with the pathway previously proposed for thephotometabolism of benzoate is discussed. 4. The photometabolism of benzoate isaccomplished by a novel reductive pathway involving its reduction to cyclohex-I -ene-l-carboxylate, followed by hydration to 2-hydroxycyclohexanecarboxylateand after dehydrogenation to 2-oxocyclohexanecarboxylate further hydrationresults in ring-fission and the production of pimelate. 5. Attempts were madeto prepare cell-free extracts capable of dissimilating benzoate.

From extensive studies on the oxidative meta-bolism of aromatic compounds by aerobic micro-organisms, many of the pathways and mechanismsinvolved have been described (Evans, 1963;Ribbons, 1965; Dagley, 1967). Bacteria thatdissimilate aromatic compounds under anaerobicconditions have also been reported. Tarvin &Buswell (1934) and Barker (1956) described severalaromatic acids that were utilized under strictlyanaerobic conditions by the methanogenic bacteria;Harary (1956) isolated a Cto8tridium sp. capable ofanaerobic fermentation of nicotinate; and Scher &Proctor (1960) isolated several strains of non-

sulphur photosynthetic bacteria that utilizedbenzoate anaerobically in the light.

Clark & Fina (1952) showed that cultures ofmethanogenic bacteria grown on benzoate failed tometabolize catechol or protocatechuate, andRoberts (1962) reported that similar benzoate-induced cultures produced methane without a lagwhen incubated with cyclohexanecarboxylate,butyrate or propionate, whereas long inductionperiods were required with fumarate, acrylate or

2-methylpropionate, which suggested a method ofaromatic metabolism different from the knownaerobic type and one that possibly is reductive. The

* Present address: Department of Biophysics andPhysical Biochemistry, Johnson Research Foundation,University ofPennsylvania, Philadelphia, Pa. 19104, U.S.A.

first stage of the anaerobic fermentation of nicotin-ate by a Clo8tridium sp. has been shown to involvehydration to form 6-hydroxynicotinate (Harary,1957a,b), and several of the later intermediates havebeen described (Tsai, Pastan & Stadtman, 1966). Instudies on the photometabolism of benzoate by a

Rhodop8eudomona8 sp. Proctor & Scher (1960)proposed a pathway that is typically oxidative,which invoked protocatechuate and catechol as

intermediates. The absence of enzymes of theseoxidative pathways from R. palu8tri8 grown photo-synthetically on aromatic substrates has, however,been demonstrated (Hegeman, 1967; Dutton &Evans, 1967), and the present paper providesfurther evidence by describing a new reductiveroute for the metabolism of the benzene ring.Preliminary accounts of this work have beenpublished (Dutton & Evans, 1967, 1968a,b).

MATERIALS AND METHODSCulture techniqueB

Rhodop8eudomona3 palustri8 (Scher strain), a gift fromDr J. Turner (Department of Biochemistry, University ofLiverpool), was obtained as a Rhodope?udomonas sp. butidentified from its microscopic appearance (van Niel, 1944)and requirement for p-aminobenzoate (Hutner, 1950) as

Rhodop8eudomona8 paludtri8. It was grown photosynthetic-ally under 02-free N2 or aerobically in normal laboratorylight conditions; in either case the following medium was

525

P. L. DUTTON AND W. C. EVANSused: organic substrate, 0-5g./l. (aromatic acids) or 1-5g./l.(aliphatie acids); (NH4)2SO4, 1.0g./l.; K2HPO4, 0-5g./l.;MgSO4,7H20, 0-1g./l.; FeSO4,5H20, 25mg./l.; p-amino-benzoate, 0-1mg./l.; biotin, 0-05mg./l.; thiamin hydro-chloride, 1-Omg./l.; nicotinic acid, 1.0mg./l. The mediumwas made up with tap water, adjusted to pH7-2-7-4 withNaOH and autoclaved for 20min. at 201b./in.2.The organism was grown photosynthetically at 30-32° in

5 1. Pyrex bottles in a glass-sided tank illuminated on bothsides by a bank of eight to ten 60w tungsten lamps. After2 days' growth on media containing benzoate a further1-5g. of sodium benzoate was usually added, the pH of theculture being adjusted at the same time with NaOH topH7-2; the cells were generally harvested after 65-72hr.The culture, maintained in a benzoate-mineral saltsmedium, was transferred, by using a 5% inoculum, every

3-4 days. Cultures grown with hydroxybenzoates as

substrates were started with 250ml. of inoculum from a

culture grown photosynthetically on acetate. The cellswere harvested by centrifugation at 5000g for 5min. at 00

in an MSE High Speed 18 centrifuge and the pellet was

washed once with about 10vol. of 0-05m-tris-HCl or

0 05M-phosphate (K2HPO4-NaOH) buffer, pH7-2, centri-fuged again and used immediately.For aerobic growth, baffled flasks (11.) containing 250ml.

of medium were inoculated from a slope and after 3-4 daysin a rotary shaker (Gallenkamp Orbital Shaker) (130strokes/min.) the contents were transferred to a 51. or 101. culturevessel. After 3-4 days' forced aeration at 300, towards theend ofthe exponential growth phase, the cells were harvestedin the Sharples Super centrifuge, washed once with 0-05M-phosphate buffer, pH 7 2, and stored at -20°. The aerobic-ally growing cultures were not kept in the dark; Lascelles(1956) has noted that normal laboratory light conditionshave little or no effect on photosynthetic bacteria growingaerobically.

Analytical techniques

Spectrophotometric determinations were carried out inthe Beckman DB or Unicam SP. 800 recording spectro-photometers.

Benzoic acid and the phenolic acids in culture media were

determined as follows: after centrifugation, the clearsupernatant (5-Oml.) was acidified with 0-2ml. of 1-5M-H2SO4 and extracted twice with 10ml. of ether. Thecombined extracts were washed with 5ml. of distilled water(acidified to pH< 3) and the aromatic acid was extractedfrom the ether into 5-Oml. of 4% (wlv) NaHCO3; theextinction was measured at 270nm. for benzoate and atsuitable wavelengths for the phenolic acids. Phenoliccompounds were also determined with the Folin-Ciocalteureagent.

Bacterial dry weight was determined from the extinctionat 660nm., by reference to a standard calibration curve.

Protein was determined as described by Warburg &Christian (1942).Manometric experiments were conducted in the con-

ventional Warburg constant-volume apparatus at 300; 02

uptake was measured by the direct method (Dixon, 1952).Radioactive materials that were coloured or insoluble

were assayed as a solid film of standard thickness on

aluminium planchets by using a Geiger-Muller tube inconjunction with an I.D.L. scaler. Soluble radioactive

compounds were assayed in an NE 8304 automatic liquid-scintillation spectrometer (Nuclear Enterprises Ltd.,Edinburgh). The scintillation fluids employed were NE 213and NE 220 (Nuclear Enterprises Ltd.). The maximumefficiency for counting 14C was approx. 90%, correctionsfor quenching being made when necessary.

Values for melting points are uncorrected.

ReagentsChemicals. Benzoic acid, sodium benzoate, the mono-

hydroxybenzoic acids, 3,4- and 2,6-dihydroxybenzoic acidand catechol were obtained from British Drug Houses Ltd.,Poole, Dorset; the other dihydroxybenzoic acids (except2,3-dihydroxybenzoic acid, which was prepared by themethod of Perkin & Robinson, 1914), pimelic acid, cyclo-hexanecarboxylic acid and 2,4-lutidinic acid came fromKoch-Light Laboratories Ltd., Colnbrook, Bucks.; 2-oxo-pimelic acid came fromK & K Laboratories Inc., Plainview,N.Y., U.S.A., and cyclohex-3-ene-1-carboxylic acid fromRalph N. Emanuel Ltd., London. Analytical-gradereagents were used where available.

In the preparation of cyclohex-1-ene-1-carboxylic acid,the cyclohexanone cyanohydrin, prepared by the methodof Ruzicka & Brugger (1926), was converted into cyclo-hexene-1-carbonitrile by the method of Chakravarti (1947).This was hydrolysed by refluxing for 3ihr. with 10vol. of18m-H2SO4-acetic acid-water (1:1:1, by vol.) (Babior &Bloch, 1966). The product was recrystallized from lightpetroleum (b.p. 40-60°) and had m.p. 36-38° (reportedm.p. 36-37°).

Cyclohex-2-ene-1-carboxylic acid was prepared asdescribed by Boorman & Linstead (1935) and purified bydebromination of the crystallized 2,3-dibromocyclohexane-carboxylic acid, which had m.p. 167-169° (reportedm.p. 167°).The preparation of 2-oxocyclohexanecarboxylic acid

(m.p. 75-76°, decomp.; reported m.p. 79-81°, decomp.) andits reduction to 2-hydroxycyclohexanecarboxylic acid(m.p. 109-110°; reported m.p. 1110) were as described byGardner, Perkin & Watson (1910).

trans-4-Hydroxycyclohexanecarboxylic acid was a giftfrom Dr F. Dickens, Dr N. R. Campbell and J. H. Hunt.

Radiochemicals. [ring-U-14C]Benzoic acid (specific radio-activity 395 1lc/mg.) and [carboxy-14C]benzoic acid (specificradioactivity 160,uc/mg.) were obtained from The Radio-chemical Centre, Amersham, Bucks. These were purified bytwo-dimensional t.l.c. on silica gel G developed by solvents(a) and (c) (for short-exposure experiments), or solvent (e) inboth directions (for isotope-dilution experiments). Thebenzoic acid spot was located by radioautography, closelycut out, eluted and taken up into a few millilitres of 0-05M-tris-HCl buffer, pH7.2, and used, undiluted, immediatelyor on the following day.

Solvents. Solvents used for extraction or preparativepurposes were generally redistilled. Ether was redistilledover 'reduced iron'; ethanol was refluxed for 6hr. overNaOH pellets (40g./I.) and Zn dust (20g./l.) and thendistilled.

ChromatographyAnalytical chromatography was performed by t.l.c.

according to the methods of Randerath (1963). The glassplates (20cm. x 20 cm.) were coated to a thickness of

526 1969

AROMATIC METABOLISM BY R. PALUSTRIS0-25mm. and the following systems were used. With silicagel G (E. Merck A.-G., Darmstadt, Germany): (a) benzene-methanol-acetic acid (45:8:4, by vol.) and (b) (45:8:2, byvol.); (c) toluene-ethyl acetate-formic acid (5:4: 1, by vol.)and (d) (15:6:1, by vol.); (e) benzene-dioxan-acetic acid(45:5:2, by vol.); (f) light petroleum (b.p. 40-600)-diethylether-acetic acid (50:50:1, by vol.) and (g) (80:20:1, byvol.); (h) light petroleum (b.p. 40-60°)-diethyl ether (9:1,v/v); (i) methanol-chloroform-water (20:75:3, by vol.) and(j) (25:65:4, by vol.); (k) ethanol-aq. NH3 (sp.gr. 0-88)-water (25:1:6, by vol.) and (1) (16:1:3, by vol.); (m) butan-1-ol-ethanol-water (7:1:2, by vol.).With cellulose powder (Whatman Chromedia): (n)

phenol-water (18: 7, w/v); (o) butan-1-ol-acetic acid-water(4:1:5, by vol.); (p) 2-methylbutan-2-ol-5M-formic acid(1:1, v/v); (q) benzene-acetic acid-water (125:72:3, byvol.); (r) sodium formate (5%, w/v)-formic acid (200:1,v/v); (s) ethanol-aq. NH3 (sp.gr. 0-88)-water (16:1:3, byvol.).The following compounds were detected by the spray

reagents: acids by 0-1% Bromocresol Green in ethanol(Lugg & Overall, 1948); phenolic compounds by diazotizedp-nitroaniline or sulphanilic acid followed by 10% (w/v)Na2CO3 (Smith, 1960); x-carboxypyridines by 3% (w/v)FeSO4; phosphates by a method based on one described byFeigl (1966). The plates were sprayed with ammoniummolybdate [5% (w/v) in aq. 35% (v/v) HNO3], dried at 700,sprayed again with benzidine-HCI [0.05% in aq. 10% (v/v)acetic acid], dried at room temperature, and sprayed withsaturated aq. sodium acetate. Phosphate-containing spotswere deep blue.

Radioactive areas on chromatograms were located byradioautography. The t.l.c. plate was carefully placeddirectly on to the X-ray film (Ilfex No-Screen X-ray film;Ilford Ltd.) in a light-proof holder. The chromatogramwas marked in two or three places with radioactive ink tofacilitate the location of the radioactive areas on thechromatogram after the film had been developed (PhenisolX-ray developer; Ilford Ltd.).

Experimental procedures

Photoasimilation experiments. Experiments designed tofollow the disappearance of aromatic substrates in bufferedcell suspensions anaerobically in the light were normallyperformed in 100ml. conical flasks equipped with gas inletand outlet tubes, the latter being led into a 20 cm. head ofwater to provide a slight positive pressure within thesystem. The vessels were shaken in a Warburg apparatusfitted with a Perspex water bath at 30°, illuminated frombeneath by a bank of 40w tungsten lamps (B. Braun,Melsungen, W. Germany).

Short-exposure experiments with radioactive benzoate. Theapparatus for short-exposure experiments was similar tothat described by Knight (1962). The vessel was constructedfrom a 100ml. medical flat bottle, the neck of which wasattached by a thick rubber tube to an assembly of inlet andoutlet tubes for a cooling system and gas. The outflowing gaswas led through a trap containing m-NaOH (50ml.) andinto a cylinder of water about 30 cm. in depth. A hole wascut into the bottom of the bottle to accommodate a tap(4mm. bore) for taking samples, and a second hole, throughwhich additions were made, was cut into the shoulder. Theflask was illuminated from the front by a 500w Photoflood

lamp (Phillips Electrical Ltd., London) placed 20cm. fromthe vessel. The contents of the vessel were stirred vigorouslyby a magnetic stirrer revolving on the back wall, andthe temperature was maintained at 340 by regulation ofthe flow rate of tap water through the cooling system.Aluminium reflectors were constructed round the vessel,and these also acted as shields to prevent light shining onthe dial of a top-loading balance placed directly under thesampling tap to gauge the approximate weight ofthe sampletaken during the experiment.

Cells grown photosynthetically on benzoate, suspendedin 100ml. of 0-05M-tris-HCI buffer, pH7-2 (4-6mg. drywt./ml.), were equilibrated in the light under a rapid flowofN2 (02-free) for 10min. Benzoate (50ttLmoles) was addedand was allowed to disappear over the next 32-38min.; thispreincubation period enabled the system to metabolizemuch smaller amounts of benzoate immediately and rapidlywith the result that the specific radioactivity of the labelledbenzoate was not diminished by dilution with unlabelledbenzoate. At 42 min. a further 2-5,umoles of benzoate wereadded, followed 2 min. later by 140-150juc of [14C]benzoate.Five samples (approx. 20g.) were taken over thenext 60sec., these being run directly into 250ml. conicalflasks containing 90 ml. of ethanol at -20° to arrest meta-bolic activity. Each sample was weighed, and all com-parisons between samples were made on the basis of radio-activity per unit weight of sample.

After storage overnight at -20° the samples were warmedup to 500; the insoluble matter was sedimented by centri-fugation and extracted by shaking with 80% (v/v) ethanol(lOOml.) at room temperature for 60min., and again with96% (v/v) ethanol (lOOml.) for a further 60min. Theinsoluble material was designated 'cell debris'. The extractswere combined and concentrated under reduced pressure ina rotary evaporator (temp. <350) to a volume of about10ml. With the removal of the ethanol a quantity ofpigmented lipids formed a fine precipitate. This wassedimented by centrifugation at 105000g for 60min. at 20and washed by shaking with 20ml. of water for 60min.followed by centrifugation again; the pellet was designated'lipid fraction'. The supernatant and the washings werecombined, acidified to pH < 3 with 0-5 ml. of 75% formicacid and extracted three times with 35ml. of ether. Thecombined ether extracts were shaken three times with30ml. of4% NaHCO3; the ether-soluble neutral compounds('EN fraction') remained in the ether phase whereas theether-soluble acids ('EA fraction') were extracted into theNaHCO3 phase. The NaHCO3 extract was then acidified(pH < 3) with 1-5M-H2SO4 and the EA fraction extractedback into ether (three times with 70ml. of ether). Theaqueous phase from the first ether extraction was furtherextracted at pH < 3 with ether for 24hr. in a liquid-liquid-extraction apparatus, the ether extract from this beingdesignated the 'E24 fraction' and the aqueous phase the'W fraction'.The whole of each EA fraction was prepared for quantita-

tive application to a chromatogram. To avoid loss ofvolatilecompounds during the evaporation of the large volume ofether, which is significant when only trace quantities arepresent, the free acids were converted into their non-volatileammonium salts by aerating for 5 min. with N2 that hadbeen passed through aq. NH3 (sp.gr. 0-88). The ether wasthen evaporated under reduced pressure and the residueacidified with 0-2 ml. of 1-5M-H2SO4; the flask was washed

Vol. 113 5'27

P. L. DUTTON AND W. C. EVANSwith small volumes of ether until free from radioactivematerial. The washings (<lOmi.) were combined, con-centrated to a volume suitable for chromatography on awarm-water bath and all applied to a silica gel G t.l.c.plate, which was developed two-dimensionally by solvent (a)followed by solvent (c). Each E24 fraction was treatedsimilarly although the treatment with N2+NH3 wasomitted, since there were no significant losses of radio-activity during evaporation of the ether. The EN fractionswere not, after preliminary studies, investigated bychromatography. Each W fraction was evaporated underreduced pressure in a rotary evaporator to a volume of1-2ml. A portion of this equivalent to about 3g. of theoriginal cell suspension was resolved into three main areasby t.l.c. on cellulose powder developed unidirectionally bysolvent (n). The frontal area (Rp 0-65- 1-0) that ran abovethe 'tris area' was eluted and separated further by two-dimensional t.l.c. on cellulose powder developed by solvents(p) and (o). The area from the origin to the 'tris area' wasdesignated 'phosphate area'.

I8otope-diltution experiments. Experiments were per-formed in the same equipment used for the photoa#simila-tion experiments described above. Cells grown photo-synthetically on benzoate were suspended in 15ml. of0-OSM-tris-HCI buffer, pH 7-2 (8-9mg. dry wt./ml.). After10min. equilibration, with illumination under an atmo-sphere ofargon, [14C]benzoate (51tc, 22,umoles) was addedtoeach flask followed by 0-5mg. of a possible intermediate inthe unlabelled form. After a further 90min. each cellsuspension was centrifuged, the supernatant acidified topH < 3 with 1-5M-H2SO4 and extracted three times with15ml. of ether. The combined extracts were treated in thesame way as the EA fraction in the previous section, followedby two-dimensional t.l.c. on silica gel G developed in bothdirections by solvent (e). With the less-stable compoundsthe chromatographic procedure was omitted; isolation wasaccomplished by addition of more carrier to the extractfollowed by crystallization of the compound or a suitablederivative.

RESULTSUnder anaerobic photosynthetic conditions,

cultures grew on benzoate, m-hydroxybenzoateand p-hydroxybenzoate, but not on o-hydroxy-benzoate, protocatechuate or nicotinate. Thegrowth rate was highest on benzoate, and slightlyhigher on m-hydroxybenzoate than on p-hydroxy-benzoate. The organism did not grow on benzoate,o- and m-hydroxybenzoate and catechol underaerobic conditions, but grew rapidly on p-hydroxy-benzoate and protocatechuate.

Aerobic metabolism of p-hydroxybenzoate

Sequential-induction data. The oxygen con-sumption by R. palu8tris grown aerobically onp-hydrioxybenzoate with various substrates isshown in Table 1. p-Hydroxybenzoate and proto-catechuate were immediately oxidized without aninduction period, but none of the other compoundsstimulated oxygen uptake above that of the

Table 1. Oxygen consumption with various substratesby R. palustris grown aerobically on p-hydroxy-benzoate

Each Warburg flask contained, in 3-Oml. of 0-05M-phosphate buffer, pH7-2: cells, approx. 8mg. dry wt./ml.;substrate (added from side arm), 3,umoles. The centre wellcontained 0-2ml. of 20% (w/v) KOH. Temperature, 30°;atmosphere, air.

SubstrateNoneCatecholBenzoate2,4-Dihydroxybenzoateci8-is-Muconatep-HydroxybenzoateProtocatechuate

02 consumed (p1.)

After 10min.2014151917

11786

After 20min.3931373740

221155

endogenous uptake. Cells grown aerobically onacetate only oxidized p-hydroxybenzoate or proto-catechuate after a lag period.

Oxidation of protocatechuate by crude ceU-freeextracts. Extracts from cells grown aerobically onp-hydroxybenzoate, supplemented with Fe2+(1mM), did not oxidize p-hydroxybenzoate, butreadily oxidized protocatechuate with the con-sumption of 1 ,umole of oxygen/,mole of substrate.The cell-free extracts, prepared by disruptingsuspensions (25-35mg. dry wt./ml.) in 0-05m-phosphate buffer, pH 7-2, at 0-4' in a Mullard 60wultrasonic generator for 20min., followed bycentrifugation at 250OOg for 20min. at 20, were aclear pink from the presence of pigmented particles,probably chromatophore fragments. Removal ofthe particles by further centrifugation at 105 OOOgfor 60min. at 20 produced a colourless supernatantofsimilar activity to the 25 OOOg supernatant, whichshowed that the enzyme is soluble.During the oxidation of protocatechuate the

reaction mixture became yellow. This colourpersisted after the oxygen uptake had ceased, andfaded over the following 20-30min. without furtheroxygen uptake. If the extracts were dialysed for18 hr. at 0-4' against tap water and supplementedwith Fe2+, the yellow compound accumulated. Thematerial, yellow at neutral and high pH values andcolourless at low pH values, possessed spectralproperties identical with those described by Dagley,Evans & Ribbons (1960) for y-carboxy-m-hydroxy-muconic semialdehyde. This structure was con-firmed by its property of cyclizing in the presenceof NH4+ to form a compound possessing spectraland chromatographic properties and colour re-actions that were identical with those of 2,4-lutidinic acid. These findings show that the enzymeresponsible for ring-fission was a protocatechuate4,5-oxygenase.

528 1969

AROMATIC METABOLISM BY R. PALUSTRIS

With undialysed crude cell-free extracts the dis-appearance of protocatechuate and the ring-fissionproduct was accompanied by the transient accumu-lation of an acid that produced a blue-green colourin the Rothera (1908) test, suggesting the presenceof an a-oxo acid. The accumulation was made morecomplete by the addition of sodium arsenite(Dagley, Chapman, Gibson & Wood, 1964). The2,4-dinitrophenylhydrazone of the oxo acid wasprepared from a reaction mixture that contained, in8 ml. of 0-05 M-phosphate buffer, pH 7-2: crude cell-free extract (55mg. of protein), Fe2+ (1 mM), sodiumarsenite (5mM) and 30,umoles of protocatechuate(added in three 10,umole amounts, the second andthird additions being made after the disappearanceof the yellow ring-fission compound). The producthad RF values similar to those of the 2,4-dinitro-phenylhydrazone of authentic pyruvic acid, andmixtures of the two derivatives were not resolvedby one-dimensional t.l.c. on silica gel G developedby solvents (c), (1) and (m).

Cell-free extracts, with added Fe2+, prepared fromcells grown aerobically on acetate or photosynthetic-ally on p-hydroxybenzoate were barely active onprotocatechuate.

120 180Time (miii.)

Fig. 1. Photoassimilatioii of various substrates by R.paluatri8 grown photosynthetically on benzoate. Eachflask (100ml.) contained, in 30ml. of 0-05m-tris-HCI buffer,pH7-2; cells, 5-6mg. dry wt./ml. and substrate, 20,umoles.Temperature, 300; atmosphere, argon. 0, Benzoate;0, o-hydroxybenzoate; A, m-hydroxybenzoate; o, p-hydroxybenzoate; El, 2,3-dihydroxybenzoate; A, proto-catechuate; V, catechol and the other dihydroxybenzoates.

Photometaboli8m of benzoate and other aromaticcompound8

Photoassimilation of aromatic compound8. Theability of cells grown photosynthetically on

benzoate to photoassimilate various aromaticcompounds is shown in Fig. 1. The following were

utilized in order of decreasing rates: benzoate,m-hydroxybenzoate, o- and p-hydroxybenzoateand protocatechuate. Catechol was not utilizedover the 4hr. experimental period; nor were any

of the other dihydroxybenzoates except 2,3-dihydroxybenzoate, the uptake of which ceasedafter a period of slow assimilation. Treatment ofthe cell suspensions with chloramphenicol (100 pug./ml.), an inhibitor of protein synthesis, for 15min.under argon before the addition of the substrates,affected the rates of assimilation of neither themonohydroxybenzoates nor benzoate, indicatingthat the enzymes involved were already present inthe system.When grown photosynthetically on p-hydroxy-

benzoate, the cells were able to photoassimilate themonohydroxybenzoates and benzoate at similarrates whereas protocatechuate disappeared more

slowly (Fig. 2a). Grown photosynthetically on

m-hydroxybenzoate, the organism was able toutilize benzoate and to a smaller extent proto-catechuate (Fig. 2b).

Cells grown photosynthetically on acetate as

carbon source were barely active on benzoate,m-hydroxybenzoate and p-hydroxybenzoate.

Effect8 of air and darkne8s on the photoa&oimilationof benzoate. Buffered suspensions of cells, grownphotosynthetically on benzoate, were allowed toassimilate benzoate photosynthetically for 45min.,at which time air was admitted to two flasks. Oneflask was left in the light and the other was removedto another water bath at the same temperature butin the dark. After 40min. both flasks were returnedto photosynthetic conditions by flushing withargon and vigorously shaking each vessel for a fewseconds. The results (Fig. 3) show that admissionof air in the dark or the light caused an immediatecessation of benzoate utilization. Return tophotosynthetic conditions allowed the assimilationof benzoate to proceed unimpaired. A third flaskwas subjected to a period of darkness underanaerobic conditions, during which time no ben-zoate was metabolized. On return of the flaskto photosynthetic conditions recovery was un-expectedly slow. It was also shown that when theorganism was maintained anaerobically in the darkin the absence of benzoate for 40min. added ben-zoate was photometabolized normally; but, whenbenzoate was added before the anaerobic-darkperiod, benzoate photometabolism was initiallyimpaired. At present there is no explanation for thiseffect.

Short-expo8ure experiment8. Experiments wereperformed to follow the time-course of the photo-assimilation of benzoate. The patterns of uptakeof [ring-U-14C]benzoate (ring experiment) and[carboxy-14C]benzoate (carboxy experiment) were

Vol. 113 15. C

P. L. DUTTON AND W. C. EVANS

20

l 5

I0.- 10

iQSce5

0 20 40 60 80 100 0

Timie (min.)60 120

Fig. 2. Photoassimilation of various substrates by R. palwstris grown photosynithetically on (a) p}-hydroxy-benzoate or (b) m-hydroxybenzoate. Details were as given in Fig. 1.

I--

OD

4)

0

4)

0 20 40 60 80 100 120 140

Time (min.)

Fig. 3. Effects of air in the light and in the dark and ofdarkness under anaerobic conditions on the photometa-bolism of benzoate by R. palustri8. Each flask (250ml.)contained, in 60ml. of 0O05M-tris-HCl buffer, pH7-2: cells,"6mg. dry wt./ml.; benzoate, 55,tmoles. Temperature,30°; atmosphere, argon. In the period between the arrows

(45-85min.) air was admitted to two flasks, one flask (o)being kept in the light and the other (A) in the dark; thethird flask (E) was maintained in the dark under argon.

compared. In both experiments there was rapidincorporation of 14C into the cell debris and Wfraction, with a relatively low influx into the lipidfraction and the E24 fraction. Incorporation of14C into the EN fraction was also low in both

experiments and the presence of a small amount oflipid, not removed during the centrifugation pro-cedure, probably accounted for the major part of it;the addition of catechol (in the ring experiment) tothe ether as a carrier followed by shaking withsaturated lead acetate solution, which removed thecatechol into the aqueous phase, caused no signifi-cant decrease in the radioactive content ofthe etherphase, indicating the absence of labelled catechol.Chromatography of the EA fraction revealed four

radioactive areas with RF values lower than thebenzoic acid spot, but which contained only littleradioactivity. The E24 fraction also comprised twomain areas of activity similar to those of the EAfraction. The series of radioautograms of the twofractions from the ring and carboxy experimentswere similar. Co-chromatography of the separatedcomponents of the EA fraction with all the mono-hydroxybenzoic acids and dihydroxybenzoic acidsshowed no coincidence of the labelled compoundswith those areas developed by diazotized p-nitro-aniline, which proved the absence of radioactivehydroxybenzoic acids.

Resolution of the W fraction by cellulose t.l.c.developed by solvent (n) produced three main areas(Fig. 4a): area I, containing phosphates coincidentwith radioactive areas; area II, containing the'tris'; area III, in which there was heavy and earlyincorporation of labelled material, most of whichran close to the solvent front. The components ofarea III were resolved as shown in Fig. 4(b), theseries of radioautograms prepared from the ringand carboxy experiments being again strikingly

530 1969

AROMATIC METABOLISM BY R. PALUSTRIS

similar. The major area A was composed of twoclose-running spots, and in these experiments wasassayed as one; it was clear, however, from theradioautograms that the two components werepresent in approximately equal amounts through-out the experimental period.Each radioactive area was carefully removed,

eluted into water and assayed; the remaining areaswere also assayed so that the total radioactivity oneach chromatogram could be determined. Theradioactivity of each area was expressed as a

(a) (b)

o A

~~~~~j 2 Methylbutan-2-oI-formic acid-water

Fig. 4. Radioautograms of the separation by cellulose t.l.c.Of labelled water soluble compounds from R. palustri8 ex-posed to [ring U '40]benzoate for 14sec. with illuminationunder nitrogen. (a) Separation of W fraction into areas I,II and III; solvent (n). (b) Further separation of area III,solvents (p) and (o).

so

50Pb 404._

0CacSo 30ac0,4)*+ 20

I0P010

percentage of the total radioactivity, phosphatearea included, and this relative radioactivity plottedagainst time, as shown for the two experiments inFig. 5(a) and 5(b). In both analyses the curves ofthe assayed spots behaved similarly; area Apossessed negative slopes, which shows that thefirst detectable water-soluble compound to beformed from benzoate is present in the area; thecurves of C both rose to a peak and then fell away,slopes characteristic of early intermediates. Thelow relative radioactivities of the B and D areasobscured any effect of this kind.Under the experimental conditions with low

concentrations of substrate of high specific radio-activity, the intermediary pool sizes of the water-soluble compounds were generally much larger thanthose of compounds that were soluble in ether(Table 2).

Isotope-dilution experiments. The identificationof labelled compounds possibly involved in theearly reactions of benzoate photometabolism wasachieved by examining the supernatants of bufferedcell suspensions that had been photometabolizinghigh concentrations of [14C]benzoate for 90min. inthe presence of suspected intermediates (0.5mg.)acting as carrier. Each carrier compound was testedfor incorporation of 14C from [carboxy-14C]benzoate,except with 2-oxocylohexanecarboxylic acid when[ring-U-14C]benzoate was used.

Cyclohexanecarboxylic acidwas separated (Fig. 6)by chromatography and was shown to contain14C. Further two-dimensional t.l.c. on silica gel G

Time (sec.)

Fig. 5. Time-course of incorporation of (a) [ring-U-14C]benzoate and (b) [carboxy-14C]benzoate by illuminatedsuspensions of R. palustris into W fraction. At zero time 140-150/LC of [14C]benzoate was added to lOOml. of0 05M-tris-HCl buffer, containing cells, 5-6mg. dry wt./ml., and unlabelled benzoate, 2-5/cmoles. Temperature,340; atmosphere, nitrogen. Components: o, A; A, B; l, C; A, D; *, 'phosphate area'.

Vol. 113 531

P. L. DUTTON AND W. C. EVANS

Table 2. Time-course of incorporation of [ring-U-14C]benzoate by illuminated 8uspen8ions of R.palustris into major components of ether-soluble(EA plus E24) and water-soluble (W) fractions

Experimental details are given in Fig. 5.

10-3 X Incorporation of 14C (c.p.m./g. ofcell suspension)

Time(sec.)6

14-5233652

Major components of Compounds A, B, CEA+ E24 fractions and D ofW fraction

11-411-49-87-86-1

56-272-776-669-1

101-0

Fig. 6. Separation by silica-gel t.l.c. of the compoundslabelled in the isotope-dilution experiments. Details are

given in Table 4. The components were: E, trans-2-hydroxy-cyclohexanecarboxylic acid; F, unknown; G, pimelic acid;H, benzoic acid; I, 'reduced benzoic acids'.

developed by solvent (d) followed by solvent (k)and by solvent (h) followed by solvent (i) failed toresolve the radioactivity from the acid; the RFvalues of cyclohex- 1 -ene- 1 -carboxylic acid on thesesystems, however, are similar to those of cyclo-hexanecarboxylic acid. The radioactive materialwas diluted with 532mg. of cyclohexanecarboxylicacid, whichwas converted into the amide (m.p. 186-1870; reported m.p. 185-186°) and crystallizedfrom water, ethanol, ethanol-water and ethanol-ether to constant specific radioactivity (Table 3).The value was about 25% of that expected ifall the labelled material isolated was associatedwith cyclohexanecarboxylic acid.

Cyclohex-l-ene-l-carboxylic acid was similarlyseparated (Fig. 6) and the area it occupied on the

chromatogram was shown to be strongly andcoincidently labelled. The material was dilutedwith 950mg. of cyclohex-l-ene-l-carboxylic acidand from this the amide (m.p. 129-131°; reportedm.p. 127-128°) and the trans-2-bromocyclohexane-carboxylic acid (m.p. 109'; reported m.p. 1080) wereprepared. After repeated crystallization of theamide from water and ethanol, and of the hydro-bromide from light petroleum (b.p. 60-80°) mixedwith benzene, ethanol or ether, both compoundshad the same molar specific radioactivity, thevalue being about 75% of that expected if all theradioactivity isolated was due to cyclohex-l-ene-carboxylic acid (Table 3).

Cyclohex-2-ene-1-carboxylic acid was, owing toits instability, not separated by chromatography.A 300mg. sample of the acid was added directly tothe ether extract, the ether was evaporated and theacid dissolved in a little chloroform. A slightexcess of bromine was added to form 2,3-dibromo-cyclohexanecarboxylic acid (m.p. 167-169°; re-ported m.p. 1670), which was thoroughly washedwith water and crystallized from 90% formic acidto constant specific radioactivity. As shown inTable 3, the specific radioactivity was lower thanthat of either of the above compounds, the totalradioactivity being about 6% of that located in thecyclohexane-l-carboxylic acid.

Cyclohex-3-ene-1-carboxylic acid was treatedsimilarly and was brominated to give 3,4-dibromo-cyclohexanecarboxylic acid (m.p. 810; reportedm.p. 850). After four crystallizations the compoundcontained no detectable radioactivity.

trans - 2 - Hydroxycyclohexanecarboxylic acid,separated by chromatography (Fig. 6) from theother ether-soluble material, was coincident with aradioactive area, and further two-dimensional t.l.c.on silica gel G developed by solvent (c) followed bysolvent (1), andby solvent (j) followed bysolvent (b),failed to resolve the carrier and radioactivity.trans - 2 - Hydroxycyclohexanecarboxylic acid(200mg.) was added and this was crystallized toconstant specific radioactivity from ether andbenzene, and afterwards its p-bromophenacyl ester(m.p. 139-140°) was crystallized from ethanol,ethanol-water, ethanol-benzene and methanol;both compounds possessed the same molar specificradioactivity, the value of which (Table 3) showedthat all the radioactivity isolated with the carrierwas trans-2-hydroxycyclohexanecarboxylic acid.

2-Oxocyclohexanecarboxylic acid (300mg.) wasadded directly to the ether extract. The acidiccompounds were separated from any labelledneutral compounds such as cyclohexanone byextraction with 4% sodium hydrogen, carbonatefollowed, after acidification, by extraction back intoether. The ether was evaporated and the acidmixture heated at 85° for 20min. to decarboxylate

u H

o

qE

u

B d ad.+ OBezn-ixn-ctcai

532 1969

AROMATIC METABOLISM BY R. PALUSTRIS

cis 00~~~00

0

0 ~~ ~ ~ ~ ~ ~

00 aq

0 -~~~~~~~~~~~~~~041~ --CO

444 c~~~~0 0 10~~~~~~~~~cl ~ 00atmDE-i ~~~~~COI~ `

0

~60

4))

00

)'10 00

Ca~~ ~ ~~ 0

0 a 1-4 0 Ca

p.

co4) (L" (") '41

NN

04 Ca4

~~~~~~~~~~~~~~~~~~~~~~~~~~~C

0

104

44~~~~~on

0~~~~~~~~~~~~~

0~~~~~~~~~~~~~~~~~7'

co C .-c

oN 0 0

Ca "A1~4~4~ 0 doce

)~~~~

-~~~~~~c

0 0 C.4 co ~ ~

P-Q, e~4 N C)

-b -k 0 0coco Ca

~~ ~~ ~ ~~~~~~~~~~0 00oi.

Vol. 113 533

P. L. DUTTON AND W. C. EVANSthe 2-oxocyclohexanecarboxylic acid to formcyclohexanone, from which cyclohexanone thio-semicarbazide was prepared (m.p. 159-160;reported m.p. 165-167°); the thiosemicarbazide ofauthentic cyclohexanone and a mixture of bothpreparations also had m.p. 159-160°. The crudeproduct was thoroughly washed with water andthen crystallized to constant specific radioactivity.As shown in Table 3, the value was low but theradioactive compound co-crystallized with thecyclohexanone thiosemicarbazide from water,ethanol, ethanol-water and benzene.

Pimelic acid was separated by chromatography(Fig. 6) and was found to be coincidently labelled.Further two-dimensional t.l.c. on silica gel Gdeveloped by solvent (c) followed by solvent (k),and by solvent (j) followed by solvent (f), and oncellulose powder developed by solvent (o) followedby solvent (s), and by solvent (q) followed bysolvent (t), did not separate radioactivity from thepimelic acid. Further pimelic acid (200mg.) wasadded to the radioactive material and was crystal-lized from water, benzene and benzene-ethanol, andafterwards its di-p-bromophenacyl ester (m.p. 137-1380; reported m.p. 136-137°) was prepared andcrystallized from ethanol, benzene, ethanol-benzeneand chloroform; both compounds possessed thesame molar specific radioactivity, the value ofwhich showed that virtually all the radioactivityisolated was associated with pimelic acid (Table 3).

Neither 2-oxopimelic acid (isolated from thereaction mixture as the 2,4-dinitrophenylhydra-zone) nor trans-4-hydroxycyclohexanecarboxylicacid, as shown by radioautography, was detectablylabelled.

Attempted preparation of cell-free extracts active onbenzoate. Suspensions (40-50mg. dry wt./ml.) ofcells grown photosynthetically on benzoate in 10ml.of 0 05M-tris or 0.05M-phosphate buffer, pH7*2,were disrupted at 0-4° in a Mullard 60w ultrasonicgenerator for 10min. or in a Hughes (1951) press.The disrupted cell suspensions were centrifuged at25 OOOg for 15min. at 20 to remove the cell debris,producing a clear deep-red supernatant. Additionof the supernatant to a vessel illuminated undernitrogen at 300 and containing 20,umoles of ben-zoate in 20ml. of 0-05M-tris or 0-05M-phosphatebuffer, pH 7 2, failed to cause any detectable break-down of benzoate. The addition of various com-binations of Fe2+, Mg2+, NADH, ATP and GSHmade no difference. Extracts that had beenprepared under nitrogen were also inactive, evenwhen supplemented with these cofactors, as werethe preparations that were added directly afterthe ultrasonic treatment or still frozen from theHughes press, with the centrifugation procedureomitted. Extracts from which the chromatophorefragments had been removed by centrifugation

under nitrogen at 105 OOOg for 90min. at 20 andsupplemented with Fe2+, Mg2+ and ATP did notdissimilate benzoate.

It was subsequently found that the addition ofsmall amounts of disrupted cell suspensions towhole cells actively photometabolizing benzoatecaused a drastic decrease in the rate of benzoateutilization (Dutton & Evans, 1968a).

DISCUSSION

Aerobic metaboliam of p-hydroxybenzoate. Theonly aromatic compounds found capable ofsupport-ing aerobic growth of R. palustri8 were p-hydroxy-benzoate and protocatechuate; similar observationswith strains of the same species have been describedby Leadbetter & Hawk (1965) and Hegeman (1967).The intermediates shown to be involved in theaerobic metabolism of p-hydroxybenzoate areconsistent with the conversion of p-hydroxy-benzoate into protocatechuate, its cleavage by aprotocatechuate 4,5-oxygenase (Dagley et al. 1960)to form y-carboxy-a-hydroxymuconic semialde-hyde, and with subsequent breakdown involvingpyruvate. The presence of p-hydroxybenzoate (orpresumably protocatechuate) and oxygen wasobligatory for the induction of protocatechuate4,5-oxygenase.Hegeman (1967) has reported that extracts from

a strain of R. palustris grown aerobically on p-hydroxybenzoate were capable of oxidizing y-carboxy-a-hydroxymuconic semialdehyde in anNADP-dependent step to the correspondingmuconic acid. This reaction is analogous to the onedescribed by Nishizuka, Ichiyama, Nakumura &Hayaishi (1962) in which extracts from a Pseudo-monas sp. grown on catechol oxidized a-hydroxy-muconic semialdehyde to oc-hydroxymuconic acidin an NAD-dependent step; the reaction, however,contrasts with the pathway described by Dagleyet al. (1964), who showed that extracts from anotherPseudomonas sp. converted y-carboxy-ac-hydroxy-muconic semialdehyde into y-hydroxy-y-methyl-oc-oxoglutarate and formate in the absence ofeither NAD or NADP.

Photometabolism, of benzoate and other aromaticacids. In earlier studies of the photometabolism ofbenzoate by a Rhodopseudomonas sp., Proctor &Scher (1960) reported that this organism, grownphotosynthetically on benzoate, was capable, inthe dark, of the aerobic oxidation of benzoate viaprotocatechuate, catechol and an a-oxo acid. Theinference was made (Proctor & Scher, 1960) thatmolecular oxygen could replace a proposed photo-synthetically generated oxidant, assumed to beinvolved in the photometabolism of benzoate.Evidence that is in conflict with these proposals maybe summarized as follows.

534 1969

AROMATIC METABOLISM BY R. PALUSTRIS

(a) The oxygen uptake exhibited by R. palu8tri8grown photosynthetically on benzoate, m-hydroxy-benzoate or p-hydroxybenzoatc when incubatedwith various compounds did not reflect the abilityof the organism to assimilate the same compoundsunder photosynthetic conditions. Irrespective ofthe compound utilized for growth, an oxygenuptake was observed only with p-hydroxybenzoateand protocatechuate, significantly the only com-pounds able to support aerobic growth of thisorganism (Dutton & Evans, 1967). (b) Proto-catechuate 4,5-oxygenase, induced during aerobicgrowth on p-hydroxybenzoate, was virtually absentwhen the organism was grown photosyntheticallyon this substrate. (e) The photometabolism ofbenzoate was totally inhibited by oxygen. (d) Underphotosynthetic conditions, cells grown photo-synthetically on benzoate did not utilize catecholand utilized protocatechuate only slowly. Proto-catechuate also failed to support photosyntheticgrowth of the organism. (e) The short-exposureexperiments with [carboxy-14C]benzoate and [ring-U-14C]benzoate indicated that early decarboxyla-tion does not occur. Patterns of incorporation of14C from the ring and carboxyl group of benzoatewere very similar; further, release of the carboxylgroup as carbon dioxide would probably haveresulted in a higher incorporation of radioactivityinto the 'phosphate area' in the experiment with[carboxy-14C]benzoate (see Knight, 1962). Meta-bolites such as catechol or any of the monohydroxy-or dihydroxy-benzoates were not detected inthese experiments.

Isotope-dilution experiments have led to theidentification of several compounds from which thepathway shown in Scheme 1 can be proposed for thephotometabolism of benzoate by R. palu8tri8. Thisconstitutes a completely new method of aromaticring metabolism: the aromatic nucleus is firstreduced, producing an aliphatic cyclic acid, and itappears that ring-fission and subsequent meta-bolism is accomplished by a sequence similar to thef-oxidation of fatty acids. Benzoate is reducedto cyclohex- 1-ene-1-carboxylate (I). Hydrationproduces 2-hydroxycyclohexanecarboxylate (II),which is dehydrogenated to form 2-oxocyclohexane-carboxylate (III), and hydration of this results inring-cleavage and the formation of pimelate (IV).Strong evidence has been presented for the identi-fication of compounds (I), (II) and (IV), whereasradioactive 2-oxocyclohexanecarboxylate was pre -sent in such small quantities that the specificradioactivity of the carrier was rather low; however,after its conversion into cyclohexanone, ninecrystallizations of the thiosemicarbazide failed toremove the radioactivity. Labelled cyclohex-2-ene-1 -carboxylate and cyclohexanecarboxylate werealso detected, buit in smaller quantities than the

CO2H CO2H

N 4H4 H20

(I)

C02H

C02H

CO2H

W~~~~~~~~~~.,

535C(02H

OH

(II)

S, 2H

CO2H

OH

(IV) (III)Scheme 1. Proposed pathway for the photometabolism ofbenzoic acid by R. palu8tri8.

cyclohex-1-ene-1-carboxylate (6% and 22%respectively). Neither the nature of the firstintermediate nor the roles of cyclohex-2-ene- 1-carboxylate and cyclohexanecarboxylate can bedecided with any certainty from these experiments;however, it seems reasonable to suppose that thefirst intermediate is a cyclohexadienecarboxylate,possibly cyclohexa- 1 ,5-diene- 1 -carboxylate, sincecyclohex-3-ene- 1 -carboxylate was not detectablylabelled and cyclohex-2-ene-1-carboxylate mayarise as a side product by reduction of the diene inthe a-position. Similarly cyclohexanecarboxylatemay be a side product, although it is also possiblethat this compound is a precursor ofcyclohex-1 -ene-1-earboxylate, benzoate being completely reducedfollowed by specific dehydrogenation in thea-position. Future work concerned with the identi-fication of the water-soluble compounds, especiallythe components of area A (Fig. 5b; short-exposureexperiments), and the unknown ether-soluble acid(Fig. 6; isotope-dilution experiments), and furtherstudies with cell-free extracts, should lead to aclarification of the early reductive reactions andalso those subsequent to pimelate formation. So far,attempts to prepare cell-free extracts capable ofbenzoate breakdown have failed. This may beconnected with the observation (Dutton & Evans,1968a) that long-chain fatty acid componentsassociated with the pigmented particles in the cellextract, but not completely removed from theextract with the particles, inhibited the photo-assimilation of benzoate by whole cells. Theinhibition appeared to be specific to aromaticphotometabolism, there being no effect on eitherphotosynthetic growth on acetate or malate, or theaerobic utilization of p-hydroxybenzoate. It isknown that long-chain fatty acids inhibit a varietyof metabolic reactions, including electron transport,but, since saturated fatty acid homologues as lowas butyrate and propionate (but not acetate) are

Vol. 113

,5)36 P. L. DUTTON AND W. C. EVANS 1969effective inhibitors (P. L. Dutton & W. C. Evans,unpublished work), the cause may be duie tocompetition for cofactors, such as CoA, requiired byboth fatty acid and benzoate metabolismn.

In the aromatization of cyclohexanecarboxylateto benzoate by liver enzymes, Babior & Bloch (1966)showed that this reversal of benzoate reduction alsoinvolves cyclohex-l-ene-l-carboxylate, but cyclo-hex - 2 - ene - 1 - carboxylate and cyclohex - 3 - ene -l-carboxylate did not appear to participate directlyin the aromatization reactions. It was also shownthat the intermediates are present in the form oftheir CoA thiol esters.

Since the anaerobic breakdown of benzoateappears to be closely associated with the reactionin the light, the reduction of aromatic substratesby R. paluatri8 may be catalysed by reductasescoupled to a low-redox-potential component of thelight-induced electron-transport system, such asferredoxin. The enzymes involved in the photo-metabolism of aromatic acids are inducible andappear to lack substrate-specificity, as shown by theability of the organism grown photosyntheticallyon benzoate or m- or p-hydroxybenzoate to photo-assimilate a variety of related compounds. Cellsgrown on p-hydroxybenzoate, for example, wereable to utilize all the monohydroxybenzoates andbenzoate at similar rates.

This novel reductive pathway of aromatic ringdissimilation, employed by R. palustri8 anaerobic-ally in the light, contrasts with the well-knownoxidative mechanisms ofthe obligate aerobic micro-organisms. It seems reasonable to consider thatfermentation of aromatic compounds by non-photosynthetic anaerobic micro-organisms, espec-ially the methanogenic bacteria (Clark& Fina, 1952;Roberts, 1962), might also be accomplished by areductive method of aromatic metabolism.

P. L. D. is grateful to the Ministry of AgricultureFisheries and Food, and the Scientific Research Council, forfinancial support. We also thank Dr M. Knight of thisDepartment, and Dr. G. D. Hegeman and Mr. M. Guyer ofthe University of California at Berkeley, whose workusing different approaches has also indicated a reductivepathway, for helpful discussions during the preparation ofthe paper.

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