butane metabolism by butane-grown

7/28/2019 Butane Metabolism by Butane-grown

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Printed in Great Britainicrobiology (1999>, 145, 1 173-1 1 80

Butane metabolism by butane-grown'Pseudomonas butanovora 'Daniel J . ArpTel: + 1 541 737 1294. F a x : + 1 541 737 3.573. e-mail: a r p d t g bcc.orst.edu

Laboratory fo r N i t rogenFixation Research,Depar tment of Botany andPlant Pathology, OregonState University,2082 Cordley, Corvallis,OR 97331, USA

The pathway of butane metabolism by butane-grown Pseudomonasbutanovora' was determined to be butane+ -butanol+ butyraldehyde+butyrate. Butane was initia lly oxidized at the term inal carbon to produce 1-butanol. Up to 90% of t he bu tane consumed was accounted fo r as I-buta no lwhen cells were incubated in the presence of 5 mM I-propanol (to blocksubsequent metabo lism of I-butanol). No production of the subterminaloxidation product, 2-butano1, was detected, even in the presence of 5 mM 2-pentanol (an effective inh ibitor of 2-bu tanol consumption). Ethane, propaneand pentane, but not methane, were also oxidized. Bu tane-grown cellsconsumed I- bu tano l and other term inal alcohols. Secondary alcohols, including2-butano1, were ox idized to the corresponding ketones. Butyraldehyde wasfurthe r oxidized to butyrate as demonstrated by blocking butyrate metabolismwith 1mM sodium valerate. Buty rate also accumulated from butane whencells were incubated with 1mM sodium valerate. The pathway intermediates(butane, I-butanol, butyraldehyde and butyrate) and 2-butanol stimulated 0,consumption by butane-grown cells. I-Butanol, butyraldehyde and butyra tesupported growth of 'P. butanovora', as did 2-butano l and lactate.

INTRODUCTION

Keywords : butane metabolism, al kane metabolism, 'Pseudomonas butanovora ', alkaneoxidation

A number of bacteria have been isolated that are capableof growth on butane. Most of these bacteria aremembers of the Rh od obac er-No card ia-A rthro bac er-Corynebacterium group of Gram-positive bacteria(Ashraf et al., 1994; McLee et al., 1972; Perry, 1980).However, two Gram-negative bacteria which can growon butane, 'Pseudomonas butanovora ' and Pseu-d o m o n a s sp. strain CRL 71, have been described ( Ho u e tal., 1983; Takahashi, 1980). Butane-oxidizing bacteriacan be considered as part of a larger group of bacteriawhich are characterized by their ability to grow ongaseous alkanes such as ethane and propane, but notmethane (Ashraf et al., 1994; Klug & Markovetz, 1971).This larger group is also dominated by Gram-positivebacteria, although some Pseudomonas spp. will grow onC,-C, alkanes (Hou et al., 1983). Bacteria that grow onone gaseous alkane will generally grow on other gaseousor volatile alkanes ; or example, Myco bacteri um vaccaewill grow on propane (Vestal & Perry, 1969) or butane(Phillips & Perry, 1974). Bacteria that can grow onmethane, methanotrophs, generally do not use otheralkanes as growth substrates (Murrell, 1992) though

they can often carry out the hydroxylation of gaseousalkanes (Burrows e t al., 1984; Colby et al. , 1977).As pointed out by Ashraf et al. (1994) in a recent reviewof the subject, the pathways for the metabolism of thelight n-alkanes (ethane, propane and butane) havereceived little attention compared to those of methaneand liquid n-alkanes. The pathway of butane metab-olism has not previously been established directly forany butane-oxidizing bacterium. Butane, as with otheralkanes, is generally assumed to be harvested by amonooxygenase, which results in hydroxylation of thealkane (Ashraf et al., 1994; Perry, 1980). However,production of 1-butanol or 2-butanol f rom butane hadnot been directly demonstrated for any butane-grownbacterium. The subsequent metabolism of either 1-butanol, the terminal oxidation product, or 2-butanol,the subterminal oxidation product, would be expectedto require different pathways. Evidence for pathwaysconsistent with both oxidation products has beenpresented (Lukins & Foster, 1963; Phillips & Perry,1974; van Ginkel et al., 1987). Terminal oxidation ofbutane by M. vaccae JOB5 was proposed because cellsgrown on either butane or butyrate expressed isocitrate

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lyase activity, which is required to as similate the 2-carbon compounds formed as a resul t of fur thermetabolism of butyrate (Phillips & Perry , 1974) . Incontras t , ce l ls grown on butanone d id not produceisocitrate lyase activity. Butanone was subsequentlydecarboxyla ted to propionate and fur ther metabol izedby the methylmalonate-succinate pathway (Phillips &Perry, 1974). Buta ne-grow n Nocard ia T B 1 p r o d u c e dbutyra te f rom n-butane while in the presence of arseni teand a pathway of butane to 1-bu tano l to bu ty ra ldehydeto butyra te was suggested (van Ginkel et al . , 1987) .However , p roduc t ion of the f i rs t two proposed in ter-media tes (1-butanol and bu tyra ldehyde) was no t dem -onstrated directly and the possibility of a concur ren tpathway in i t ia ted by subterminal oxidat ion of bu tanewas not e l iminated . Subterminal oxidat ion was in-d ica ted fo r p ropane -g rown M y c o b a c t e r i u m s m e g m a t i s422, which accumulated butanone when exposed to n-butane (Lukins & Foster , 1963) .As with butane , the pathw ay of propane m e tabol i sm hasreceived l imited a t tent ion . Support for both terminaland subterminal oxidat ions of propane has been pre-sented (Perry , 1980) . Accum ulat ion of acetone ( f rom 2-propanol) supported the conclusion that propane oxi-dat ion was pr imari ly subterminal in propane-ut i l iz ingbacteria such as M . vaccae JOB5 (Luk ins & Foster ,1963; Perry , 1980) . However , consumption of thete rmina l ox ida t ion p roduc t , 1 -p ropano l , was a l so dem-ons t ra ted fo r propane -g rown M . v ac ca e JOB5 (Pe r ry ,1968). Fur therm ore , som e propane-grow n s tra ins ofArthrobac t e r consumed 1-propan ol ( the terminal oxi-da t ion p roduc t of propane ) bu t no t 2 -p ropano l , wh ileother s t ra ins consumed both isomers (Stephens &Dal ton , 1986) . Bo th 1 -p ropano l and 2 -p ropano l wereprodu ced by cell-free extra cts of Arthrobac t e r sp . CRL-60 , P s e u d o m o n a s fluorescens NRRL-B-1244 and Brevi-bac t e r ium sp. NRRL B-11319 (Pate1 et al . , 1983) .Current evidence indicates tha t both terminal andsubterminal hydroxyla t ions of propane can occu r .' P . b u t a n o v o r a 7grow s on C,-C, n-alkan es as well as ona number of alcohols and organic ac ids (Takahashi ,1980). However , g row th on a lkenes and suga rs was n o tobserved. We recent ly dem onstra ted th at th is bacter ium,when g row n o n bu tane , cou ld in i t i a te the deg rada t ion ofa num ber of chlor inated a l iphat ic compoun ds, inc ludingc h l o r o f o r m ( H a m a m u r a et al., 1997) , and th is degra-da t ion appea red to be in i t ia ted by butane mono-oxygenase. In th is wor k , the pathw ay of bu tane me tab -ol ism by th is Gram-negat ive bacter ium was e lucidated .METHODSCell growth and preparation of cell suspensions. Cells of'Pseudomonas butanovora ' (AT CC 43655) were grown aspreviously described (Hamamura et al., 1997). The basalmedium (1 ) contained 8 g (NH,),HPO,, 1.9g Na,HPO,.7H,O, 2 g KH,PO,, 0.5 g MgSO, .7H,O, 0.06 g CaC1,. 2H,O,0-05 g yeast extract and 1 ml trace element solution (Wiegant& de Bont, 1980) at p H 7-1. Cells were cultured in 160 mlserum vials containing 50 ml basal medium and capped withbutyl rubber stoppers and aluminium crimp seals. Each vial

received 5 ml CO , as an overpressure. Butane (10 ml) wasadded to the vial as an overpressure for growth on bu tane. Forgrowth on 1-butanol, butyraldehyde or 2-butanol, appropriatevolumes of each pure liquid were added directly to the sterilemedium. For growth on butyrate or lactate, appropriateamounts of stock solutions (1 M) of sodium butyrate orsodium lactate were added to the medium. Cultures wereshaken at 160 oscillations min-l and maintained a t 30 "Cduring growth and harvested after 2 or 3 d. T he limitingnutrient for growth was 0, ;butane-grown cultures typicallyreached an OD,,, of 0.6 upon exhaustion of the 0,.Cells were harvested by centrifugat ion (10 min at 12000g;10 "C) and resuspended in 1 ml buffer [8 g (NH,),HPO,, 1.9 gNa,HPO, .7H,O, 2 g KH,PO,, 0-5 g MgSO, .7H,Op H 7-11. Cell suspensions were typically prepared fresh dailyand used within 6 h. However, cell suspensions retainedbutane consumption activity for at least 30 h when stored onice without agitation. Typical protein concentrations for thecell suspensions were 5-7 mg protein (ml suspension)-'.Measurement of cell activities. Butane consumption wasmeasured in a 1 ml gas-tight syringe (Hami lton 1001 RN) withthe needle removed. T he reaction mixture consisted of 0.7 or0.8 ml 0,-saturated buffer, 0.1 or 0.2 ml butane-saturatedbuffer, and addition of a cell suspension (typically 0.025 ml),other compounds (e.g. 1-propanol) as indicated, and ad-ditional buffer for a total volume of 1 ml. A glass bead in thesyringe facilitated mixing of the components. Additions to thesyringe were made by injection through the opening into thebody of the syringe. N o gas phase was present in the syringe.Movement of the plunger facilitated addition and removal ofsamples without introducing a gas phase. Samples of the liquid(10 pl) were removed periodically and analysed for butanecontent by GC as described below. In some instances, 1-butanol was also analysed. Consumption of other alkanes(0.14.2 pM) was measured similarly. The reactions werecarried out a t room temperature (20Consumption and accumulation of alcohols, butyraldehydeand butyrate were measured in 7 ml serum vials capped withbutyl rubber stoppers and aluminium crimp seals. Thereaction mixture consisted of the substrate with or withoutinhibitor (at the indicated concentrations), cell suspension(10-100 pl), and buffer to a total of 1 ml. Butane (1 m l) orpropane (1ml) gas, where indicated, was added as anoverpressure to the headspace. Vials were shaken at 100oscillations min-l in a 20 "C water bath during the reactions.Liquid samples (2-10 pl) were removed periodically and theconcentrations of substrates and products were determined byGC .0, consumption by cells in the presence of various compoundswas measured with a Clark-style 0, electrode inserted into a1.6 ml chamber sealed with a capillary inlet through whichadditions were made. The contents of the electrode chamberwere stirred with a magnetic stir bar. The reaction mixtureconsisted of air-saturated buffer with substrates and cellsadded as indicated, The reactions were carried out at roomtemperature (20 1 "C).Analytical techniques. Concentrations of alkanes, aldehydes,ketones and organic acids in the liquid phase of reactionmixtures were determined by G C with a Shimadzu GC-8A gaschromatograph equipped with a flame-ionization detector anda 30 cm long by 0.1 cm i.d. stainless steel column packed withPorapak Q. The oven temperature was varied depending onthe compound analysed. The following temperatures wereused for each set of compounds: 25 "C, methane and ethane;70 "C, propane; 80 "C, butane and methanol; 100"C, ethanol;

1 "C).

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Butane metabolism

I

1 . 1 . 1 . 1 . .10 20 30 40 50

r'

WaJ0ac,

-D-0a,mN

0.8

0.6

0.4

0.2

Time (min)' 0 40 60 80 100I . I . I . I . 1

mTime (min)

fig. 1. Metabolite consumption and production by butane-grown ' P . butanovora'. (a) Consumption of butane ( 0 ) ndproduction of 1-butanol (u) n the presence of 5 mM 1-propanol. The reaction mixture (1 ml) contained buffer, 230 nmolbutane, 915 nmol 0,, 5 pmol 1-propanol and 20 pl cell suspension (147 pg protein). (b) Consumption of 1-butanol ( 0 )and production of butyraldehyde (W ) with 1 pmol 1-butanol and 25 pl cell suspension (133 pg protein). (c) Consumptionof 2-butanol (a)and production of butanone (a)wi th 1 pmol 2-butanol and 25 pI cell suspension (133 pg protein). (d)Production of butyrate from butane in the presence of 1 mM valerate wit h 6 ml air, 1 ml butane (added as anoverpressure), 1 pmol sodium valerate, and either 10pl cell suspension (47 pg protein) (m ) or 40 pI cell suspension(188 pg protein) (a). or the sample wi th the higher cell density, the init ial valerate concentration (1 mM) had decreasedto 0.04 mM by 50 min. (b-d) Reactions were carried out in sealed 7 ml vials wi th 1 ml reaction mixture containing bufferand the indicated concentrations of substrate and ce l l suspension. (a-d) Liquid samples were removed a t the indicatedtimes and analysed by GC.

110 "C, pentane, butanone, acetone and butyraldehyde;120 "C, 2-butanol, 1-propanol, 2-propanol, propionaldehydeand butanone; 130"C, 1-butanol and butyraldehyde; 150"C,1-pentanol, 2-pentanol and 2-pentanone; 160 "C, butyric acidand valeric acid. Concentrations of compounds were de-termined by comparison of peak heights from samples to peakheights of standards of known concentration. Concentrationsof gaseous compounds in standards were calculated usingHenry's law and appropria te Henry's law constants (Smith&Baresi, 1989). Identities of products were determined bycomparison of retention times and peak shapes to those ofauthentic compounds.The protein contents of cell suspensions were determined bythe Biuret assay (Gornall et al., 1949) after cells weresolubilized in 3 M NaOH for 30 min at 65 "C. BSA was usedas a standard.Chemicals and gases. All chemicals were of reagent grade.Acetylene was generated from CaC, by addition of H,O.Propane and butane were purchased from Airgas; ethane waspurchased from Matheson ; methane was purchased fromAirco.

RESULTSAlkane oxidation by butane-grow n ' . butanovora'N o n e of the predicted products of either terminaloxidation of butane (1-butanol or butyraldehyde) orsubterminal oxidation (2-butanol or butanone) weredetected in culture supernatants of butane-grown 'P .butanovora'. Likewise, n o 1 - or 2-butanol w as detectedin resting cell suspensions during consumption ofbutane. Therefore, inhibitors of the subsequent metab-olism of predicted products from each metabolic stepwere identified to c ause the produ cts t o accumulate.1-Propanol was then included in the reaction mixturesas a structural analogue of 1-butanol to compete for theenzyme(s) tha t further metabolized 1-butanol, therebyinhibiting 1-butanol consumption. Upon addition of5 m M 1-propanol , 0-35 m M 1-butanol was producedafter 60 min (1 ml reaction mixture, 7 ml vial, appro x.250 pg cell protein). Concentrations of 1-propanol fro m

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Table 1. inhibition of substrate consumption bysubstrate analogues or products in butane-grown'P. butanovora '

Subst rate (m M ) Inhibi tor (m M ) Inhibi tion ('/o)'1-Butanol (1.0)1-Butanol (0.1)2-Butanol (0.1)2-Butanol (0.2)2-Butanol (1.0)Butanone (1.0)Butyrate (0.1)Butyrate (0.1)

1-Propanol (5.0)1-Propanol (5.0)1-Propanol (5.0)2-Pentanol (5.0)Butanone (1.0)2-Butanol (1.0)Propionate (1.0)Valerate (1.0)

767147988

1006 386

"- Percentage inhibit ion relat ive to rate of subs t r a t e consumpt i onat the same concent rat ion in the absence of inhibitor.

Table 2. Rates of alcohol consumption by butane-grown'P. butanovora 'Reactions were carried out with cell suspensions containing13 3 pg protein. The init ial concentration of each alcohol was1 .0 mM . Th e r a t e of consumption is measured as nmol alcoholconsum ed min-l (m g protein)- '. ND , Non e detected.

Subst rate Rate of Accumulatedconsumpt ion product

Met hano lEthanol1-Propanol2-Propanol1-Butanol2-Butanol1-Pentanol2-Pentanol

< 1022 523 51.583411503752.54

NDND

PropionaldehydeAcetoneButyraldehydeButanone2-Pentanone

ND

0-1 o 15 mM were tested; 5 mM 1-propanol led to thegreatest accumulation of 1-butanol. A time course ofbutane consumption was inversely proportional to thetime course of 1-butanol accumulation (Fig. la ). Whenbutane oxidation ceased (due to 0, depletion), 1-butanolproduction also ceased. The ratio of 1-butanol con-sumption to butane production in this experiment (Fig.l a ) was 0.83. Because most of the butane consumedcould be accounted for as I-butanol , terminal oxidationof butane to 1-butanol was indicated as the predominantroute of butane oxidation. The ratio of 1-butanolproduction to butane consumption varied with cellpreparations from a low of 0.2 to a high of 0.9 with amean value of 0.6+0-2. Butane consumption in thepresence of 1-pentanol (5 mM ) also led to accumulationof 1-butanol. When butane was omitted from thereactions with 5 mM 1-propanol, or when acetylene(2% , v / v ) , an inactivator of butane oxidation activity,was included in the reactions with butane and 5 mM 1-propanol, no 1-butanol was formed. These resultsindicate tha t I-butanol was produced from the oxidationof butane.The difference between the amount of 1-butanol thataccumulated and the amount of butane consumed wasmost likely due to incomplete inhibition of the sub-sequent metabolism of 1-butanol. The inhibition of 1-butanol metabolism by 1-propanol was examined di-rectly and, though substantial, was not complete, evenwhen the 1-butanol concentration was only 2 % of the 1-propanol concentration (Table 1). Therefore, somefurther metabolism of 1-butanol by resting cells wouldbe expected even in the presence of 5 mM 1-propanol.The difference between the amounts of butane con-sumed and 1-butanol formed was not likely to be due t osubterminal oxidation of a portion of the butane. No 2-butanol was detected (detection limit approx. 0.01 mM )in the reaction mixture, even in the presence of 2-pentanol (5 mM), an effective inhibitor of 2-butanolconsumption (Table 1). Butane oxidation occurredprimarily (if not exclusively) a t the terminal carbon.

' P . butanovora' grows on a variety of alkanes(Takahashi, 1980). Therefore, the consumption ofseveral alkanes by butane-grown cells was examined.Cell suspensions (128 pg protein) consumed ethane[203 16 nmol min-' (mg protein)-'], propane[148&23 nmol min-l (mg protein)-'], butane[119+ 8 nmol min-l (mg protein)-'] and pentane[214+42 nmol min-' (mg protein)-']. Methane con-sumption was not detected. Methane also was not agrowth substrate for this bacterium (Takahashi, 1980).The products of propane oxidation in the presence of5 mM 1-butanol (to inhibit 1-propanol consumption)were examined. Production of 1-propanol was readilyobserved (time course not shown) . Th e concentration of1-propanol produced (0.13 mM produced in 60 min byapprox. 120 pg protein) was similar t o the amount of 1-butanol (0.14 mM ) produced by this cell suspensionwhen inhibited with 5 mM 1-propanol. However, no 2-propanol production was detected in cells incubatedwith 5 mM I-butanol or 5 mM 2-butanol (detectionlimit, 0-02 m M 2-propanol). As with butane, onlyterminal hydroxylation of propane was observed.

Alcohol consumption by butane-grow n 'P .butanovora 'Butane-grown resting cell suspensions readily consumed1-butanol (Fig. 1 ). Th e predicted produc t of an alcohol-dehydrogenase-catalysed reaction, butyraldehyde,accumulated as 1-butanol was consumed and was thensubsequently consumed. The a mount of butyraldehydeproduced was no t stoichiometric with the amount of 1-butanol consumed. Apparently some consumption ofbutyraldehyde took place prior to complete consump-tion of the 1-butanol, although the possibility thatanother pathway of 1-butanol consumption was func-tioning simultaneously has not been ruled out.Consumption of other terminal alcohols was alsoexamined (Table 2). Th e pattern paralleled that of

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Butane m etabolismalkane oxidat ion , with a l l a lcohols being consumedexcept methano l. Interestingly, oxida tion of ethanol d idnot result in accum ulation of acetaldehyd e and o xi-dat ion of 1-pentanol d id not resul t in the accum ulat ionof valeraldehyde, while oxid ation of 1 -pro pan ol didresult in accumulation of propionaldehyde.Although sub terminal oxidat ion of bu tane to 2 -bu tano lwas not detec ted , 2-butanol wa s consumed by butane-grown cells (Tables 1 and 2 ) . Th e consumpt ion o f 2 -butanol was accompanied by the accumulat ion ofbutanone, the predicted product of the oxidation of 2-butanol (Table 2) . A t ime course of 2-butanol con -sumption and butanone accumulat ion revealed rapidinitial rates followed by a decrease in both rates asbutanon e accumulated (Fig . lc ) . T he ra t io of butano neaccumulated to 2-butanol consumed decreased from0-92 (a t 6 m in) to 0.30 (at 102 min). In spite of the rap idin i t ia l ra te of 2-butanol consumption , complete con-sumption of the 2-butanol was not observed even after102 min (Fig . lc ) because th e butano ne tha t accum ulatedinhib i ted the consumption of 2-butanol (Table 1).2-Butanol a lso inhib i ted the oxid at ion of butanon e (T able1). Two other secondary a lcohols , 2-pentanol and 2-prop ano l, were also oxidized by resting cells of butane -g r o w n P . butanouora . 2-Propanol was oxid ized es-sentially stoichiometrically to acetone. For example ,af ter 30 min with cells (0.3 mg prote in) the concen-tra t ion of 2-propanol in the react ion mixture haddecreased 0 .26 m M (from 1 mM) while the concen-tra t ion of acetone increased to 0-27 m M . 2-Pentanol wasoxidized to pentanone. For both of these secondaryalcohols, the time courses of consumption were notlinear. The rates decreased with time as the corre-sponding ketone accumulated , which is s imilar to theresul t with 2-butanol . Thus, while secondary a lcoholswere not produced in substant ia l amounts f rom ei therbutane or prop ane , secondary alcohols w ere nonethelessconsumed by butane-grown cells.Aldehyde, keton e and organic acid consum ption by . butanovoraWhen resting cells of P . butanouora were incubatedwith 1 mM butyra ldehyde, the butyra ldehyde wasreadi ly consumed a t a ra te of 353 nmo l min- (mgprotein)-. Th e consum ption rate was essentially con-s tant unt i l a l l the butyra ldehyde was consumed. Thepredicted product of the oxidat ion of butyra ldehyde isbutyra te . However , no butyra te accumu lated dur ing theoxidation of butyraldehyde. Nonetheless, butyrate con-sumption was readily observed. With 1 m M b u ty r at e ,consumption ra tes ranged from 28 to 71 nmo l min- (m gpro tein ) - . T o demons t ra te tha t bu ty ra ldehyde wasindeed oxidized to butyrate, an inhibitor of butyra teconsumption was sought . Sodium valerate (1 m M ) w asfound to be an effective inhibitor of butyra te con-sumpt ion (Tab le 1). When cells (56 pg prote in) wereincubated with 1 mM bu ty ra ldehyde and 1 m Mvalerate, production of butyrate was observed. After48 min, the butyra te concentra t ion reached 0.5 m M .

The resul ts indicated that butyra ldehyde was indeedoxidized to butyrate and the cells further metabolizedbutyrate in the absence of a metabolic inhibitor.When cells were incubated in the presence of butane(0.2 atm , 0.26 m M in so lu tion ) and 1m M va le ra te ,butyra te product ion was observed (Fig . I d ) .T h e specificra te of butyra te product ion was 38 nmo l min-l (mgprotein)-. Th is rate is lower than typical rates of butaneconsumption [90-160 nm ol min- (m g protein)-],which could indicate tha t va lera te does n ot completelyblock butyra te consu mption (perhaps because of in-eff ic ient uptake ) , or th a t som e in termedia tes o ther th anbutyra te a lso accumulate . With a low prote in con-centra t ion (0.05mg protein ml-), the rate of butyratep roduc t ion was cons tan t fo r 30 min (Fig . Id) . When theprotein co ncentratio n was increased fourfold, the initialra te of butyra te product ion increased fourfo ld . How-ever, a maximu m concentra t ion was reached a t 30 minfol lowed by complete consumptio n of the butyra te overthe nex t 45 min. Butyra te consumption occurred be-cause the cells consu med the valera te, thereby relievingthe inhib i t ion . At 50 min, the valera te concentra t ion haddecreased to 0.04 mM . N o bu ty ra te was de tec ted w hen(1)bu tane was omi t ted , (2 ) va le ra te was omi t ted o r ( 3 )cells were pretreated wit h acetylene (3Yo , v/v , acety lene;15 min) . These resul ts fu r ther confirm that the path wayof butane o xidat ion includes product io n of butyra te .T he consum ption of ketones by butane-grown P .butanouora was examined. Because 2-butanol wasconsumed by butane-grown cells to produ ce butanone ,it was of interest to determine the ra te of bu tanoneconsumption . Rest ing ce l ls of P . butanouora con-sumed bu tanone (1m M ) at a ra te of 120 nmol min-(m g protein)-. P . butanouora produced acetone f rom2-p ropano l (Tab le 2 ) . When ace tone consumpt ion wasexamined in the absence of 2-propan ol , a s low ra te ofconsum ption [9 nmol min- (m g prote in)-] was ob-served. Recently, Ensign and coworkers (Ensign et al.,1998) demo nstra ted th at for som e bacter ia the f i rs t s tepin the metabolism of ketones involves a carboxylationreact ion . For example , the consumption of acetone byresting cells of Xanthobacter Py2 was stimulated byaddit ion of CO, (Sluis et al., 1996) . However , nost imula t ion in the ra te of butanone or acetone con-sumption by butane-grown P . butanouora was ob -served upon add i t ion of 2 % CO, a n d N a H C O , ( t o5 m M ) .0, consumption and cell gro wt h associated w ithproposed metabolites of butaneOxida t ions of butane and the products of butanemetabolism require the consumption of 0, eitherdirec tly by butane monooxygenase or for oxidat ion ofthe reduced e lec tron carr iers (e.g. NA DH ). Th e addi t ionof butane o r i ts metabol i tes to ce l ls of P . butanovora isexpected to stimulate 0, consum ption . However , cel lsof P . butanouora had a high initial rate of 0,consumption (endogenous respira t ion) , even without

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Table3.Rates of 0, consumption by butane-grown' P . butanovora' in the presence of various metabolitesAssays were carried out with 25 p1 resuspended cells (100pgprotein) in a 1.6 ml elect rode chamber at 20 "C. The se cellsconsumed butane at a rate of 160 nm ol min-' (m g protein)-'.0, consumpt ion i s measured as nmol 0, consumed min- '(mg protein)-' . T he rate & standard deviat ion for threereplicates is indicated.

Metabol i te added Concn( m M )

0, consumpt ionIni tial rate Rate af ter

10 m inNoneButane 0.241-Butanol 1.00Butyraldehyde 1.00Butyrate 1.00Lactate 1.002-Butanol 1.00

163f 1* 52& 14155& 11 216f 7378f 7 429k 9274& 35 371f 7137&5 290& 9268f 228 f 4300k 166f 1

''Th is init ial rate in the absence of added m etaboli te continued for3 4 min then decayed with a half-life of abou t 3-4 min.

addition of substrates (Table 3). This activity was notlikely to be due to residual butane because C,H, was notinhibitory or due to other exogenous substrates becauseadditional washings did not substantially diminish theactivity. The endogenous respiration rate was constantfor 3-5 min then decayed exponentially with a half-lifeof 3-4 min (data not shown).When butane was added tocell suspensions, no increase above the initial rate ofendogenous respiration was observed (Table 3). How-ever, the rate increased with time rather than decreased,which indicates that butane did support 0, consump-tion. When butane was added at different time pointsafter the endogenous respiration rate had decayed,progressively lower rates of 0 , consumption wereobserved. This loss of activity may indicate an in-activation of the butane monooxygenase when exposedto 0, in the absence of an oxidizable substrate. For 1-and 2-butanol and butyraldehyde, a stimulation in theinitial rate of 0, consumption was observed. For 2-butanol, the rate of 0, consumption decreased withtime, which was expected given the time course of 2-butanol consumption (Fig. lc ). Butyrate exhibited apattern of 0, consumption similar to that of butane.While the time courses of 0, consumption were complexand influenced by changes in endogenous respirationand possibly other factors (e.g. accumulation of meta-bolites), stimulation of 0, consumption was evident forall the compounds tested.' P . butanovora' might also be expected to grow on eachof the proposed intermediates. Therefore, cell growthon 1-butanol (8.8 mM) , butyraldehyde (10 and 2 mM)or butyrate (11.4 mM) was examined. Lactate (19 mM)and 2-butanol (8.8 mM) were included for comparison.All five compounds supported growth. The following

OD,,, values were reached for each substrate after 2 dgrowth: butane, 0.59; 1-butanol, 0.70; butyrate, 0.99;lactate, 0.65; Zbutanol, 0.54; and basal medium (con-taining yeast extract), 0.07. Butyraldehyde at 10 mMwas toxic to the cells but supported growth at 2 mM(OD,,, = 0.21 after 1d). While growth on 1-butanol,butyraldehyde and butyrate is consistent with the role ofthese compounds as intermediates in butane metab-olism, growth on 2-butanol points out the shortcomingsof this approach.DISCUSSIONIn this work, the pathway of butane oxidation in ' P .butanouora' was found to be butane to 1-butanol tobutyraldehyde to butyrate. For the oxidation of any n-alkane, a significant question is whether the oxidationoccurs at the terminal or subterminal carbons, o r both.The site of oxidation determines the products, which, inturn, influence the pathways required to metabolizethese products. In the case of ' P . butanovora', theterminal oxidation product of butane oxidation, 1-butanol, was readily observed (Fig. la ) , while noproduction of the subterminal oxidation product, 2-butanol, was detected. While a low rate of 2-butanolproduction cannot be ruled out (eg .


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Butane m etabolismIPropa no1OH Valerate0

FurthermetabolismHa-C H r C H r H3 CHa- H r CH2- hH2 a H3- H r Hz- g H 0 CH3- C H r H- CO O-Butane I-Butanol Butyraldehyde Butyrate

2-Pentanol 2-ButanolButanone 0HFurtherC Ha -C H rC H - -Ha CHs-CH2-g-CHa 0 etabolism

2-Butano! Butanone... .......... ... ...... ..... .......................... . .... . .................... .................... ..... ............. .......... ........ .... .................................................... ............................ . ........ .......................................................

Fig. 2. Scheme of butane and 2-butanol metabolism by butane-grown P. butanovora. The upper pathway follows fromthe terminal oxidation of butane. The lower pathway shows the oxidation of 2-butano1, although no subterminaloxidation of butane was detected. Inhibitors of each transformation are indicated above the arrows.

oxidation depicted in Fig. 2. This pathway is supportedby direct observatio n of the prod ucts of each step of thepathway. For conversion of butane to 1-bu tano l andbutyra ldehyde to butyra te , i t was necessary to us einhib i tors to s low the consum ption of the product suchthat the product accumulated in the react ion mixture .Th e pathw ay was fur ther confirmed by demo nstra t ionof: (1) consumption of each of the proposed in ter-mediates, ( 2) enhan ceme nt of 0, consumption by eachof the proposed in termedia tes and (3) g r o w t h of thebacter ium on each of the intermediates. Because theamoun t o f subs t ra te consumed was a lways more th anthe amoun t of product tha t accumulated , the possib il i tyremains that o ther pathways are funct ioning s imul-taneously. However, none of the inhibitors completelyblocked the subsequent oxidat ion of the p roduct , w hichmay also account for some, if not all, of the differencebetween the am ount of substra te consumed an d theamount of product tha t accumulated . At present , i t isnot known if there are specific uptake systems foralcohols such as 1- and 2-butanol or carboxylic ac idssuch as butyrate and valerate. Inefficient uptake ofinhib i tors could a lso expla in the incomplete inhib i t ion .Whether o r not a l ternat ive pathw ays exist , the results doindicate th a t the proposed pathw ay is the predominantpathway. Up to 90% of the butane consumed could beaccounted for as 1-butanol . Substant ia l accumulat ionsof butyraldehyde and butyrate are also consistent withthe p roposed pa thway as the dom inan t pa thway .Th e proposed pathway for butane m etabolism is ident-ical to that proposed by van Ginkel e t a f . (1987) forNocardia TB1. Product ion of butyra te f rom butane(when Nocardia TB 1 cel ls were inhib i ted with arseni te)indicates a terminal oxidat ion of butane . However ,product ion of 1 -butanol ( f rom butane) or butyra ldehydefrom ei ther butane or I -butanol was not demonstra teddirec t ly . The proposed pathway of butane metabolismfo r Nocardia TB1 was fur ther supported by measure-ments of activities associated with th e intermed iates. 1-Butanol-dependent 0, consumption was rapid inbutane-grown cells and only slightly above the en-dogenous ra te in succinate-grown Nocardia TB1. Cellsare expected to be ab le to consum e in termedia tes in a

catabol ic pathw ay, provided the co mpo unds are readi lytransported in to the ce lls and are no t toxic to cells at theconcentra t ions used for consum ption assays . Therefore ,consumption of in termedia tes provides support for aproposed pathway. However , 2-butanol-dependent 0,consumption was a lso observed in butane-grown N o -cardia TB 1 but n ot in succinate-grown cel ls . This resul tcould indicate tha t subterminal oxidat ion of butaneoccurs s imultaneously with terminal o xidat ion , tha t 1-butanol and 2-butanol a re oxid ized by the sam e enzyme,o r tha t pa thways fo r ox ida t ion of both 1- and 2-butanolare induced by butane . In the present w ork , i t w as a lsodemons t ra ted tha t bo th 1 -bu tano l and 2 -bu tano l s t imu-lated 0 , consumpt ion ra te s (Tab le 3) and both wereconsumed by butane-gro wn P. butanovora (Fig. l b , c ) .Al though bu tane -g rown P . butanovora d id no t p ro -duce detectable levels of 2-butanol from butane, 2-butanol was nonetheless readily consumed by butane-grown cells. Therefore, the ability to consume a sec-ondary a lcohol does not ensure that a subterminaloxidat ion of the a lkan e occurs . Future w ork will focuson iso lation an d purification of the enzymes involved inbutane metabolism and ident if ica t ion and character-ization of the genes which enc ode these enzymes.

This research was supp or ted by Na t iona l Ins t itu tes of He a l t hg ran t n o . GM.56128 and the O regon A gr icu ltu ra l Exper imen tSta t ion .

Ashraf, W., Mihdhir, A. & Murrell, 1. C. (1994). Bacterial oxidationof propane. FEMS Microbiof Lett 122, 1-6.Baptist, 1. N., Gholson, R. K. & Coon, M. 1. (1963). Hydrocarbonoxidation by a bacterial enzyme system. I . Products of octaneoxidation. Biochim Biophys Acta 69 , 4 0 4 7 .Burrows, K. J., Cornish, A., Scott, D. & Higgins, 1.1. (1984).Substrate specificities of the soluble and particulate methanemono-oxygenases of Methyfosinus trichosporiurn OB3 b. GenMicrobiof 130,3327-3333.Colby, J., Stirling, D. 1. & Dalton, H. (1977). Th e soluble methanemono-oxygenase of Methyfococcus capsufatus(Ba th). Its ability

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to oxygenate n-alkanes , n-alkenes , e thers , and a l icyclic , arom aticand heterocycl ic compou nds . Biochem J 165, 395-402.Ensign, S. A., Small, F. J., Allen, 1. R. & Sluis, M. K. (1998). N e wroles for CO , in the microbial metabolism of al iphat ic epoxidesand ketones . Arch Microbiol 169, 179-187.van Ginkel, C. G., Welten, H. G. J., Hartmans, 5. & de Bont,J. A. M. (1987). Metabolism of t rans-2-butene and butane inNocardia T B 1 . J Gen Microbiol 133, 1713-1720.Gornall, A. G., Bardawill, C. J. & David, M. M. (1949). Deter-minat ion of serum proteins by means of the Biuret reaction. J BiolC h em 177,751-766.Hamamura, N., Page, C., Long, T., Semprini, L. & Arp, D. J. (1997).Chloroform cometabolism by butane-grown CF8, Pseudomonasbutanovora, a n d Mycobacterium vaccae JOB5 and m e thane -grown Methylosinus trichosporium OB3b. Appl Environ Micro-b i o l 6 3 , 3607-3613.Hou, C. T., Patel, R., Laskin, A. I., Barnabe, N. & Barist, 1. (1983).Product ion of methyl ketones from secondary a lcohols by cellsuspensions of C, t o C, n-alkane-grown bacteria . Appl EnvironMicrob io l46 , 178-184.Hyman, M. R., Murton, I. B. & Arp, D. J. (1988). Interact ion ofammonia monooxygenase from Nitrosomonas europaea withalkanes , a lkenes , and alkynes . Appl Environ Microbiol 54,Klug, M. 1. & Markovetz, A. J. (1971). Utilization of al iphat ichydrocarbons by micro-organisms. Adv Microb Phys io l5 , 1-43.Lukins, H. B. & Foster, 1. W. (1963). Methyl ketone metabolism inhydrocarbon-ut i l iz ing mycobacteria . J Bacteriol85, 1074-1087.McLee, A. G., Korm endy, A. C. & Wayman, M. (1972). Isolationand characterizat ion of n-butane-ut il iz ing microorganisms. C an JMicrobiol 18, 1191-1195.Murrell, J. C. (1992). Genetics and molecular biology of m ethano-trophs . FEMS Microbiol Rev 88, 233-248.

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Patel, R. N., Hou, C. T., Laskin, A. I., Felix, A. & Derelanko, P.(1983). O x i d a t i o n of a lkanes by o rgan i sm s grown on C2-C4alkanes . J Appl Biochem 5 , 107-120.Perry, J. 1. (1968). Substrate specificity in hydrocarbon-utilizingmicroorganisms. Antonie Leeuwenhoek 34, 27-36.Perry, J. 1. (1980). Propane ut i l izat ion by microorganisms. A d vAppl Microbiol26, 89-115.Phillips, W. E. & Perry, J. 1. (1974). Metabolism of n-bu tane and 2-bu tanon e by Mycobacterium vaccae. J Bacteriol 120, 987-989.Sluis, M. K., Small, F. J., Allen, J. R. & Ensign, 5. A. (1996).Involvement of an ATP -dependent ca rboxylase in a C0 , -dependen t pa thway of acetone metabolism by Xanthobacterstrain Py2. J Bacteriol 178, 4020-4026.Smith, M. R. & Baresi, L. (1989). Meth ane e s t im a t ion fo r m e thano-genic and methanotrophic bacteria . In Gases in Plant andMicrobial Cells, pp . 275-308. Edited by H. F. Linskens & J. F.Jackson. London : Springer.Stephens, G. M. & Dalton, H. (1986). T h e r o le of the t e rm ina l andsubterminal oxidat ion pathways in propane metabolism bybacteria. J Gen Microbiol 132, 2453-2462.Takahashi, 1. (1980). Product ion of intracellular and extracel lularprotein from n-butane by Pseudomonas butanovora sp. nov. Adz!Appl Microbiol26, 117-127.Vestal, 1. R. & Perry, 1.1. (1969). Divergent metabolic pathwaysfor prop ane and p rop iona te ut il ization by a soi l isola te . J BacteriolWiegant, W. W. & de Bont, 1. A. M. (1980). A new rou te fo rethylene glycol metabolism in Mycobacterium E44. J GenMicrobiol 120, 325-331.

99 , 216-221.

. . .. . . . . . . . . . . .Received 13 October 1998; revised 12 January 1999; accepted 21 January1999.

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