JOURNAL OF BACrERIOLGY, May 1970, p. 430-437Copyright a 1970 American Society for Microbiology

Anaerobic Degradation of the Benzene Nucleus bya Facultatively Anaerobic Microorganism1

BARRIE F. TAYLOR,' WILLIAM L. CAMPBELL,' AND IRA CHINOYDepartment of Microbiology, The University of Texas at Austin, Austin, Texas 78712

Received for publication 16 February 1970

A bacterium was isolated by elective culture with p-hydroxybenzoate as substrateand nitrate as electron acceptor. It grew either aerobically or anaerobically, by nitraterespiration, on a range of aromatic compounds. The organism was identified as apseudomonad and was given the trivial name Pseudomonas PN-1. Benzoate andp-hydroxybenzoate were metabolized aerobically via protocatechuate, followed bymeta cleavage catalyzed by protocatechuic acid-4,5-oxygenase, to yield a-hydroxy-'y-carboxymuconic semialdehyde. Pseudomonas PN-1 grew rapidly on p-hydroxy-benzoate under strictly anaerobic conditions, provided nitrate was present, eventhough protocatechuic acid-4,5-oxygenase was repressed. Suspensions of cellsgrown anaerobically on p-hydroxybenzoate oxidized benzoate with nitrate andproduced 4 to 5 umoles of CO2 per Mmole of benzoate added; these cells did notoxidize benzoate aerobically. The patterns of the oxidation of aromatic substrateswith oxygen or nitrate by cells grown aerobically or anaerobically on different aro-matic compounds indicated that benzoate rather than protocatechuate was a keyintermediate in the early stages of anaerobic metabolism. It was concluded that thepathway for the anaerobic breakdown of the aromatic ring is different and quitedistinct from the aerobic pathway. Mechanisms for the anaerobic degradation of thebenzene nucleus by Pseudomonas PN-1 are discussed.

The aerobic degradation of aromatic com-pounds by microorganisms has been studied indetail by several groups of investigators (2, 5, 12).Molecular oxygen is required for cleavage, andfor most aromatic molecules molecular oxygen isalso required in the preparation of the benzenering for cleavage. Oxygen is incorporated directlyinto the degradative products and is essential forthe catabolism of aromatic compounds by thesemechanisms. The breakdown of aromatic com-pounds by microorganisms under anaerobic con-ditions has received, in comparison, little con-sideration. Tarvin and Buswell (18), 26 years ago,obtained from the sludge of anaerobic digestiontanks of sewage plants mixed cultures of micro-organisms which fermented a range of aromaticcompounds to a mixture of CO2 and CH4. Clarkand Fina (1), using mixed cultures from a similarsource, confirmed that benzoate was degraded toa mixture of CO2 and CH4. Fina and Fiskin (6)

'Contribution No. 1182 from the Institute of Marine andAtmospheric Sciences, Rosentiel School of Marine and Atmos-pheric Sciences, University of Miami.

2Present address: Rosensteil School of Marine and Atmos-pheric Sciences, University of Miami, Miami, Fla. 33149.

8 Present address: Department of Microbiology, SouthwesternMedical School, Dallas, Tex.

used mixed cultures enriched from rumen fluidor anaerobic digestion tanks, and proved that14CH4 was produced from benzoate-J-14C addedduring fermentative growth on benzoate. Notting-ham and Hungate (11) have recently confirmedthis observation using a methanogenic enrich-ment culture from sewage sludge. Mixed cultures,with or without prior exposure to benzoate,formed 14CO2 and 14CH4 from benzoate uniformlylabeled with 14C in the aromatic ring.Some members of the family Athiorhodaceae

use benzoate as a carbon source during photo-heterotrophic growth. The anaerobic metabolismof benzoate by Rhodopseudomonas palustris,which grows either aerobically in the dark oranaerobically in the light on p-hydroxybenzoate(pHBz), has been investigated by several groupsof workers (4, 8, 9; A. Hawk and E. R. Lead-better, Bacteriol. Proc., p. 22, 1965). The an-aerobic photometabolism of benzoate involves areduction of the aromatic ring to a cyclohexanederivative prior to cleavage (4, 8).The present investigation was initiated with a

search for microorganisms which might growanaerobically, by nitrate respiration, on aromaticcompounds. By enrichment culture techniques

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VOL. 102, 1970 ANAEROBIC DEGRADATION OF THE BENZENE NUCLEUS

with pHBz as a substrate, a bacterium was iso-lated which grew either aerobically or anaerobi-cally on several aromatic compounds. Its facul-tative nature made the organism ideal for acomparison of the aerobic and anaerobic modesof degradation of aromatic compounds. The com-parison revealed that the anaerobic degradativepathway was quite distinct and independent ofthe aerobic pathway. Mixed cultures of bacteriahave been reported to grow anaerobically bynitrate respiration on aromatic compounds (13),but this is the first report of a pure culture of amicroorganism possessing this ability.

MATERIALS AND METHODSIsolation and cultivation of the organism. The me-

dium (adjusted to pH 8.5 with NaOH) contained:Na2HPO47H20, 7.9 g; KH2PO4, 1.5 g; NH4CI, 0.3 g:KNO3, 2.0 g; MgSO4.7H20, 0.1 g; trace metal solu-tion (19), 5.0 ml; and water to 1 liter. The tracemetals and magnesium sulfate were sterilized sep-arately. KNO3 was omitted for aerobic cultivation.Neutralized solutions of aromatic compounds wereadded at a final concentration of 5 mm. Media wereusually autoclaved, but solutions of thermolabilecompounds, e.g., catechol and protocatechuate, weresterilized by filtration (mean pore size, 0.45 jim;Millipore Corp., Bedford, Mass.).The organism was isolated from soil by an enrich-

ment technique. Screw-cap tubes (capacity, 20 ml)were filled with anaerobic medium, containing pHBzas substrate, and were inoculated with soil samples.After several weeks of incubation at 30 C in the dark,one tube became turbid and vigorously evolved a gas.After several transfers through liquid anaerobicmedium, the culture was streaked on solid anaerobicmedium and incubated in a desiccator (desiccantomitted). The desiccator was evacuated and contained,in a small beaker, chromous chloride solution to re-move residual oxygen. A pure culture was obtainedby repeated streaking of isolated colonies, and finallya single colony was reinoculated into liquid anaerobicmedium.The culture was maintained on pHBz at 30 C in

screw-cap tubes filled with liquid anaerobic mediumand was subcultured every 1 to 3 days. Stock cultureswere preserved as stabs in solid anaerobic medium at5 C. Inocula (1%, v/v) grown anaerobically on pHBzwere used to seed bulk cultures. Cells were grownaerobically in 500-ml portions of medium (con-tained in 2-liter Erlenmeyer flasks) with rotary shak-ing at 30 C. Crops of cells for manometric studieswere grown anaerobically at 30 C in 500-ml screw-capbottles filled with medium. Cell-free extracts of cellsgrown anaerobically were prepared from culturesgrown under strictly anaerobic conditions. Prior toincubation, these cultures were gassed for 15 min withargon which had been passed through chromouschloride solution and hot copper wire to removetraces of oxygen.

Cells were harvested, in the later stages of exponen-tial growth, by centrifugation at 16,000 X g for 20

min at 5 C and then were washed twice in one-tenththe culture volume of a cold buffer containing thephosphate constituents of the medium adjusted topH 8.0 with NaOH (T3 buffer). The washing bufferfor cells grown anaerobically, and destined foranaerobic manometric studies, was supplemented with0.2% (w/v) KNO. Cells were either resuspended inT3 buffer for manometric experiments or stored at-20 C for later preparation of cell-free extracts.Manometry. The oxidation of aromatic substrates

was measured at 30 C by use of a Warburg apparatusfitted with a gassing manifold (Gilson Medical Elec-tronics, Middleton, Wis.). Warburg flasks contained:tris(hydroxymethyl)aminomethane (Tris)-hydrochlo-ride buffer (pH 8.0), 100 jAmoles; KNOs, 40.umoles;aromatic substrate, 10 jAmoles; cell suspension (in 1.0ml of T3 buffer) or cell-free extract; and water to 3.0ml. KNO3 was omitted in aerobic experiments.The center well contained 0.10 ml of20% (w/v) KOH,but in experiments to measure CO2 production thiswas omitted and 0.20 ml of 2 N H2S04 was tipped in,from a side arm, at the end of the reaction to liberatebound CO2. After equilibration, the substrates andKNO3 were added from a side arm at zero time. Inanaerobic experiments, the flasks were gassed for 10min with argon.

Cell-free extracts. Cells were suspended, in theratio of 1 g (wet weight) to 3 to 5 ml of 0.1 M Trisbuffer (pH 8.0), and, after the addition of an equalweight of Ballotini beads, were disrupted by exposureto ultrasonic oscillations, for 5 min at 5 C, from anultrasonic disintegrator (Measuring and ScientificEquipment, London, England). Suspensions of dis-integrated cells were centrifuged at 20,000 X g for 20min at 5 C to prepare a crude extract. The crude ex-tract was then centrifuged at 96,000 X g for 2 hr at5 C, and the supernatant, or "soluble," fraction wasretained. The "soluble" fraction usually contained5 to 10 mg of protein per ml.

Enzymatic assays. Standard procedures were usedto assay catechol-2,3-oxygenase (3) and protocate-chuic acid-4,5-oxygenase (3) with a Gilford Model2000 multiple sample recording spectrophotometer(1-cm light path). Each cuvette contained: Trisbuffer (pH 8.0), 150 pmoles; catechol or sodiumprotocatechuate, 0.5 jumole; various amounts of"soluble" extract; and water to 3.0 ml. The substratewas added last. The formation of a-hydroxy-y-car-boxymuconic semialdehyde (HCSMA) was followedat 410 nm, and the production of a-hydroxymuconicsemialdehyde, at 373 nm. A molar extinction coeffi-cient of 2.90 X 104 cm2 per mole was used to calcu-late the amount of HCMSA formed (3). Specificactivities were expressed as micromoles of HCMSAformed per minute per milligram of protein.

Other methods. Nitrite was estimated by diazotiza-tion with sulfanilic acid and coupling with a-naph-thylamine (15). pHBz was measured by the methodof Folin and Ciocalteau (7). Protein was determinedby the method of Lowry et al. (10), with bovineserum albumen as the standard. Spectra of products,in 0.4 N HCI or 0.4 N NaOH, from manometric andcell-free extract experiments were measured against

431

TAYLOR, CAMPBELL, AND CHINOY

water by use of a Cary model 14 recording spectro-photometer.

RESULTSDescription of the organism. The organism was

a small, short, motile, gram-negative rod, almostovoid in shape. Cells were often paired and, es-pecially during aerobic growth, chains of pairedcells were sometimes observed. Cells grownaerobically were larger than those grown an-aerobically. Motility was characteristic of polarflagellation, and a positive result was obtainedwith the N,N'-dimethyl-p-phenylenediamine oxi-dase test (17). On the basis of these characteris-tics and because of its facultative denitrifyingability, the organism was tentatively identified asa pseudomonad and was given the trivial namePseudomonas PN-1. Under anaerobic conditions,colonies on pHBz-agar were small (1 to 2 mm indiameter) and beige in color, but after 2 to 3weeks of incubation they turned yellow and in-creased in size (3 to 4 mm in diameter). Coloniesformed under aerobic conditions were of a similarsize but were white in color. Aerobically oranaerobically grown colonies were circular andraised, and had sharp margins.

Nitrate was essential for the anaerobic growthof the organism on pHBz. Either in the light ordark, no growth occurred on media lackingKNO3 or on a malate-yeast extract mediumsuitable for the propagation of purple nonsulfurbacteria. Isolated colonies of Pseudomonas PN-1grown anaerobically on pHBz plates were inocu-lated into aerobic liquid pHBz medium. Thesecultures grew after aerobic incubation but re-tained their potential for anaerobic growth. Thebuoyant density of deoxyribonucleic acid (DNA)extracted from aerobically and anaerobicallygrown cultures of Pseudomonas PN-1 was deter-mined by the CsCl density gradient method (14).The density of the DNA in both cases was iden-tical at 1.726 g/cc, corresponding to a guanineplus cytosine content of 67.3%. No satellitebands ofDNA were detected. Pseudomonas PN-1had a generation time of 4 hr during anaerobicgrowth on pHBz. Growth was proportional topHBz utilization, and nitrite accumulated duringthe early stages of exponential growth (Fig. 1).Nitrite later disappeared, and a gas, presumablynitrogen, was evolved. Nitrite accumulated to atoxic level if the concentration of KNO3 wasraised from 0.2 to 0.5% (w/v).Growth experiments. Pseudomonas PN-1 grew,

both aerobically and anaerobically, after 1 to 2days incubation on pHBz, benzoate, and m-hy-droxybenzoate (mHBz). The organism also de-veloped rapidly on protocatechuate under an-

E2.

z

i

E

1I0 I-Ih2ri2

14 Is 22 35HOURS

FIG. 1. Anaerobic growth of Pseudomonas PN-1 onp-hydroxybenzoate-nitrate medium. Symbols: 0,p-hydroxybenzoate used (,moles/ml); E0, cell protein(ptg/mi); A, nitrite formed (pmoles/ml).

aerobic but not aerobic conditions. After a lag of9 to 11 days, growth under both conditions alsooccurred on hydrocinnamic acid but only anaero-bically on o-hydroxybenzoate (oHBz). No growthoccurred, either aerobically or anaerobically, onphenol, catechol, benzyl alcohol, 6-hydroxynico-tinic acid, cyclohexane carboxylate, cyclohex-3-ene carboxylate, or a mixture of cis and transisomers of 1 :2-cyclohexane diol.

Aerobic metabolism of aromatic compounds.Cells grown aerobically on pHBz oxidized pHBzand protocatechuate, but not benzoate, catechol,mHBz, oHBz, or cyclohexane carboxylate (Table1). The oxidation of protocatechuate produced ayellow intermediate which absorbed maximallyat 410 nm in alkali but was colorless in acid.These spectral properties are characteristic ofHCMSA which is produced by meta cleavage ofprotocatechuate (3). ,r-Ketoadipate, an inter-mediate on the pathway from ortho cleavage ofprotocatechuate, was not detected either insupernatant fractions from cell suspension experi-ments or in the spent media from cells grownaerobically on pHBz. "Soluble" cell fractions ofcells grown aerobically on pHBz oxidized proto-catechuate with the consumption of about equi-molar amounts of 02, but produced no CO2(Table 2). The "soluble" cell fraction containedprotocatechuic acid-4, 5-oxygenase, and theproduct from spectrophotometric assays exhibiteda spectrum, in acid and alkali, identical with thatof HCMSA. These results were consistent with ameta cleavage mechanism of protocatechuate.

Cells grown aerobically on mHBz oxidizedmHBz, protocatechuate, and pHBz, but not

432 J. BACTERIOL.

VoL. 102, 1970 ANAEROBIC DEGRADATION OF THE BENZENE NUCLEUS

TABLE 1. Aerobic oxidation of aromatic sub-strates by Pseudomonas PN-1 grown aero-bically on different aromatic compounds"

Growth substrateb

Substratep-Hydrox- m-Hydrox- Bensoateybenzoate ybenzoate

None .............5...1 5c 1.7 2.1p-Hydroxybenzoate... 2.7 2.5 10.6Protocatechuate. 6.4 3.9 9.0Benzoateo. 1.7 1.7 6.0Catechol. 1.3 1.3 1.8m-Hydroxybenzoate.. 1.7 8.6 20.5o-Hydroxybenzoate... 1.6 1.3 2.9Cyclohexane carbox-

xylate.1.4 NT NT

aWarburg flasks contained in 3.0 ml: 100,umolesof Tris buffer (pH 8.0), cells, 10 ,moles of sub-strate, 0.10 ml of 20%/ KOH (center well), and airas the gas phase.

b The amounts of cells were as follows: p-hydroxy-benzoate, 5.12 mg of protein per Warburg flask; m-hydroxybenzoate, 4.37 mg of protein per Warburgflask; benzoate, 2.22 mg of protein per Warburg flask.

- Micromoles of 02 consumed during incubationat 30 C for 60 min after the substrates were tippedin. NT = not tested.

catechol, benzoate, or oHBz (Table 1). A yellowintermediate accumulated during the oxidation ofprotocatechuate, and presumably mHBz wasmetabolized via protocatechuate with meta cleav-age to HCMSA.

Cells grown aerobically on benzoate oxidizedmHBz, pHBz, protocatechuate, and benzoate,but not catechol or oHBz (Table 1). A yellowintermediate was formed during the oxidation ofprotocatechuate. A "soluble" extract from cellsgrown aerobically on benzoate contained proto-catechuic acid-4,5-oxygenase, but not catechol-2, 3-oxygenase.

Anaerobic metabolism of aromatic compounds.Cells grown anaerobically on aromatic com-pounds oxidized aromatic substrates with either02 or KNO5. This provided a convenient meansof comparing both aerobic and anaerobic modesof metabolism of cells grown anaerobically ondifferent aromatic compounds. Cells grownanaerobically on pHBz oxidized, with 02, bothpHBz and protocatechuate, but not benzoate(Table 3). A yellow intermediate, indicative ofmeta cleavage, accumulated during protocate-chuate oxidation. Under anaerobic conditions,these cells oxidized pHBz and benzoate but notprotocatechuate (Table 3). Catechol and oHBzwere not oxidized with either 02 or KNO3.

Alicyclic derivatives (cyclohexane carboxylate, amixture of cis and trans isomers of 1:2-cyclo-hexane diol, 3-cyclohexene carboxylate) were notoxidized under anaerobic conditions with KNOi.

Cells grown anaerobically on benzoate oxidizedwith 02 a range of aromatic compounds similarto those oxidized by cells grown aerobically onbenzoate (Table 4). The oxidation of protocate-chuate again produced a yellow intermediate.However, under anaerobic conditions, these cellsoxidized only benzoate (Table 4). pHBz wasoxidized with KNO3 after a long lag period (1.5to 2 hr), but protocatechuate was never oxidizedunder anaerobic conditions.A different pattern of substrate oxidation with

02 or KNO3 was also evident with cells grownanaerobically on protocatechuate (Table 5). Cellsincubated aerobically oxidized protocatechuate

TABLE 2. Stoichiometry of 02 uptake and C02production from protocatechuate (PCA) by a

"soluble" extract of cells grown aerobicallyon p-hydroxybenzoatea

PCA Total 02 Total C02 02 consumed C02 evolvedadded uptake evolved per umole per jmole

of PCAb of PCAbi.moles pmolcs pmoles Pmolcs pmoles

0 0.00 2.95 4.5 3.1 0.9 0.010 8.6 3.0 0.9 0.015 12.8 3.3 0.9 0.0

a Warburg flasks contained in 3.0 ml: Tris buffer(pH 8.0), 100 Amoles; "soluble" extract, 3.71 mg ofprotein; and PCA as indicated.

b Endogenous (no substrate) values subtracted.

TABLE 3. Oxidation of aromatic compounds with02 or KNO3 by Pseudomonas PN-1 grown

anaerobically on p-hydroxybenzoatea

Substrate 02 consumedb N2 evolvedc

pmoles pmolcsNone................ 3.2 0.6p-Hydroxybenzoate .... 32.4 20.0Protocatechuate ..... . 20.8 0.5Benzoate .............. 4.9 17.1m-Hydroxybenzoate.... 5.9 0.5Catechol .............. 3.0 0.5

a Warburg flasks contained in 3.0 ml: Tris buffer(pH 8.0), 100 ;moles; cells, 4.62 mg of protein; sub-strate, 10;moles; 20%o KOH (center well), 0.10 ml;and KNO3 (anaerobic flasks), 40 pmoles.

b Gas phase, air; 60 min of incubation at 30 Cafter tipping in substrates.

Gas phase, argon; 60 min of incubation at 30 Cafter tipping in substrates.

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TAYLOR, CAMPBELL, AND CHINOY

TABLE 4. Oxidation of aromatic compounds with02 or KNO3 by Pseudomonas PN-I grown

anaerobically on benzoatea

Substrate 02 consumedb N2 evolved'

pmoks pmoles

None................ 55.6 1.9Benzoate .............. 7.8 11.5p-Hydroxybenzoate .... 17.3 3.0Protocatechuate ....... 8.0 1.7m-Hydroxybenzoate.... 8.9 2.2o-Hydroxybenzoate .... 7.9 2.6Catechol .............. 4.0 2.1

a Warburg flasks contained in 3.0 ml: Tris buffer(pH 8.0), 100 jAmoles; cells, 4.12 mg of protein; sub-strate, 10,umoles; 20%o KOH (centerwell), 0.10 ml.;KNO3 (anaerobic flasks), 40,umoles.

b Gas phase, air; 90 min of incubation at 30 Cafter tipping in substrates.

c Gas phase, argon; 90 min of incubation at 30 Cafter tipping in substrates.

TABLE 5. Oxidation of aromatic compounds with02 or KNO3 by Pseudomonas PN-1 grown

anaerobically on protocatechuatea

Substrate 02 consumed5 N2 evolvedc

,moles JAmolesNone................ 1.7 2.6Protocatechuate ....... 8.5 8.1Benzoate .............. 2.7 16.6p-Hydroxybenzoate .... 4.9 8.0m-Hydroxybenzoate.... 3.9 9.2o-Hydroxybenzoate .... 3.4 4.3Catechol .............. 1.2 2.1

a Warburg flasks contained in 3.0 ml: Tris buffer(pH 8.0), 100 ,moles; substrate, 10 ,umoles; 20%KOH (center well), 0.10 ml; and KNO3 (anaerobicflasks), 40 j;moles.

b Gas phase, air; cells, 0.99 mg of protein; incu-bated for 60 min at 30 C.

c Gas phase, argon; cells, 6.02 mg of protein;incubated for 60 min at 30 C.

much faster than benzoate, but the reverse situa-tion applied under anaerobic conditions. Theoxidation of protocatechuate with 02 but notwith KNO3, resulted in the formation of a yellowintermediate.

Cells grown anaerobically on mHBz oxidizedaerobically pHBz, mHBz, and protocatechuatemore rapidly than benzoate. Under anaerobicconditions, however, benzoate, pHBz, and mHBzwere oxidized more rapidly than protocatechuate(Table 6). A yellow intermediate was againformed only during the aerobic oxidation ofprotocatechuate.

Cells grown anaerobically on pHBz oxidized

benzoate with KNO3 and evolved 4 to 5 ,umolesof CO2 per ,umole of benzoate (Table 7). Benzo-ate was not oxidized aerobically by cells grownanaerobically on pHBz, thus eliminating the pos-sibility of interference from aerobic metabolism.Enzyme levels. The level of protocatechuic acid-

4, 5-oxygenase in "soluble" extracts of cellsgrown on pHBz was considerably higher inaerobic cells (specific activity, 0.52) than inanaerobic cells (specific activity, 0.01).

DISCUSSIONPseudomonas PN-1 grew either aerobically or

anaerobically, by nitrate respiration, on a rangeof aromatic substrates. Cells grown aerobicallyon pHBz oxidized protocatechuate, forming ayellow intermediate with spectral propertiescharacteristic of HCMSA. "Soluble" extracts ofthese cells contained significant levels of proto-

TABLE 6. Oxidation of aromatic compounds with02 or KNO3 by Pseudomonas PN-l grown

anaerobically on m-hydroxybenzoatea

Substrate 02 consulmedb N2 evolved'

jumoles umoles

None................ 3.5 2.2m-Hydroxybenzoate 9.6 5.5Protocatechuate ....... 9.8 4.5Benzoate .............. 6.7 5.8p-Hydroxybenzoate ... 11.8 5.7o-Hydroxybenzoate ... 5.9 4.4Catechol .............. 3.0 2.3

a Warburg flasks contained in 3.0 ml: Tris buffer(pH 8.0), 100 jAmoles; substrate, 10 jsmoles; 20%0KOH (center well), 0.10 ml; and KNO3 (anaerobicflasks), 40 jAmoles.

b Gas phase, air; 2.56 mg of cell protein; incu-bated for 90 min at 30 C.

c Gas phase, argon; 4.37 mg of cell protein incu-bated for 90 min at 30 C.

TABLE 7. Stoichiometry of CO2 and N2 productionfrom benzoate (Bz) oxidation with KNO3 by

Pseudomonas PN-J grown anaerobicallyon p-hydroxybenzoatea

Bz added Total N2 Total CO2 N2 evolved C02 evolvedevolved5 evolved5 perBmzol Bezml

Amoles jsmoks pmoles pmoles pmoles

1 3.8 5.5 3.8 5.52 6.8 9.6 3.4 4.83 8.5 16.3 2.9 5.4

a Warburg flasks contained in 3.0 ml: cell sus-pension, 6.60 mg of protein; Tris buffer (pH 8.0),100,umoles; KNO3, 40 tmoles; and Bz as indicated.

b Endogenous (no substrate) values subtracted.

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VOL. 102, 1970 ANAEROBIC DEGRADATION OF THE BENZENE NUCLEUS

catechuic acid-4, 5-oxygenase and catalyzed theoxidation of protocatechuate with the consump-tion of equimolar amounts of 02 without liberat-ing CO2. The aerobic metabolism of pHBz there-fore followed the well-established pathway involv-ing meta cleavage (3). Cells grown aerobically onbenzoate oxidized protocatechuate but notcatechol, and soluble extracts contained proto-catechuic acid-4,5-oxygenase but not catechol-2,3-oxygenase. The metabolism of benzoate viaprotocatechuate instead of catechol is unusual buthas been observed in Aspergillus nidulans (C. J.Shepherd and J. R. Villaneuva, J. Gen. Micro-biol. 20:vii, 1959) and Pseudomonas testosteroni(20). Cells grown aerobically on benzoate oxidizedboth pHBz and mHBz, and this may reflect abroad specificity for the mixed function oxi-dases involved in the formation of proto-catechuate. Alternatively, benzoate may induceseveral specific aromatic acid hydroxylases.The objective of the present work was to estab-

lish that the anaerobic metabolism of aromaticcompounds by Pseudomonas PN-1 was definitelyanaerobic. The aromatic ring, in the absence ofmolecular oxygen, must be disrupted by a mech-anism different from the known aerobic processes,and cell suspension experiments were designed toestablish this point. Differences in the aerobic andanaerobic metabolism of aromatic compoundswere most easily discerned with cells grown onbenzoate or pHBz (Table 8). Furthermore, cellsgrown anerobically on pHBz metabolized ben-zoate only under anaerobic conditions. The dif-ferences in the patterns of substrate oxidation,with 02 or KNOI, by cells grown on mHBz orprotocatechuate are not so clear. However,protocatechuate was always oxidized morerapidly than benzoate under aerobic conditions,whereas under anaerobic conditions the conversesituation existed. Protocatechuate oxidation withoxygen always produced a yellow intermediate(HCMSA), but this was never visible duringanaerobic metabolism of protocatechuate.The initial stages in the aerobic metabolism of

aromatic compounds by Pseudomonas PN-1 con-verge on protocatechuate, which is then con-verted to HCMSA by meta cleavage catalyzed by

TABLE 8. Summary of substrate oxidation, with 02or KNO3, by Pseudomonas PN-1 grown onbenzoate (Bz) or p-hydroxybenzoate (pHBz)

Growth Substrates oxi-substrate dized withONOS

Bz Bz Protocatechuate, pHBz,mHBz, Bz

pHBz Bz, pHBz Protocatechuate, pHBz

protocatechuic acid-4, 5 oxygenase; in contrast,the anaerobic pathway of metabolism convergeson benzoate (Fig. 2).The complete oxidation of benzoate with KNO3

can be expressed by the equation:

C7H602 + 6KNO3 -. 7C02 + 3N2 + 6KOH

Pseudomonas PN-1 grown anaerobically onpHBz oxidized benzoate with KNO3, with theevolution of 4 to 5 ,umoles of CO2 and about 3umoles of N2 per ,umole of benzoate. The libera-tion as CO2 of 60 to 70% of the carbon of ben-zoate indicated that the benzene ring had beendisrupted and degraded under anaerobic condi-tions.The repression of protocatechuic acid-4, 5-

oxygenase, under strictly anaerobic conditions,and the continued rapid growth of PseudomonasPN-1 on pHBz (mean generation time, 4 hr)further supports the idea that a pathway distinctfrom known aerobic mechanisms is operatingunder anaerobic conditions.The anaerobic growth by nitrate respiration of

mixed cultures of microorganisms on proto-catechuate was reported by Oshima (13). Anaero-bic growth on protocatechuate required a com-bination of a denitrifying pseudomonad and anonmotile, gram-negative isolate which had"rather anaerobic tendencies" (13). These or-ganisms produced N2 from nitrate while growingsingly on nonaromatic carbon sources but did notdevelop anaerobically on protocatechuate in pureculture. Cell-free extracts of mixed cultures,grown anaerobically on protocatechuate, pro-duced ,B-ketoadipate from protocatechuate duringanaerobic incubation with protocatechuate andKNO3. 180 incorporation from '80-labeledKNO3 was greater in the cells grown anaero-bically with protocatechuate than with succinate.

mHBz

benzoate

\\HBpHBzprotocatechuate -_ HCMSA

protocatechuate

pHBz -' benzoate -- CO1 + H,O

mHBzFIG. 2. Aerobic (upper) and anaerobic (lower) me-

tabolism ofaromatic compounds by Pseudomonas PN-1.mHBz and pHBz = m- and p-hydroxybenzoate;HCMSA = a-hydroxy--y-carboxymuconic semialde-hyde.

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TAYLOR, CAMPBELL, AND CHINOY

Oshima (13) concluded that, during the anaerobicgrowth on protocatechuate, the oxygen atoms ofnitrate were incorporated in a manner similar tothat for molecular oxygen in the aerobic pathwayinvolving an oxygenase, and that the aerobic andanaerobic pathways had common intermediates.The present work does not reveal the mode of

cleavage of the aromatic ring by PseudomonasPN-1 under anaerobic conditions, but it does indi-cate that the pathway is different from the aerobicroute of metabolism. Furthermore, the anaerobicpathway of degradation requires anaerobiosis forits operation because, for example, benzoate ismetabolized anaerobically but not aerobically bysuspensions of cells grown anaerobically onpHBz.The pathway of anaerobic degradation of

benzoate by R. palustris has recently been par-tially elucidated (4, 8). Benzoate is photometabo-lized with reduction to cyclohex-1-ene carboxylatefollowed by hydration to 2-hydroxycyclohexanecarboxylate. Dehydrogenation then yields 2-keto-cyclohexane carboxylate which is thiolyticallycleaved to pimelate. Dutton and Evans (4) sug-gested that methanogenic fermentation of aro-matic compounds may also proceed via reducedintermediates. This unifying hypothesis may wellextend to Pseudomonas PN-1. However, Pseudo-monas PN-1 did not grow on cyclohexane car-boxylate, and neither was this compound oxidizedwith KNO3 by cell suspensions grown anaerobi-cally on pHBz. Mutants of R. palustris blocked atvarious stages in the pathway of photometabolismof benzoate retained their potential for photo-synthetic growth on pHBz (8). Probably separategroups of enzymes are synthesized for the an-aerobic photometabolism of benzoate and pHBzby R. palustris. In contrast, our results suggestthat Pseudomonas PN-1 metabolizes pHBz viabenzoate. These differences indicate that a differ-ent pathway, and perhaps a different mechanism,operates in Pseudomonas PN-1. A reductivemechanism may not be involved; furthermore,such a mechanism raises the question of thesource of reducing power, which is presumablygenerated photochemically in R. palustris. Analternative mechanism may involve the additionof water to the aromatic ring (Fig. 3). This typeof mechanism was suggested by Stanier (16) asone of several possibilities for the aerobic metab-olism of aromatic compounds. The addition ofwater would form a polyhydroxy-derivative ofcyclohexane carboxylate which could then bedehydrogenated and thiolytically cleaved aspostulated in the pathway for R. palustris.

COCH COOH COOH

--- -

SENZOIC ACID ON OHTRIHYDROXY DIHYDROXYCYCLOHEXANE CYCLOHEXAN -2 -ONE- I -CARSOXYLIC ACID CAROOXYLIC ACID

+H,OCOOH

CO, + H,_04- _ COOHOH

DIHYDFIOXY PIMELIC ACID

POSITIONS OF HYDROXYL GROUPS ARE ASITRRILY ASIGNED

FIG. 3. Hypothetical scheme for the saturation ofthe aromatic ring and its subsequent cleavage byPseudomonas PN-1.

ACKNOWLEDGMENTS

This work was financed by grants from the Robert A. WelchFoundation and the National Science Foundation (GB-8173).We thank Derek S. Hoare for his interest and encouragement

during this investigation, and we also gratefully acknowledge theassistance of Margaret Hensley. We thank Manley Mandel, of theM. D. Anderson Hospital and Tumor Institute, Houston, Tex., forthe DNA analyses. Ira Chinoy was a participant in a programsupported by the National Science Foundation for talented highschool students.

LITERATURE CITED

1. Clark, F. M., and L. R. Fina. 1952. The anaerobic decomposi-tion of benzoic acid during methane fermentation. Arch.Biochem. Biophys. 36:26-32.

2. Dagley, S. 1967. The microbial metabolism of phenolics, p.287-317. In A. D. McLaren and G. H. Peterson (ed.), Soilbiochemistry. Marcel Dekker, New York.

3. Dagley, S., W. C. Evans, and D. W. Ribbons. 1960. New path-ways in the oxidative metabolism of aromatic compoundsby microorganisms. Nature (London) 188:560-566.

4. Dutton, P. L., and W. C. Evans. 1969. The metabolism ofaromatic compounds by Rhodopseudomonas palustris. A.new, reductive, method of aromatic ring metabolismBiochem. J. 113:525-536.

5. Evans, W. C. 1963. The microbiological degradation of aro-

matic compounds. J. Gen. Microbiol. 32:177-184.6. Fina, L. R., and A. M. Fiskin. 1960. The anaerobic decom-

position of benzoic acid during methane fermentation. II.

Fate of carbons one and seven. Arch. Biochem. Biophys.91:163-165.

7. Folin, O., and V. Ciocalteau. 1927. On tyrosine and trypto-phane determinations in proteins. J. Biol. Chem. 73:627-650.

8. Guyer, M., and G. Hegeman. 1969. Evidence for a reductivepathway for the anaerobic metabolism of benzoate. J. Bac-teriol. 99:906-907.

9. Hegeman, G. D. 1967. The metabolism of p-hydroxybenzoateby Rhodopseudomonas palustris and its regulation. Arch.Mikrobiol. 59:143-148.

10. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

11. Nottingham, P. M., and R. E. Hungate. 1969. Methanogenicfermentation of benzoate. J. Bacteriol. 98:1170-1172.

436 J. BACTERIOL.

VOL. 102, 1970 ANAEROBIC DEGRADATION OF THE BENZENE NUCLEUS

12. Ornston, L. N., and R. Y. Stanier. 1964. Mechanism of 6-keto-adipate formation by bacteria. Nature (London) 204:1279-1283.

13. Oshima, T. 1965. On the anaerobic metabolism of aromaticcompounds in the presence of nitrate by soil microorgan-isms. Z. Allg. Mikrobiol. 5:386-394.

14. Schildkraut, C. L., J. Marmur, and P. Doty. 1962. Deter-mination of the base composition of deoxyribonucleic acidfrom its buoyant density in CsCl. J. Mol. Biol. 4:430-443.

15. Smith, A. J., and D. S. Hoare. 1968. Acetate assimilation byNitrobacter agilis in relation to its "obligate autotrophy."J. Bacteriol. 95:844-855.

16. Stanier, R. Y. 1948. The oxidation of aromatic compounds byfluorescent pseudomonads. J. Bacteriol. 55:477-494.

17. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966. Theaerobic pseudomonads: a taxonomic study. J. Gen. Micro-biol. 43:159-271.

18. Tarvin, D., and A. M. Buswell. 1934. The methane fermenta-tion of organic acids and carbohydrates. J. Amer. Chem.Soc. 56:1751-1755.

19. Vishniac, W., and M. Santer. 1957. The thiobacilli. Bacteriol.Rev. 21:195-213.

20. Wheelis, M. L., N. J. Palleroni, and R. Y. Stanier. 1967. Themetabolism of aromatic acids by Pseudomonas testosteroniand P. acWdovorans. Arch. Mikrobiol. 59:302-314.

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