JOURNAL OF BACTERIOLOoY, June 1982, p. 1172-11820021-9193/82/061172-11$02.00/0

Vol. 150, No. 3

Metabolism of Cyclohexaneacetic Acid andCyclohexanebutyric Acid by Arthrobacter sp. Strain CAl

HELEN J. OUGHAM AND PETER W. TRUDGILL*Department ofBiochemistry, University College of Wales, Aberystwyth, Dyfed, SY23 3DD, United Kingdom

Received 17 November 1981/Accepted 3 February 1982

A strain of Arthrobacter was isolated by enrichment culture with cyclohexane-acetate as the sole source of carbon and grew with a doubling time of 4.2 h. Inaddition to growing with cyclohexaneacetate, the organism also grew withcyclohexanebutyrate at concentrations not above 0.05%, and with a variety ofalicyclic ketones and alcohols. Oxidation of cyclohexaneacetate proceededthrough formation of the coenzyme A (CoA) ester followed by initiation of a (-oxidation cycle. 3-Oxidation was blocked before the second dehydrogenation stepdue to the formation of a tertiary alcohol, and the side chain was eliminated asacetyl-CoA by the action of (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase. Thecyclohexanone thus formed was degraded by a well-described route that involvesring-oxygen insertion by a biological Baeyer-Villiger oxygenase. All enzymes ofthe proposed metabolic sequence were demonstrated in cell-free extracts. Arthro-bacter sp. strain CAl synthesized constitutive (-oxidative enzymes, but furtherinduction of enzymes active toward cyclohexaneacetate and its metabolites couldoccur during growth with the alicyclic acid. Other enzymes of the sequence, (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase and enzymes of cyclohexanone oxida-tion, were present at neglWble levels in succinate-grown cells but induced bygrowth with cyclohexaneacetate. The oxidation of cyclohexanebutyrate wasintegrated into the pathway for cyclohexaneacetate oxidation by a single (-oxidation cycle. Oxidation of the compound could be divided into two phases.Initial oxidation to (1-hydroxycyclohexan-1-yl)acetate could be catalyzed byconstitutive enzymes, whereas the further degradation of (1-hydroxycyclohexan-1-yl)acetate was dependent on induced enzyme synthesis which could be inhibitedby chloramphenicol with the consequent accumulation of cyclohexaneacetate and(1-hydroxycyclohexan-1-yl)acetate.

Cyclohexyl n-alkanes are components ofcrude oils. Their metabolism by bacteria is gen-erally initiated by hydroxylation of the terminalmethyl group to yield a primary alcohol, whichis subsequently oxidized to a fatty acid anddegraded by (-oxidation (1). Removal of acetyl-coenzyme A (CoA) units results in the formationof either cyclohexane carboxylic acid or cyclo-hexaneacetic acid (cyclohexaneacetate), de-pending on whether the hydrocarbon chain car-ries an odd or even number of carbon atoms (1).Cyclohexane carboxylic acid is not metabolical-ly recalcitrant, and many bacteria are capable ofutilizing it as a sole source of carbon and energy(1, 3, 4, 15, 24, 28). Degradation by (-oxidationhas been described (4, 24), but examples of analternative route involving hydroxylation at the4-position, dehydrogenation, and aromatizationto form p-hydroxybenzoate, which is then de-graded by well-described routes (7, 20), havebeen reported (3, 15, 28). Cyclohexaneacetatepresents a contrasting situation since the posi-

tion of the ring, relative to the carboxyl group,would be expected to block (3-oxidation.Perhaps as a consequence of this, previous

attempts to isolate organisms capable of growthwith cyclohexaneacetate have been unsuccess-ful (1), and the accumulation of cyclohexaneace-tate by microorganisms growing at the expenseof dodecylcyclohexane (C12 side chain) has beenreported (1).We have isolated a strain of Arthrobacter,

capable of growth with cyclohexane-acetic acidand cyclohexanebutyric acid as sole sources ofcarbon, which is also able to function as acomponent of a mixed culture system capable ofdegrading cyclohexyl n-alkanes with an evennumber of carbon atoms in the side chain (10).This paper presents evidence for a pathway ofcyclohexaneacetate oxidation (Fig. 1) suggestedin a preliminary report (21) and for the integra-tion of cyclohexanebutyrate metabolism intothis pathway by a single (-oxidation cycle cata-lyzed by constitutive enzymes.

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CYCLOHEXANE ACIDS METABOLISM BY ARTHROBACTER 1173

COO CO .SCoA CO .SCoA

CH2 ATP CH 2H ,2e CH H202 'bCoA2 '

0 ~~~~c0CYCLOHEXANEACETYL-CoA CYCLOIIXYLIDENE

ACRTYL-CoA

CO .SCoA

HO CH2

(1-lHYDROXYCYCLOIEXAN-1-YL)ACETYL-CoA

CH3CO.SCoA

HaO ° °AP2NADP

2 6-OZOIEXANOATI

NAD

NAD12

COO,'

6-HYDROXY-HIXANOATE

NADP6-CAPRW- 120LATW

FIG. 1. Pathway of oxidation of cyclohexaneacetate by Arthrobacter sp. strain CAl.

MATERIALS AND METHODS

Orpnisms. Arthrobacter sp. strain CAl was isolat-ed from field soil, contaminated with aviation fuel, byenrichment on mineral salts medium containing cyclo-hexaneacetate (0.1%) as the sole source of carbon.The organism was maintained on nutrient agar slants.Growth of . Liquid cultures of Arthrobacter

sp. strain CAl were obtained by inoculating mineralmedia of the following composition (grams/liter):KH2PO4, 2.0; Na2HPO4, 4.0; (NH4)2SO4, 1.0; andtrace element solution (25), 4 ml. Carbon sources weresupplied at 0.1% [cyclohexaneacetate, (1-hydroxycy-clohexan-1-yl)acetate, and adipatel, 0.05% (cyclohex-anebutyrate and octanoate), and 0.2% (succinate), andmedia were adjusted to pH 7.1 with 2.5 M NaOHbefore autoclaving. Volatile substrates (cyclohexanol,cyclohexanone, and related compounds) were sup-plied from diffusion tubes as previously described (5).The sequence of transfers used to grow batches ofcells was typically as follows. Medium (20 ml) in a 100-ml Erlenmeyer flask was inoculated with a loopful ofcells from a nutrient apr slant and grown with shakingfor 48 h at 30'C, and 10 ml of this was used to inoculate100 ml of medium in a 250-ml Erlenmeyer flask. Aftergrowth to late logarithmic phase, this was used in tototo inoculate 900 ml of medium in a 2-liter flask. Thisculture was grown to late logarithmic phase and eitherharvested or added to 10 liters of medium in a 14-literfermentor (New Brunswick Scientific Co., NewBrunswick, N.J.), supplied with air at 0.7 liter/min,and stirred at 250 rpm. Cultures were harvested eitherby centrifugation at 15,000 x g for 15 min (smallvolumes) or in an Alfa-Laval (Alfa-Laval Ltd., Mid-dlesex, U.K.) continuous-flow centrifuge at a flow rateof 0.5 to 0.8 liter/min at ambient temperature. The cellpaste was washed by suspension in 42 mM Na+K+-phosphate, pH 7.1, followed by further centrifugationat 15,000 x g, and the washed cell pellet was finallyresuspended in 1.5 vol of the same buffer and eitherused directly or stored at -18°C until required.

Culture dendty. Culture density was followed bymeasuring absorbance at 580 um and diluting samplesas necessary so that Asm never exceeded a value of1.0.

Cegl-ftee extracts. Cell-free extracts were preparedfrom suspensions of harvested cells in 42 mM Na+K+-phosphate buffer (1 g [wet weight] of cells per 1.5ml of buffer) by a single passage through a Frenchpressure cell (American Instrument Co., Silver Spring,Md.) with a pressure difference at the orifice of 138MPa. DNase (0.2 mg/ml) was added, and the superna-tant extract was obtained by centrifuation at 20,000 xg for 20 min at 4°C. Protein content was measured bythe modified biuret method (12), and cell extractstypically contained 10 to 15 mg of protein per ml.RePiraty Oxygen consumption was mea-

sured manometrically with constant-volume manome-ters (Braun, Melsungen, Germany) and polarographi-cally with a Clark oxygen electrode (Yellow SpringsInstrument Co., Yellow Springs, Ohio) at 30°C.Enzyme asys. Cyclohexanone 1,2-monooxygen-

ase, cyclohexanol dehydrogenase, 6-oxohexanoate de-hydrogenase, 6-hydroxyhexanoate dehydrogenase,and 1-oxa-2-oxocycloheptane (6-caprolactone) hydro-lase were assayed as previously described (19). Cyclo-hexaneacetyl-CoA synthetase was assayed by an ad-aptation of the method of Overath et al. (22) asmodified by Blakley (4). Cyclohexylideneacetyl-CoAdehydrogenase was determined by following the rateof 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazoliumbromide reduction at 545 nm or polarographically inthe presence of phenazine methosulfate, essentially asdescribed for fatty acyl-CoA dehydrogenases (19a). (1-Hydroxycyclohexan-1-yl)acetyl-CoA lyase, whichcleaves its substrate to form cyclohexanone and ace-tyl-CoA, was assayed routinely by following the de-crease in absorbance at 340 mm due to NADPHoxidation when 0.2 pmol of (1-hydroxycyclohexan-1-yl)acetyl-CoA was added to a 1-cm light-path cuvettethat contained, in a final volume of ml: 0.1 mmol ofTris-hydrochloride buffer, pH 8.0, 0.75 ,umol of

CYCLOHEXANEACETATE

cooI

ADIPATI CYCLOHEXANONI

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1174 OUGHAM AND TRUDGILL

NADPH, 20 ,u g of cyclohexanone 1 ,2-monooxygenasepurified from Nocardia globerula sp. strain CL1 (8),and test extract (0.5 to 2 mg of protein). Cyclohexyli-deneacetyl-CoA hydratase was detected by coupling itwith (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase andpurified N. globerula cyclohexanone 1,2-monoox-ygenase. Rates were determined by measuring thedecrease in absorbance at 340 nm when 0.05 ,umol ofcyclohexylideneacetyl-CoA was added to a 1-cm light-path cuvette that contained, in a final volume of 1 ml;80 pmol of Na+K+-phosphate buffer, pH 7.4, 0.25,umol of NADPH, 15 p,g of purified N. globerulacyclohexanone 1,2-monooxygenase (0.3 U), and testextract (0.1 to 1 mg of protein).Chromatography. Aldehydes and ketones were

identified by thin-layer chromatography of their 2,4-dinitrophenylhydrazone derivatives in: solvent A,light petroleum-diethyl ether (7:3 by volume); solventB, toluene-tetrahydrofuran (19:1 by volume); or sol-vent C, n-hexane-ethyl formate (7:3 by volume). Re-verse-phase paper chromatography on liquid paraffin-impregnated Whatman no. 4 chromatography paperwas performed by using the upper phase of solvent D,an equilibrium mixture of methanol-chloroform-water-liquid paraffin (20:20:12:3 by volume) (30).

Gas-liquid chromatography (GLC) of alicyclic alco-hols, ketones, and methyl esters of carboxylic acidswas performed on a Pye 104 gas chromatograph (Pye-Unicam Instruments Ltd., Cambridge, U.K.) fittedwith one of the following analytical columns: 1.25%diglycerol on Chromosorb W (1.9 m by 4 mm), 10%diethylene glycol succinate on Chromosorb W (1.3 mby 4 mm), or Porapak PS (1.3 m by 4 mm). Ajudiciouscombination of column choice, operating temperature,and carrier gas flow rate allowed all compounds ofpotential interest to be separated from each other.Gas htography and ma spectral analysis.

Analyses were performed on a Pye 104 gas chromato-graph equipped with a column of 1.5% diglycerol onChromosorb W coupled to an Associated ElectricalIndustries MS 30 mass spectrometer.

Synthesis of c ls. 6-Hydroxyhexanoic acid wasprepared by alkali hydrolysis of 6-caprolactone aspreviously described (19). (1-Hydroxycyclohexan-1-yl)acetic acid was routinely prepared by the method ofWatanabe et al. (30), using lithium-naphthalene tosynthesize the compound from cyclohexanone andacetic acid. (1-Hydroxycyclopentan-1-yl)acetic acid,(1-hydroxycycloheptan-1-yl)acetic acid, and, occa-sionally, (1-hydroxycyclohexan-1-yl)acetic acid wereprepared by the Reformatsky reaction as described byNatelson and Gottfried (18). Cyclohexylideneaceticacid was prepared from (1-hydroxycyclohexan-1-yl)-acetic acid by dehydration with acetic anhydride ac-cording to the procedure of Beesley et al. (2).

Synthesis of CoA esters. N-Hydroxysuccinimide es-ters of the cycloalkylacetic acids were prepared by themethod of Lapidot et al. (16) and recrystallized fromhot ethanol. CoA esters were then prepared from theN-hydroxysuccinimide esters as follows: 0.5 ml of 0.1M thioglycolic acid was added to 20 mg of reducedCoA in 0.7 ml of distilled water; solid NaHCO3 wasthen added to adjust the pH to 7.5 to 8.0, the reactionmixture was agitated by a gentle stream of nitrogen,and 150 p,mol of the N-hydroxysuccinimide ester,dissolved in 1.5 ml of tetrahydrofuran, was then add-ed. When free -SH groups were no longer detectable

(29), the N2 flow rate was increased to drive offtetrahydrofuran, and after cooling to 40C, insolubledebris was removed by centrifuging at 1,000 x g. ThepH of the CoA ester solution was adjusted to 6.5 with0.1 M HCI and stored at -18°C until required.

Chemicals. Chloramphenicol was obtained from Al-len and Hanburys Ltd., London, U.K. Cyclohexane-acetic acid and cyclohexanebutyric acid were suppliedby Aldrich Chemical Co., Milwaukee, Wis. CoA,NAD, NADH, NADP, and NADPH were supplied byBoehringer Corp., London, U.K. Cycloheptanone,cyclooctanone, and 2-hydroxycyclohexanone weresupplied by Ralph Emanuel, Wembley, U.K. Adipicacid, 6-caprolactone, cyclohexanol, and cyclopentan-one were supplied by Koch-Light Laboratories, Coln-brook, U.K. Acetyl-CoA, 3-[4,5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide, and S-succinyl-CoA were supplied by Sigma Chemical Co., St. Louis,Mo. 6-Oxohexanoic acid was a gift from N. A. Don-oghue, and cyclohexaneglycolic acid was a gift fromR. Howe of I.C.I. Pharmaceuticals, Alderley Park,U.K.Prit of chemicas. Cyclohexanone was purified by

redistillation, and the purity of cyclohexaneaceticacid, (1-hydroxycyclohexan-1-yl)acetic acid, and cy-clohexylideneacetic acid was assessed by GLC afterconversion to the methyl esters by the procedure ofMetcalfe and Schmitz (17).Biohemical supplies. Citrate:oxaloacetate lyase

(CoA acetylating; EC 4.1.3.7; citrate synthase), threo-D.-isocitrate:NAD oxidoreductase (decarboxylating;EC 1.1.1.41; iso-citrate dehydrogenase), and L-ma-late:NAD oxidoreductase (EC 1.1.1.37) were suppliedby Boehringer Corp. Bovine serum albumin (Cohnfraction V), citrate (isocitrate) hydrolyase (EC 4.2.1.3;aconitase), and DNase were supplied by Sigma.

RESULTSCyclohexaneacetate metabdism. (i) Growth ex-

periments. The mean generation time of Arthro-bacter sp. strain CAl at 30°C was 4.2 h in 0.1%cyclohexaneacetate mineral salts medium. Theyield of cells was approximately 1.5 g (wetweight)/g of substrate supplied. Strain CAl wasalso capable ofgood growth with a wide range ofalicyclic compounds including (1-hydroxycyclo-hexan-1-yl)acetate, (1-hydroxycycloheptan-1-yl)acetate, cyclohexylideneacetate, cyclohexanol,cyclohexanone, cycloheptanol, cycloheptanonecis, trans-cyclohexane-1,2-diol, 2-hydroxycy-clohexanone, and 2- and 3-methyl cyclohexan-ols. In addition, good growth was obtained with6-caprolactone, 5-valerolactone, 6-hydroxyhex-anoate, 6-oxohexanoate, pimelate, adipate, glu-tarate, acetate, and homogentisate. Growth withcyclohexanebutyrate and octanoate was ob-tained only at substrate concentrations of 0.05%or lower. It is of interest that strain CAl wasincapable of growth with n-alklanes, cyclohexyln-alkanes, cyclohexanepropionate, cyclohexanecarboxylate, and all tested derivates of cyclo-pentane.

(li) Oxidation studies with cyclohexaneacetate-

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CYCLOHEXANE ACIDS METABOLISM BY ARTHROBACTER 1175

5 -

4

do

0

I~~~~~I0 30-

I~~~~~~~~

at a rate significantly above the endogenouslevel, suggesting that enzymes for the 1-oxida-tion offatty acids were synthesized constitutive-ly by Arthrobacter sp. strain CAl.

(iv) Metabolite accumulation. A 1-liter cultureof Arthrobacter sp. strain CAI growing on cy-clohexaneacetate (0.1%) secreted into the medi-um a compound that reacted with 2,4-dinitro-phenylhydrazine to give a neutral derivative,suggesting it to be an aldehyde or a ketone.Paper chromatography and TLC of this deriva-tive (Table 1) and GLC analysis of the freecompound on 1.5% diglycerol and Porapak PScolumns showed it to be cyclohexanone.

Quantitative assay of the amount of cyclohex-anone produced during growth of the culturewas carried out enzymatically, using purified N.globerula cyclohexanone 1,2-monooxygenaseand measuring total substrate-stimulatedNADPH oxidation, coupled to cyclohexanone

90 120 oxygenation. There was short-term accumula-tion of cyclohexanone, which reached a maxi-miiim nf 'a A mM{1Uztll/, f thp ftk,%rPtL-n1 mayi-

FIG. 2. Respiratory activities of cyclohexaneace-tate-grown Arthrobacter sp. strain CAl. Each War-burg flask contained, in a volume of 1.9 ml: Na+,K+-phosphate buffer (pH 7.1), 70 ,umol; cell suspension(5.3 mg [dry weight] of bacteria); and 3 Fmol ofsubstrate. The center well contained 0.1 ml of 20%oKOH. Reactions, at 30°C, were initiated by tippingsubstrates from the side arms. Cyclohexanebutyrate(0), cyclohexaneacetate (U), cyclohexylideneacetate(0), (1-hydroxycyclohexan-1-yl)acetate (0), cyclo-hexanol (V), cyclohexanone (A), 6-caprolactone (A),6-hydroxyhexanoate (V), p-hydroxyphenylacetate(C), 2,5-dihydroxyphenylacetate (*). Endogenousrespiration (2 pLmol of 02 per h) was subtracted.

grown Ardhrobacter sp. strain CAl. Cyclohexan-eacetate, cyclohexanebutyrate, (1-hydroxycy-clohexan-1-yl)acetate, cyclohexylideneacetate,cyclohexanone, 6-caprolactone, and6-oxohexanoate were oxidized immediately andrapidly (Fig. 2). Adipate was oxidized slowly,whereas 3,4-dihydroxyphenylacetate (homopro-tocatechuate), 2,5-dihydroxyphenylacetate (ho-mogentisate), p-hydroxyphenylacetate, and cy-clohexaneglycolate caused no initial stimulationof oxygen consumption, although the initiationof oxygen uptake after 40 to 60 min was indica-tive of enzyme induction by the aromatic com-pounds.

(i) Oxidation studies with succinate-growncelLs. Whole cells oxidized cyclohexaneacetateand cyclohexanebutyrate at rates that were ini-tially only slightly above rates of endogenousoxygen consumption, followed by a markedincrease in oxidation rate after 40 min (Fig. 3).Only the growth substrate and the straight-chainfatty acid octanoate were immediately oxidized

MUM UL .0.UVI JIJ1/ ULLV LH1OU CLIGlM MUM-mum yield) after 10 h of growth (Fig. 4). Theprobability that cyclohexanone was a metabolicintermediate, rather than a side product, wasreinforced by the observed rapid disappearance

.- 2

a,0

0

0 1X

0 30 60

MIN

FIG. 3. Respiratory activities of succinate-grownArthrobacter sp. strain CAI. Conditions were identi-cal to those described in Fig. 1, and Warburg flaskscontained 6.8 mg (dry weight) of bacteria. Succinate(x), octanoate (p), cyclohexanebutyrate (0), cyclo-hexaneacetate (3), cyclohexane carboxylate (+). En-dogenous respiration (1.5 sLmol of 02 per h) wassubtracted.

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1176 OUGHAM AND TRUDGILL

TABLE 1. Chromatogaphic analysis of themetabolite accumulated in culture medium by

Arthrobacter sp. strain CAlChromatographic Rf valueTLC in given Paper

Culture solvent system chromatog-- raphy sol-

A B C vent sys-tem D

2,4-Dinitrophenylhydra-zonebof:Cyclopentanone 0.37 0.56 0.47 1.00Cyclohexanone 0.43 0.59 0.55 0.68Cycloheptanone 0.49 0.61 0.61 0.312-Methylcyclohexanone 0.53 0.63 0.59 0.323-Methylcyclohexanone 0.52 N1 0.59 0.414-Methylcyclohexanone NT 0.61 0.57 0.392-Hydroxycyclohexanone 0.22 0.49 0.31 0.64

Growth cultureZero time10 hours 0.43 0.58 0.54 0.65a Paper chromatographic values are Rx values rela-

tive to cyclopentanone.b 2,4-Dinitrophenylhydrazones were prepared by in-

cubating standard ketones and culture supernatantwith 0.1% 2,4dinitrophenylhydrazine in 2 M HCI andextracted into diethyl ether, which was dried overanhydrous Na2SO4 and evaporated to a small volumefor chromatography.

c NT, Not tested.

Ec0

z

It

0

HOURS

FIG. 4. Accumulation of cyclohexanone by Arth-robacter sp. strain CAI growing with cyclohexaneace-tate. A 1-liter culture of bacteria in a 2-liter Erlenmey-er flask was grown on a gyratory shaker (150 rpm) at30°C. Growth was followed at 580 nm (U). Ten-milliliter samples were removed aseptically at timedintervals and rapidly cooled, bacteria were removedby centrifugtion, and cyclohexanone (0) was assayedenzymatically with N. globerula sp. strain CL1 cyclo-hexanone 1,2-monooxygenase.

J. BACTERIOL.

of the compound as the culture approachedstationary phase.

(v) Enzymes of cyclohexanone oxidation. En-zymes catalyzing the established steps in theconversion of cyclohexanone into adipate (9, 19)were shown to be induced by growth with cyclo-hexaneacetate at levels comparable with thosefound in cyclohexanone-grown cells (Table 2).This evidence, in conjunction with studies per-formed on whole cells, clearly implicated cyclo-hexanone as an intermediate in cyclohexaneace-tate degradation.

(vi) Enzymology of the conversion of cyclohex-aneacetate into cyclohexanone. Two compounds,cyclohexylideneacetate and (1-hydroxy cyclo-hexan-1-yl) acetate, both structurally related tocyclohexaneacetate, are immediately oxidizedby cyclohexaneacetate-grown cells (Fig. 1). Themetabolic relationship between the three com-pounds is a clear parallel with the initial dehy-drogenation and hydration steps of a 1-oxidationcycle. However, under a variety of assay condi-tions it was not possible to demonstrate anydehydrogenase activity toward cyclohexaneace-tate, or hydration of cyclohexylideneacetate(change in absorbance at 220 nm), with crudeextract of cyclohexylacetate-grown Arthro-bacter sp. strain CAl.

(vi) Cyclohexaneacetyl-CoA dehydrogenase.Studies performed with the CoA ester of cyclo-hexaneacetate suggested the involvement of thisactivated form of the compound, since a cyclo-hexaneacetyl-CoA dehydrogenase was readilydetected in cyclohexaneacetate-grown cell ex-tract (0.03 U/mg), although a somewhat lowerlevel (0.01 U/mg) was found in extract of succi-nate-grown cells.

(viii) Cydohexaneacetyl-CoA synthetase. As-says for the presence of a CoA ester transferasein crude extracts of cyclohexaneacetate-growncells, using succinyl-CoA and acetyl-CoA aspotential donors and linking putative cyclohex-

TABLE 2. Enzymes of cyclohexanone oxidation inextracts of Arthrobacter sp. strain CAI'

Activity (U/mg of protein) in ex-tracts of cells grown on:

Enzymea Cyclohexa- Cyclo-acetate hexanone Succinate

Cyclohexanone 0.03 0.02 <0.0011,2-monooxygenase

6-Caprolactone 38.0 25.1 <0.2hydrolase

6-Hydroxyhexanoate 0.03 0.02 <0.001dehydrogenase

6-Oxohexanoate 0.47 0.37 <0.001dehydrogenase I Ia Enzymes were assayed as described in Materials

and Methods.

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CYCLOHEXANE ACIDS METABOLISM BY ARTHROBACTER 1177

aneacetyl-CoA into the CoA ester dehydroge-nase assay, proved negative. However, in as-says in which cell extract was incubated withATP, CoA, and cyclohexaneacetate in a CoAester synthetase assay system (4), the ability tosynthesize cyclohexaneacetyl-CoA was at a lev-el similar to that of octanoyl-CoA synthesis incrude extract ofoctanoate-grown cells (Table 3).The presence of significant synthetase activitytoward both cyclohexaneacetate and octanoatein extract of succinate-grown cells demonstratedthe constitutive levels of activity. An induciblecomponent, active toward cyclohexaneacetate,may also have been present, although the inher-ent insensitivity and variability of the assaysystem makes this debatable.

(ix) (1-Hydroxycydohexan-1-yl)acetyl-CoA ly-ase. An interesting feature of cyclohexaneace-tate metabolism is that although the initial stepsof a ,-oxidation cycle are permissible with thecompound as substrate, the product of the puta-tive hydration step, (1-hydroxycyclohexan-1-yl)acetyl-CoA, is a tertiary alcohol and thus notamenable to dehydrogenation to form a keto-group.However, in a consideration ofhow cyclohex-

anone might be formed from an intermediate, (1-hydroxycyclohexan-1-yl)acetyl-CoA is an ap-propriate candidate for the necessary cleavageof the side chain. Reported reactions in whichcarbon-carbon bond cleavage ofa CoA ester of a,B-hydroxy acid occurs include malyl-CoA lyase(12, 14), citramalyl-CoA lyase (6), and 3-hy-droxy-3-methylglutaryl-CoA lyase (27). In allcases, the substrate is cleaved to form acetyl-CoA and the appropriate ketone. A parallelcleavage of (1-hydroxycyclohexan-1-yl)acetyl-CoA would yield cyclohexanone and acetyl-CoA.

(1-Hydroxycyclohexan-1-yl)acetyl-CoA lyasein extracts of Arthrobacter sp. strain CAI wasassayed by a coupled system in which cyclohex-anone formed was oxygenated by purified N.globerula cyclohexanone 1,2-monooxygenase

TABLE 3. CoA ester synthetase activities inextracts of Arthrobacter sp. strain CAl

Activity (>imol of CoA-ester formed/h per mg of protein) in extracts of

Enzymea cells grown with:Cyclohexane- Octanoate Succinateacetate

Octanoyl-CoA Isynthetase 0.19 0.19 0.24

Cyclohexaneacetyl-CoA synthetase 0.20 0.07 0.11a CoA ester synthetases were assayed at pH 8 as

described in Materials and Methods.

(8), and the concomitant NADPH oxidation wasmeasured at 340 nm.The other predicted product of (1-hydroxycy-

clohexan-1-yl)acetyl-CoA lyase action was de-tected by using the coupled assay system de-scribed by P. B. Garland and P. J. Randle(Biochem. J. 91:61C, 1964), in which acetyl-CoAgenerated is condensed with oxaloacetatethrough the action of citrate synthase and, undercarefully defined assay conditions (D. J. Pear-son, Biochem. J. 95:23C, 1965), the oxaloacetateis regenerated from L-malate by the action of L-malate dehydrogenase with the concomitant re-duction of NAD. It was not possible to measureaccurately the yield of acetyl-CoA at this stagebecause of the NADH oxidase activity presentin crude extract of strain CAl.

(x) Cydohexylideneacetyl-CoA hydratase. Thepresence of hydratase activity toward cyclohex-ylideneacetyl-CoA was demonstrated by linkingthis activity with that of (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase and monitoring cyclohex-anone production in the coupled assay systempreviously described.

Addition of cyclohexylideneacetyl-CoA (5 to25 ,ul of preparation) to an assay system contain-ing strain CAl extract (0.1 to 1 mg of protein),N. globerula cyclohexanone 1,2-monooxygen-ase (0.31 U), NADPH (0.25 ,umol), and 0.1 MNa+K+-phosphate buffer, pH 7.4, in a 1-ml totalreaction volume resulted in a decrease in absor-bance at 340 nm. All components, includingmolecular oxygen, were required for activity.Although this assay system demonstrated thepresence of a cyclohexylideneacetyl-CoA hy-dratase, the specific activity measured was aminimum value as (1-hydroxycyclohexan-1-yl)-acetyl-CoA lyase may not have been present insufficient excess for a valid assay.

Purification of (1-hydroxycyclohexan-1-yl)-acetyl-CoA lyase was attempted with the objec-tives of (i) measuring accurately the yields ofcyclohexanone and acetyl-CoA and (ii) provid-ing enzyme in excess for a coupled assay systemso that the in vitro level of cyclohexylideneace-tyl-CoA hydratase could be measured.

(xi) Partial purfication of (1-hydroxycydo-hexan-1-yl)acetyl-CoA lyase. Attempts to purify(1-hydroxycyclohexan-1-yl)acetyl-CoA lyasewere frustrated by enzyme instability. Additionof 10% ethanol to buffer systems used in purifi-cation manipulations allowed the enzyme to bepurified about sixfold when crude extract (1,900mg; 0.09 U/mg of protein) was applied to acolumn of DEAE-cellulose (250-ml bed volume)and eluted with a linear gradient of KC1 (0 to 0.6M) in 2 liters of 42 mM Na+K+-phosphatebuffer. Further purification of the enzyme washampered by increasing instability. Additionalpurification steps including, for example, Sepha-

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1178 OUGHAM AND TRUDGILL

dex G-150 gel filtration resulted in a decrease inspecific activity. In addition, recovery of activi-ty from routine procedures such as ammoniumsulfate precipitation was consistently poor, anda variety of alternative procedures failed to givesignificant purification. Accordingly, prepara-

tions of (1-hydroxycyclohexan-1-yl)acetyl-CoAlyase that had been subjected to DEAE-cellu-lose chromatography, (NH4,)SO4 fractionation,and Sephadex G-150 (bed volume, 300 ml) chro-matography were used for studies of reactionstoichiometry.

(xli) Stoiometry of the (1-hydroxycydo-hexan-1-yl)acetyl-CoA lyase raction. Partially(fivefold) purified (1-hydroxycyclohexan-1-yl)-acetyl-CoA lyase was devoid of cyclohexanone1,2-monooxygenase and NADH oxidase, butsurprisingly still conained cyclohexylideneace-tyl-CoA hydratase activity, as measured by thecoupled assay system.Measurement of cyclohexanone and acetyl-

CoA formation from (1-hydroxycyclohexan-1-yl)acetyl-CoA established that the two productswere formed in equimolar amounts (Table 4).Amounts of (1-hydroxycyclohexan-1-yl)acetyl-CoA introduced into the assays were only satis-factorily estimated by either coupled assaysystem.

(xiii) Induction of enzymes catalyzing cydohex-aneacetate oidation. Established enzymes ofcyclohexanone oxidation were predictably in-duced by growth with the cyclic ketone, but ofgrt.ter interest was the observation that growthwith cyclohexanone also induced (1-hydroxycy-clohexan-1-yl)acetyl-CoA lyase (Table 5). Incontrast, 6-caprolactone, the first intermediatethat is not a six-membered alicyclic ring, did notinduce enzymes catalysing steps leading to itsformation.

TABLE 4. Yields of cyclohexanone and acetyl-CoAfrom (1-hydroxycyclohexan-1-yl)acetyl-CoA

(1-Hydroxycyclo- B, Acetyl-

hexan-1-yl)- A, Cyclohexa- coAb Raoacetyl-CoA solu- none formed formed tio,

tion added (IL1) (nmol) (nmol) A/B

0 0 0

2.5 11.3 12.9 0.875.0 26.6 24.9 1.06

10.0 54.0 50.6 1.0715.0 74.2 78.8 0.9420.0 135.5 122.7 1.11

a Cyclohexanone was assayed spectrophotometri-cally with N. globerula sp. strain CL1 cyclohexanone1,2-monooxygenase.

b Acetyl-CoA was assayed by the modified proce-dume of Pearson (Pearson, Biochem J. 91:23C, 1964;Garland and Randle, Biochem. J. 91:6C, 1964).

TABLE 5. Levels of (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase in extracts of Arthrobacter sp.

strain CAl grown with cyclohexaneacetate andmetabolic intermediatesa

(1-Hydroxycyclohexan-Growth substrate 1-yl)acetyl-CoA lyase

(U/mg of protein)

Cyclohexaneacetate ........ 0.17(1-Hydroxycyclohexan-

1-yl)-acetate ............ 0.17Cyclohexanone ...... ..... 0.186-Caprolactone ...... ..... <0.001Succinate ............... <0.001

a (1-Hydroxycyclohexan-1-yl)acetyl-CoA lyase wasassayed spectrophotometrically by coupling it with N.globerula cyclohexanone 1,2-monooxygenase.

(xiv) Reverdbilty of (1-hydroxycydobhexan-l-yl)acetyl-CoA lyase. One possible explanation ofthese observations would depend on significantreversibility of the CoA ester lyase, as has beenreported for malyl-CoA lyase (14). Growth ofArthrobacter sp. strain CAl on cyclohexanone,which yields acetyl-CoA directly during oxida-tion, would provide a situation in which thebasal level of (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase might generate significant amounts ofthe CoA ester to trigger induction of the en-zyme.The reversibility of the CoA ester lyase was

investigated according to the following proce-dure. Assay mixtures contained, in a reactionvolume of 1 ml: 90 Fmol of Na+K+-phosphatebuffer, pH 7.4, 1 Fmol of cyclohexanone, 1 to 5Fmol ofacetyl-CoA, and 5 U of partially purified(1-hydroxycyclohexan-1-yl)acetyl-CoA lyase.Reactions were incubated at 30°C on a gyratoryshaker, and samples were withdrawn at timedintervals, adjusted to pH 2 by rapid addition of 1ml of 0.1 M HCI to inactivate the lyase, readjust-ed to pH 7 by additions of 1 ml of 0.1 M NaOHand residual cyclohexanone, assayed with theN. globerula cyclohexanone 1,2-monooxygen-ase (8) as described in Materials and Methods.No detectable decrease in the concentration

of cyclohexanone in the reaction mixtures wasobserved over the 20-min reaction period, andwe concluded that under these assay conditions(1-hydroxycyclohexan-1-yl)acetyl-CoA lyasewas not significantly reversible.

Supportive data came from analysis of a reac-tion mixture which contained, in a volume of 3ml: 260 jjmol of Na+K'-phosphate buffer, pH7.4, 25 U of CoA ester lyase, 10 pmol ofcyclohexanone, and 5 pmol of acetyl-CoA. Af-ter incubation at 30°C for 30 min, the reactionmixture was adjusted to pH 11 to 12 with 1 MNaOH, incubated at 20°C for 15 min to hydro-

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CYCLOHEXANE ACIDS METABOLISM BY ARTHROBACTER 1179

loo more effective inducer. This result contrasts

with the results of CoA lyase reversibility stud-ies, an inconsistency that is currently unre-solved. The proposed pathway for cyclohexane-

to / /acetic acid oxidation that is compatible with the> 75 experimentalresults is shown in Fig. 1.> / / Cydohexanebutyrate metabolism. (i) Growth> / experiments. The mean doubling time of Arthro-

bacter sp. strain CAl at 30°C was 7.6 h on 0.05%> cyclohexanebutyrate mineral salts medium. In->/0 creasing concentrations of the compound pro-

gressively inhibited growth, with no growth oc-ag / /curring at 0.1%. Similar observations were made

with octanoate as the growth substrate, suggest-25 ing that short-chain fatty acids and their struc-

tural elements inhibit growth of this organism.(U) Oxidation studies with cyclohexanebu-

tyrate-grown cells. Cyclohexanebutyrate, cyclo-hexaneacetate, cyclohexylideneacetate, (1-hy-droxycyclohexan-1-yl)acetate, cyclohexanone,

0 25 50 75 100 6-caprolactone, 6-hydroxyhexanoate, and 6-ox-PERCENTAGE INDUCER ohexanoate were oxidized immediately and rap-

FIG. 5. Induction of (1-hydroxycyclohexan-1-yl)- idly. These results were broadly similar to theacetyl-CoA lyase during growth of Arthrobacter sp. spectrum of substrates oxidized by cyclohexan-strain CAl on mixed substrates. One-liter Erlenmeyer eacetate-grown cells (Fig. 2) and provide furtherflasks contained 500 ml of basal medium with carbon evidence that cyclohexanebutyrate is degradedsource (total, 0.1%) consisting of adipate in combina- through cyclohexaneacetate and a common met-tion with (1-hydroxycyclohexan-1-yl)acetate (0) or abolic pathway.cyclohexanone (0) on a weight percentage basis as (il) Enzyme amays on extracts of cydohexane-indicated. Cultures were inoculated with 20 ml of butyrate-gown cels. Extracts were assayed forcultures grown on the same medium and incubated at300C on a gyratory shaker (150 rpm). Bacteria were the presence of essential enzymes of the estab-

harvested after 24 h of growth, and extracts were hshed pathway of cyclohexaneacetate metabo

prepared. (1-Hydroxycyclohexan-1-yl)acetyl-CoA ly- lism. All key enzymes were present at levelsase was assayed as described in Materials and Meth- comparable with those in extracts of cyclohex-ods..The activity of the lyase in extract of cyclohexan- aneacetate-grown cells (Table 6). It thus appearseacetate-grown bacteria (specific activity, 0.17 U per likely that cyclohexanebutyrate degradation ismg of protein) was taken as 100%6.

lyze CoA esters, and acidified with 1 M -HCl;reaction products then were extracted with di-ethyl ether. GLC analysis of methyl esters,prepared according to the procedure of Metcalfeand Schmitz (17), failed to detect any methyl (1-hydroxycyclohexan-1-yl)acetate.

(xv) (1-Hydroxycydohexan-1-yl)acetyl-CoA ly-ase and the comparaive Inductive effects of cylo-hexanone and (1-hydroxycycohexan-l-yl)acetate.An alternative possibility, namely, that the CoAester lyase is induced by its product cyclohexa-none, was tested in an experiment in whichdiffering amounts of (1-hydroxycyclohexan-1-yl)acetic acid and cyclohexanone were mixedwith adipate in growth media. Cultures were

grown to the same cell density and harvested,and the (1-hydroxycyclohexan-1-yl)acetyl-CoAlyase was measured in extracts prepared fromthem. The results (Fig. 5) clearly indicated (1-hydroxycyclohexan-1-yl)acetic acid to be the

TABLE 6. Enzymes of cyclohexane acetateoxidation in extracts of Arthrobacter sp. strain CAI

Activity (U/mg of protein) in extractsof cells grown on:

Enzymey Cyohexane-

____ _ _ _ _ _ ~butyrat acetat

Cyclohexaneacetyl- 0.03 0.03 0.01CoA dehydroge-nase

Cyclohexylidenea- 0.05a 0.04a bcetyl-CoA hydra-tase

(1-Hydroxycyclo- 0.13 0.17 <0.001hexan-1-yl)-ace-tyl-CoA lyase

Cyclohexanone 1,2- 0.03 0.03 <0.001oxygenasea Represents a minimum value.b Not assayable because (1-hydroxycyclohexan-1-

yl)acetyl-CoA lyase free from cyclohexylideneacetyl-CoA hydratase is not available for coupled assays (6).

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1180 OUGHAM AND TRUDGILL

initiated by the ,3-oxidative removal of a C2 unitwith the generation of cyclohexaneacetyl-CoA.

(iv) Whole-cell oxidation studies with adipate-grown cells in the presence of chloramphenicol. Ithas already been observed that Arthrobacter sp.strain CA1, grown with succinate, showed sig-nificant substrate-stimulated oxygen consump-tion in the presence of cyclohexanebutyrate andcyclohexaneacetate, a result that is compatiblewith constitutive p-oxidative enzymes of strainCAl. The increase in oxygen consumption thatoccurred after a lag period of about 40 minwould be compatible with the induction of (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase andthe enzymes of cyclohexanone oxidation. If thisexplanation is correct, then Arthrobacter sp.strain CAl pregrown on succinate or adipate andincubated aerobically with cyclohexanebutyr-ate, in the presence of chloramphenicol, shouldaccumulate (1-hydroxycyclohexan-1-yl)acetateas a consequence of the abolition of inducedenzyme synthesis.

In preliminary experiments, it was establishedthat adipate-stimulated and endogenous oxygenconsumption by adipate-grown cells and cyclo-hexanebutyrate oxidation by induced cells werenot affected by chloramphenicol. In the absenceof effects of chloramphenicol on general oxida-tive metabolism, oxygen consumption by adi-pate-grown Arthrobacter sp. strain CAl withcyclohexanebutyrate as a substrate was fol-lowed in the presence and absence of chloram-phenicol (Fig. 6). Chloramphenicol clearly abol-ished induced oxygen consumption withoutsignificantly affecting the initial slow substrate-stimulated oxidation.

(v) Metabolite accumulation by adipate-growncells in the presence of cyclohexanebutyrate andchloramphenicol. The manometric experimentdescribed previously (Fig. 6) was repeated, andsimultaneously two larger-scale (20-fold) incuba-tion mixtures were set up on a gyratory shaker(150 rpm) at 30°C while oxygen uptake in themanometric experiment was followed. When netcyclohexanebutyrate-stimulated oxygen con-sumption had ceased, whole cells were removedfrom the larger-scale incubation mixtures bycentrifugation at 12,000 x g and 4°C for 20 min.

Neutral and acidified (pH 1) diethyl etherextractions of the supernatants were analyzedfor neutral and acidic metabolites, respectively.No neutral aldehyde or ketone metabolites couldbe detected, but GLC analysis (1.25% diglycerolcolumns at 76°C; carrier gas at 40 ml/min)showed the presence of peaks corresponding tothe methyl esters of authentic cyclohexaneace-tate (205 s) and (1-hydroxycyclohexane-1-yl) ac-etate (1100 s). There was no residual cyclohex-anebutyrate. GLC-mass spectral analysisshowed no significant differences between the

methyl esters of authentic cyclohexaneacetate(mle 156) and of (1-hydroxycyclohexan-1-yl)ace-tate (mle 172) and the accumulated metabolites.A small subsidiary peak on the trailing edge ofthe methyl cyclohexaneacetate peak (265 s) gavea mass spectrum compatible with its being amixture of methyl cyclohexaneacetate andmethyl cyclohexylideneacetate (mle 154).There was no evidence for the formation of

cyclohexanepropionate by a-oxidation, and al-though the first p-oxidation cycle, convertingcyclohexanebutyrate into cyclohexaneacetate,was complete, the incomplete conversion(=60%) of the latter compound into (1-hydroxy-cyclohexan-1-yl)acetate, even though oxygenconsumption had virtually ceased, is unex-plained.

DISCUSSIONArthrobacter sp. strain CAl is one of only two

organisms that we have isolated capable ofgrowth with cyclohexaneacetate as the solesource of carbon. There is evidence that organ-

0

.5;0

0

3 _

0 S0 100 150 200

MIN

FIG. 6. Effect of chloramphenicol on respiratoryactivities of adipate-grown Arthrobacter sp. strainCAl. Each Warburg flask contained, in a volume of1.9 ml: Na+,K+-phosphate buffer (pH 7.1), 70 Fmol;cell suspension, (5.8 mg [dry weight] of bacteria);substrate, 3 ,umol; and chloramphenicol, 500 pLg/ml.The center well contained 0.1 ml of 20%o KOH. Reac-tions, at 300C, were initiated by tipping substratesfrom side arms. Adipate, with and without chloram-phenicol (5), cyclohexanebutyrate (0), cyclohexane-butyrate with chloramphenicol (O). Endogenous respi-ration (2.1 Lmol of 02 per h) was subtracted.

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CYCLOHEXANE ACIDS METABOLISM BY ARTHROBACTER 1181

isms possessing this metabolic capability arerare in the biosphere, both from the publishedwork of others (1) and as a result of our ownexperience.The accumulation of cyclohexanone in the

growth medium in large amounts during growthon cyclohexaneacetate is an interesting phenom-enon, and it was, in fact, the odor of cyclohexa-none in culture flasks that led to its discovery. Itmay be explicable on the basis of the action ofboth constitutive and induced 3-oxidation en-zymes in conjunction with a very active (1-hy-droxycyclohexan-1-yl)acetyl-CoA lyase pre-ceding a cyclohexanone 1,2-monooxygenase oflow specific activity (Table 2).The net contribution of constitutive and any

induced 3-oxidation enzymes toward the oxida-tion of cyclohexaneacetate is not clear from theexperiments reported. The assay procedureused for measurement of acyl-CoA synthetasestypically gives very low specific activity values(4, 22); in addition, since we have been unable toseparate the cyclohexylideneacetyl-CoA dehy-drogenase from the (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase, we are unable to obtain anaccurate assay of the level ofthe former activity.However, from the results presented, it wouldappear that constitutive enzymes make a majorcontribution toward the (-oxidative steps ofcyclohexaneacetate metabolism in inducedcells.Our understanding of the pattern of enzyme

induction is incomplete. It is interesting thatcyclohexanone induces the (1-hydroxycyclo-hexan-1-yl)acetyl-CoA lyase in addition to thecyclohexanone 1,2-monooxygenase. This, pre-sumably in conjunction with the constitutive 3-oxidation enzymes, permits cyclohexanone-grown cells to oxidize cyclohexaneacetate andall the metabolic intermediates.

Logically, this would be expected to resultfrom the basal level of the CoA ester lyasecatalyzing formation of (1-hydroxycyclohexan-1-yl)acetyl-CoA from cyclohexanone and ele-vated pool levels of acetyl-CoA, which theninduces the enzyme, although the possibilitythat cyclohexanone in addition to (1-hydroxycy-clohexan-1-yl)acetyl-CoA is also a direct, butless effective inducer has not been excluded.Our inability to demonstrate reversal of thelyase is surprising in view of the establishedreversible nature of malonyl-CoA lyase (14).

Alternative theoretical pathways for the oxi-dation of cyclohexaneacetate include (i) a routethat parallels the aromatization pathway for cy-clohexane carboxylic acid oxidation (3, 15, 28)with the formation of p-hydroxyphenylaceticacid and subsequently homoprotocatechuic acidor homogentisic acid as an intermediate whichcan be cleaved by well-established routes (13,

26); (ii) the hydration of cyclohexylideneacetyl-CoA by the insertion of the elements of water inthe alternative orientation to form cyclohexan-eglycolyl-CoA which, after removal of the CoA,could by dehydrogenation, Baeyer-Villiger oxy-genation, and hydrolysis of the cyclohexyl oxa-late thus formed then yield cyclohexanol andoxalate. Whole-cell oxidation studies, metabo-lite accumulation experiments, and assay ofenzymes in cell extracts support neither of thesealternative hypotheses.Growth of Arthrobacter sp. strain CAl on

cyclohexanebutyrate is less rapid than growthwith cyclohexaneacetate and is also inhibited atconcentrations above 0.05%. This organism ap-pears to exhibit inhibition by short-chain fattyacids, as has been previously reported for yeasts(23). Growth with longer-chain cyclohexane fat-ty acids has not been investigated.

Oxidation of cyclohexanebutyrate can be di-vided into two phases. One of these, the conver-sion of the compound into (1-hydroxycyclo-hexan-1-yl)acetate (CoA ester), is catalyzed bythe constitutive (-oxidation cycle enzymes ofthe organism, which may be augmented by aparallel set of inducible specific enzymes duringgrowth with cyclohexanebutyrate and cyclohex-aneacetate. The second group of enzymes, cata-lyzing the conversion of (1-hydroxycyclohexan-1-yl)acetate into adipate, display characteristicstypical of induced catabolic enzymes, all beinginduced at least 20-fold by exposure to cyclo-hexanebutyrate and related compounds (Table6).

In view of the established involvement ofconstitutive 3-oxidizing enzymes in the initialsteps of cyclohexanebutyrate and cyclohexane-acetate oxidation by Arthrobacter sp. strainCAl and the proven ability of the organism togrow with pimelate, its inability to grow withcyclohexane carboxylate and cyclohexanepro-pionate, for which (-oxidation can facilitate ringcleavage and formation of pimelyl-CoA (4), mer-its further investigation.

ACKNOWLEDGMENTSWe are grateful to Muriel Rhodes-Roberts for identifying

Arthrobacter sp. strain CAl and to Sue Brice for typing themanuscript.

This research was supported by a grant from the ScienceResearch Council.

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