enzymerecruitment allows the biodegradation of ... · recalcitrant hydrocarbon biodegradation 717...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1979, p. 715-7220099-2240/79/10-0715/08$02.00/0

Vol. 38, No. 4

Enzyme Recruitment Allows the Biodegradation ofRecalcitrant Branched Hydrocarbons by Pseudomonas

citronellolisR. RAY FALL,* JEFFREY L. BROWN, AND TERI L. SCHAEFFER

Department of Chemistry, University of Colorado, Boulder, Colorado 80309

Received for publication 19 July 1979

Experiments were carried out to construct pseudomonad strains capable of thebiodegradation of certain recalcitrant branched hydrocarbons via a combinationof alkane and citronellol degradative pathways. To promote the metabolism ofthe recalcitrant hydrocarbon 2,6-dimethyl-2-octene we transferred the OCT plas-mid to Pseudomonas citronellolis, a pseudomonad containing the citronellolpathway. This extended the n-alkane substrate range of the organism, but didnot permit utilization of the branched hydrocarbon even in the presence of a

gratuitous inducer of the OCT plasmid. In a separate approach n-decane-utilizing(Dec') mutants of P. citronellolis were selected and found to be constitutive forthe expression of medium- to long-chain alkane oxidation. The Dec+ mutantswere capable of degradation of 2,6-dimethyl-2-octene via the citronellol pathwayas shown by (i) conversion of the hydrocarbon to citronellol, determined by gas-liquid chromatography-mass spectrometry, (ii) induction of geranyl-coenzyme Acarboxylase, a key enzyme of the citronellol pathway, and (iii) demonstration of,8-decarboxymethylation of the hydrocarbon by whole cells. The Dec+ mutantshad also acquired the capacity to metabolize other recalcitrant branched hydro-carbons such as 3,6-dimethyloctane and 2,6-dimethyldecane. These studies dem-onstrate how enzyme recruitment can provide a pathway for the biodegradationof otherwise recalcitrant branched hydrocarbons.

The utilization of n-alkanes by microorga-nisms has been the subject of many recent stud-ies, especially in relation to the environmentalimpact of oil spills (see reference 4 for a review).The biodegradation of n-alkanes proceeds pri-marily via terminal oxidation to yield the n-alkanoic acid, which is then degraded by theclassical fatty acid oxidation sequence (14, 18,26). Branched alkanes are generally less suscep-tible to biodegradation (16, 21, 22), and certainalkyl branches can result in environmental re-calcitrance (1). In this latter category are ,B-alkyl-branched alkanes, since this substitutionpattern prevents simple /-oxidation from thebranched terminus (16, 21). In an accompanyingpaper (22) we demonstrated that an anteiso-dimethyl-substituted octane such as 3,6-di-methyloctane (3,6-DMO) is not utilized by avariety of hydrocarbonoclastic microorganismsthat effectively oxidize an unbranched alkanesuch as n-octane.We also tested as growth substrate 2,6-di-

methyl-2-octene, a branched dimethyloctenewhich could potentially give rise to the isopren-oid citronellol, as shown in Fig. 1, if a suitableterminal oxidation of the anteiso-terminal

methyl were to occur. The significance of thisidea is that conversion to citronellol in microor-ganisms capable of degrading this isoprenoidcould lead to degradation of a recalcitrant car-bon skeleton. Metabolism of citronellol occursin certain pseudomonads, including Pseudomo-nas citronellolis (8, 23) and Pseudomonasaeruginosa, and some acinetobacteria (8). Sincesome strains of each of these bacteria also arecapable of n-alkane oxidation, an induction ofboth pathways could, in principle, provide amechanism for degradation of 3-methyl-branched alkanes. The studies described herefocus on attempts to produce strains of P. citro-nellolis that are capable of such pathway re-cruitment, allowing the biodegradation of oth-erwise recalcitrant branched hydrocarbons.

MATERIALS AND METHODSBacteria and growth conditions. The pseudo-

monad strains used are described in Table 1. Cellswere routinely grown in a mineral medium (H me-dium) that contained (per liter): 4.36 g of K2HPO4,1.72 g of NaH2PO4* H20, and 2.0 g of NH4Cl sterilizedtogether; and 10 ml of a filter-sterilized metals mixadded separately. The metals mix contained (per liter):300 mg of CaCl2.H20 dissolved first; and 10.0 g of

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716 FALL, BROWN, AND SCHAEFFER

[ALKANE|PATHWAY|

2,6-dimethyF2-octene 3,6-DMO 2,6-DMD

I 'recruitedlhydroxylose' IZL2L0CHOH HOH LLCHHOH2 2 2

Citronellol I- 7 fl-oxidation

A-*CH COSCoAI K

Igeranyl-COAR cqbcav ase -

COSCoA 'IC02W2

WhereR-k-l

eCO H2

RvCOSCoA

-+CH"CO HL C3 2

bOSCOAI Rs

or ,3- oxidation &

further degrtionFIG. 1. Acquired pathways for the assimilation of 3-methyl-branched hydrocarbons in P. citronellolis.

Various aspects of the alkane and citronellol pathways are discussed in the text.

TABLE 1. Pseudomonas strainsaStrain Phenotype Plasmid Source or reference

P. citronel-lolis

Fl Wild type ATCC 17643 (8)F14 Met- This studyF1404 Oct' Met- OCT Conj. AC4

F14F1437 Oct' Met- OCT Conj. P. oleovor-

ans - F14F Dec3 Dec' This studyF Dec6 Dec' This study

P. putidaAC4 Oct' Trp- OCT, A. Chakrabarty

K (9)AC9 Met- A. Chakrabarty

(9)

P. oleovorans8062 Wild type, OCT ATCC

Oct+a Abbreviations used: Met, methionine; Oct, n-oc-

tane; Dec, n-decane; Trp, tryptophan; Conj., conjuga-tion; ATCC, American Type Culture Collection.MgSO4. 7H20, 500 mg of FeSO4. 7H20, 500 mg ofascorbic acid, 100 mg of MnSO4 * H20, 20 mg ofNa2MoO4.2H20, 10 mg of CuSO4.5H20, 10 mg of

ZnS04 a7H2O, and 4 mg of H3BO3. Hydrocarbongrowth screening was carried out as described byNieder and Shapiro (19), except that H medium wasused. For some liquid cultures with hydrocarbon sub-strates, H medium supplemented with 0.01% (wt/vol)peptone and yeast extract (Difco Laboratories) wasused (HPY medium). When dicyclopropyl ketone wasadded, a filter-sterilized stock solution (10 mM) wasused. For amino acid auxotrophs, the appropriate L-amino acid was added at 20 ug/ml.

Selection ofmutants. Isolation of methionine aux-otrophs of P. citronellolis was carried out by cycloser-ine enrichment (20) after mutagenesis with ethylmethane sulfonate (Sigma Chemical Co.) as previouslydescribed (28). One such mutant, F14, was used in thisstudy. Isolation of mutant strains of P. citronellolisable to grow on decane as a sole carbon source (Dec')was readily achieved by mutagenizing cells as aboveand culturing washed survivors in H medium contain-ing 20 IL of decane per ml. Numerous Dec' cloneswere obtained, and two, F Dec3 and F Dec6, used inthese studies are described in the text.Transfer of the OCT plasmid. Two different do-

nor strains were used, including Pseudomonas oleo-vorans 8062 and Pseudomonas putida AC4, each har-boring the OCT plasmid (9). The latter strain alsocontains the transfer plasmid K. Conjugations wereset up as previously described (9) with P. citronellolisF14 as recipient. Selection was for Oct+ exconjugateson solid medium lacking the amino acid tryptophan

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RECALCITRANT HYDROCARBON BIODEGRADATION 717

(required for the donor) and supplemented with me-thionine (required for the recipient). Exconjugateclones were checked for a methionine requirement,octane utilization, and ability to utilize citronellol (8).

Materials. All materials used were reagent gradeas previously described (8). Hydrocarbon substrates,including n-alkanes, 2,6-dimethyldecane (2,6-DMD),and 3,6-dimethyloctane (3,6-DMO) were 99+% purityfrom Chemical Samples Co. 2,6-Dimethyl-2-octenewas synthesized as described elsewhere (22). Beforeuse in growth tests, each hydrocarbon was passed overa silicic acid column to remove traces of oxidizedsubstrates.

Cellular conversion of 2,6-dimethyl-2-octene.Bacteria grown in HPY-octane medium were har-vested and washed by centrifugation with H mediumcontaining no carbon source. Washed cell suspensions(10 ml) were incubated at 30°C with 120 mg of hydro-carbon for 2 h. Incubations were carried out with Hmedium supplemented with 20 mM sodium borate,pH 7.0; the presence of borate inhibits the furtheroxidation of the alcohol produced (3, 13), causing it toaccumulate and thus facilitating its subsequent iden-tification. In control experiments, cells incubated un-der these conditions with n-octane were shown toaccumulate n-octanol.

After the incubation period, cells were washed withsaline and then acidified with HCl and extracted twicewith 1 volume of ether (phases separated by centrifu-gation). The ether phase was washed twice with 1%(wt/vol) NaHCO3 and then water and evaporatedunder a stream of N2, and the residue was taken up inmethanol. The products of hydrocarbon conversionwere analyzed by gas chromatography and combinedgas chromatography-mass spectrometry with a Hew-lett-Packard 5710A gas chromatograph and 5982Adata system. Samples were injected on a Carbowax20M column (6 ft [ca. 1.83 m] by 2-mm inside diame-ter) prepared as described by Aue et al. (2) with ahelium flow rate of 3.5 ml/min, injector temperatureof 100°C, and temperature programming from 100 to3000C.Enzyme assay. Induction and assay of geranyl-

coenzyme A (CoA) carboxylase by ["4C]C02 fixationwere carried out as previously described (8), except forthe way in which induction was achieved. Strains weregrown in HPY medium supplemented with 10 mMsuccinate to an absorbance of approximately 1.0 at 660nm. The cells were harvested and washed with HPYmedium by centrifugation, suspended at an absorb-ance of 0.5 in HPY medium supplemented as indicatedin the text, and then shaken vigorously for 12 h at30°C. Cells were then harvested and assayed for ger-anyl-CoA carboxylase as previously described (8). Thismodification produced significantly greater inductionof the enzyme than previously noted (8). ["4C]NaHCO3was obtained from New England Nuclear Corp. Pro-tein content of cell extracts was measured by a micro-biuret procedure (15).

Determination of fi-decarboxymethylation.Verification of fB-decarboxymethylation was carriedout by measuring ["4C]C02 fixation into acetic acid byisolation of the acid as ['4C]acetylhydroxamate essen-tially as described by Seubert and Remberger (24).Additional verification was obtained by thin-layerchromatography of the ['4C]acetylhydroxamate on

cellulose plates (Eastman Chemical Products, Inc.),using a solvent of water-saturated ether-formic acid(3:1), and by demonstrating cochromatography withauthentic acetylhydroxamate (Rf 0.59); propionylhy-droxamate (Rf 0.83) was completely resolved in thissystem.

RESULTSAlkane specificity ofP. citronellolis. Wild-

type P. citronellolis, Fl, was unable to utilize2,6-dimethyl-2-octene as a sole carbon source(see reference 22 and Table 2). To determinewhether this could be a chain length effect, theability of P. citronellolis Fl to use n-alkanes ofvarious chain lengths was determined (Table 2),and, as shown, the organism grew with n-alkanesof 12 to 16 carbons but not with the shorter-chain alkanes. (Several growth tests shown inTable 2 will be described below.)Transfer of OCT to P. citronellolis. To

expand the chain length specificity of P. citro-nellolis Fl to shorter-chain alkanes, we madeseveral attempts to introduce the OCT plasmid,which permits growth of P. putida strains on 6-to 10-carbon n-alkanes (10, 12). As donor, wefirst examined a P. putida strain which carriesOCT and K, a transfer plasmid which mobilizesthe chromosome (9). Conjugation experimentswere carried out as shown in Table 3, using as arecipient a methionine auxotroph of P. citronel-lolis, F14. We also carried out conjugation di-rectly with P. oleovorans 8062, the original OCTstrain (11). With either, donor transfer of OCTto P. citronellolis F14 occurred at only a lowfrequency (2 x 10-8 to 5 x 10-8). However,analysis of exconjugate clones conclusively dem-onstrated that OCT was transferred to P. citro-nellolis F14 (Table 3) with retention of theability to utilize citronellol, a characteristic of P.citronellolis but not of P. oleovorans or of P.putida (8).The n-alkane specificity of exconjugate P. ci-

tronellolis F1404 and F1437 is shown in Table 2.The presence of both OCT and the endogenouslong-chain hydrocarbon oxidase system allowedthese two strains to grow with all the n-alkanestested. However, neither of these strains couldutilize 2,6-dimethyl-2-octene (Table 2). In sepa-rate growth tests where the gratuitous inducerof OCT, dicyclopropyl ketone (12, 27), was in-cluded in the plates, no growth was observed forthe two strains on 2,6-dimethyl-2-octene. Theseresults suggested that specificity of the o-hy-droxylase coded for by OCT is such that 2,6-dimethyl-2-octene is not effectively converted tocitronellol, although other explanations are pos-sible.

Selection for mutants of P. citronelloliswith an altered n-alkane specificity. In aseparate approach to promoting the oxidation of

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TABLE 2. Summary ofgrowth ofpseudomonad strains on various hydrocarbonsGrowth on hydrocarbon substrate'

Strain ~~~~~~~~~~~~~~~~~~~~~2,6-Di-n-6 n-8 n-10 n-il n-12 n-14 n-16

meth- 3,6- DMD

octene

P. citronellolisFl - - - wkb + + + - - -F14 - - - wk + + + -F1404 + + + + + + + -

F1437 + + + + + + + -F Dec3 - wk + + + + + wk wk +F Dec6 - wk + + + + + wk wk +

P. putida AC4 + + + - - - _ _ _ _

P. oleovorans 8062 + + + - - - _ _ _ _

a Abbreviations: for hydrocarbon substrates, n-6 represents n-hexane, etc.bwk, Weak growth; in these cases, reproducible weak growth was established after repeated subculture in

liquid H medium supplemented with the indicated hydrocarbon.

TABLE 3. Transfer of the OCTplasmid to P. citronellolis'

SeetdTransferDonor Recipient Selected fre- Exconjugate phenotype

quency

P. putida AC4 (OCT', K+, Trp-) P. putida AC9 (Met-) Oct+ 2 x 10-5 Oct+ Met+ (90%)Oct' Met- (10%)

P. oleovorans 8062 (OCT') P. putida AC9 (Met-) Oct+ -i0-9 Oct+ Met-P. putida AC4 (OCT+, K+, Trp-) P. citronellolis F14 (Met-) Oct+ 5 x 10-8 Oct+ Met+ (10%)

Oct+ Met- Cit- (60%)Oct+ Met- Cit+ (30%)

P. oleovorans 8062 (OCT+) P. citronellolis F14 (Met-) Oct+ 2 x 10-8 Oct+ Met- Cit+' The abbreviations used are described in Table 1, footnote a, except for Cit, which is citronellol utilization.

2,6-dimethyl-2-octene, we attempted to expandthe substrate range of the alkane-oxidizing sys-tem of P. citronellolis Fl to encompass ashorter-chain alkane such as decane. The wildtype (Fl) does not assimilate decane (Table 2).Cells were mutagenized with ethyl methane sul-fonate, and survivors were grown with a decaneenrichment. Clones which exhibited excellentgrowth on decane (Dec') were isolated fromnine different enrichment cultures. Analysis oftwo of these mutants in growth tests is shown inFig. 2A. In comparison to the wild type, P.citronellolis F Dec3 and F Dec6 are constitutivefor the assimilation of decane or hexadecane; thewild type exhibits a pronounced lag beforegrowth on hexadecane begins and is unable touse decane. Both P. citronellolis F Dec3 and FDec6 were able to utilize n-octane as a solecarbon source, although only a weak growthresponse was obtained (Table 2).These results show that the substrate range

of the medium- to long-chain alkane oxidaseoverlaps with that of the OCT system, but theinducer specificity is such that only the longer-chain alkanes (or their oxidation products), con-

taining 12 to 16 carbons, are effective inducers.That constitutive expression of the medium- tolong-chain alkane-oxidizing system allows themetabolism of recalcitrant branched hydrocar-bons is shown in Table 2. Both P. citronellolisF Dec3 and F Dec6 were able to grow slowly on2,6-dimethyl-2-octene or 3,6-DMO on solid me-dium and more extensively on the longer-chain,branched hydrocarbon 2,6-DMD (Table 2). Thewild type was unable to assimilate 2,6-DMD orthe shorter-chain, branched hydrocarbons (asmentioned above).The growth of the P. citronellolis F Dec3

mutant in liquid culture on n-decane or the threebranched hydrocarbons is shown in Fig. 2B.After an approximately 6-h lag, slow growthoccurred with either 2,6-dimethyl-2-octene or3,6-DMO, and somewhat faster growth occurredwith 2,6-DMD as substrate. The growth lag seenwith the branched hydrocarbons, but not n-de-cane (Fig. 2B), presumably represents an induc-tion period for the enzymes of the citronellolpathway (see below). Similar growth propertieswere seen with P. citronellolis F Dec6.Conversion of 2,6-dimethyl-2-octene to



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citronellol by P. citronellolis F Dec3. Sinceit seemed likely that the acquired ability of P.citronellolis F Dec3 and F Dec6 to assimilate

HOURS

6 12 18 24 30 36HOURS

FIG. 2. Growth of P. citronellolis and Dec' mu-tants. Growth studies were carried out at 30°C asdescribed in the text. (A) Growth of P. citronellolisFl on n-hexadecane (0) or n-decane (-), P. citronel-lolis F Dec3 on n-hexadecane (O) or n-decane (O),and P. citronellolis F Dec6 on n-hexadecane (A) orn-decane (A). (B) Growth of P. citronellolis F Dec3on n-decane (a), 2,6-DMD (0), 2,6-dimethyl-2-octene(U), or 3,6-DMO (O). Growth was monitored by ab-sorbance measurements at 660 nm (A64.

2,6-dimethyl-2-octene was initiated by the oxi-dation of the hydrocarbon to citronellol, we car-ried out experiments to measure the oxidationproducts of whole cells incubated with the hy-drocarbon. P. citronellolis F Dec3 was grown onan octane-containing medium as describedabove, and washed cells were allowed to metab-olize 2,6-dimethyl-2-octene under conditions inwhich alkanols (and alkenols) would accumulate(3, 13). Extracts of the alkanol-plus-alkenol frac-tion were analyzed by combined gas chromatog-raphy-mass spectrometry (Fig. 3 and 4). Figure3 shows the elution profile obtained by gas chro-matography of such an extract (the solvent-plus-hydrocarbon peak has been substracted out ofthe profile). The major peak exhibited a reten-tion time (3.9 min) identical to that for authenticcitronellol. Analysis of this major peak by mass

w

w

cLIw

0)I

I IUE

FIG. 3. Gas chromatography of the products ofoxidation of 2,6-dimethyl-2-octene by P. citronellolisF Dec3. The details of the oxidation of 2,6-dimethyl-2-octene by P. citronellolis F Dec3 cells and subse-quent gas chromatographic analysis of accumulatedalkanols and alkenols are described in the text. Themass spectrum of the major peak observed (retentiontime, 3.9 min) is displayed in Fig. 4.

I-I I.~~~ ~~~~~~~~~~__ _MIAL

'-I--1-U*-- ----- -N-

CHOH2

ci'tronellol

1I L I

140 I60

1004LUz

z:Dm

LU

OJLUJ

80

60

40

20

aiI Lw-w- w~- 11-- 11 - -I- 1-7 Iv-1- --W - * *a. 4 so Be le is.

mleFIG. 4. Mass spectrum of the major peak from Fig. 3.

.-wI - -

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spectrometry is displayed in Fig. 4. The massspectrum obtained was virtually identical withthat obtained for authentic citronellol (25),which was run separately. This confirmed thatP. citronellolis F Dec3 was capable of terminaloxidation of 2,6-dimethyl-2-octene to producecitronellol as the major product. Identificationof the other small peaks shown in Fig. 3 was notcompleted, although the peak with a retentiontime of 1.5 min was shown to be n-octanol. Thisalkanol would be expected from oxidation ofresidual n-octane still present in the cells. It hasnot yet been established whether the citronellolproduced by P. citronellolis F Dec3 is a mixtureof the DL-isomers, nor is it known whether P.citronellolis can utilize both isomers which dooccur naturally.Induction of geranyl-CoA carboxylase

and verification of fi-decarboxymethyla-tion. To establish that the citronellol producedby P. citronellolis F Dec3 from 2,6-dimethyl-2-octene is metabolized via the citronellol pathway(8, 23), we carried out experiments to measure

induction of geranyl-CoA carboxylase, a key en-

zyme of the pathway (8, 23), and to demonstratedirectly that the 6-methyl group of 2,6-dimethyl-2-octene (which becomes the fB-methyl group ofcitronellol [Fig. 1]) is removed via the ,B-decar-boxymethylation sequence (23).The induction of geranyl-CoA carboxylase by

citronellol in the wild-type strain is shown inTable 4. Exposure of the wild type to 2,6-di-methyl-2-octene resulted in no induction of theenzyme. In addition, 3,6-DMO and 2,6-DMDwere tested as potential inducers and also failedto produce induction (Table 4). In contrast, themutant strain P. citronellolis F Dec3 showedinduction of geranyl-CoA carboxylase when ex-

posed to citronellol or any of the three branchedhydrocarbons. This is consistent with the con-

version of the three branched hydrocarbons tosubstances capable of induction of the enzyme.The nature of the inducer of geranyl-CoA car-boxylase is unknown, but there apparently is nota rigid structural specificity involved since thethree branched hydrocarbons would yield very

different intermediates (Fig. 1). In this regard itis worth noting that 3-methylpentanoic (or a

metabolite thereof) is a good inducer of theenzyme (E. P. Lau and R. R. Fall, unpublishedobservation).Although the induction of geranyl-CoA car-

boxylase could be demonstrated in the wild typeor P. citronellolis F Dec3 within 2 h after ex-

posure to citronellol (data not shown), maximuminduction of the enzyme in P. citronellolis FDec3 exposed to either 2,6-dimethyl-2-octene or

2,6-DMD took much longer (6 to 8 h), presum-

ably due to a relatively slow accumulation of the


TABLE 4. Induction ofgeranyl-CoA carboxylaseand detection of 83-decarboxymethylationa

Carboxyl-ase activ- Radioac-

P. citronel- Substrate ity (nmol/ tivity inlolis strain min per acetic acid

mg of pro- (cpm)btein)

Fl Succinate 0.4 670Citronellol 14.5 35,4502,6-Dimethyl- 0.3 801

2-octene3,6-DMO 0.4 5902,6-DMD 0.7 915

F Dec3 Succinate 0.3 785Citronellol 17.8 29,7422,6-Dimethyl- 9.5 14,353

2-octene3,6-DMO 8.7 12,9592,6-DMD 15.1 17,842

a The induction conditions are described in the text.Washed cells were exposed to 10 mM succinate, or theindicated hydrocarbon or citronellol, each at a 0.05%(vol/vol) final concentration. After the induction pe-riod, cells were (i) assayed for geranyl-CoA carboxyl-ase as previously described (8) and (ii) incubated with[I4C]NaHCO3 and the indicated substrate to measurethe production of [14C]acetic acid. This latter deter-mination was carried out as described by Seubert andRemberger (24), except that [14C]NaHCO3 with a spe-cific activity of 50 mCi/mmol (New England NuclearCorp.) was used, and the [14C]acetohydroxamate wasquantitated by liquid scintillation counting of the ap-propriate region of the plate after thin-layer chroma-tography.'The average of duplicate determinations.

inducer. This probably explains the rather longgrowth lags seen in Fig. 2B.To establish that hydrocarbon degradation

was occurring by a /?-decarboxymethylation se-quence, whole cells were exposed to the appro-priate hydrocarbon and [14C]NaHCO3, and theproduction of [14C]acetic acid was monitored byconversion of the cellular acetate pool to acetyl-hydroxamate and measurement of [14C]acetyl-hydroxamate as described above. The produc-tion of [14C]acetate in the wild type was seenwhen cells were incubated with citronellol aspreviously described (8, 23), but not with succi-nate or the branched hydrocarbons (Table 4). Incontrast, P. citronellolis F Dec3 was able toeffect fi-decarboxymethylation as monitored by[I4C]acetate production when exposed to eithercitronellol or any of the three branched hydro-carbons (Table 4).These results are consistent with the degra-

dative pathways illustrated in Fig. 1 and showthat the degradative enzymes associated withthe citronellol pathway do not have rigid speci-


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ficity for the nature of the branched-hydrocar-bon terminus.

DISCUSSIONThe general resistance of alkyl-branched com-

pounds to biodegradation has been appreciatedfor several years (1, 14, 16). The particular prob-lem of hydrocarbon skeletons containing a 3-methyl branch has become recognized more re-cently from the studies of McKenna (16) andPirnik (21). McKenna showed that although avariety of hydrocarbonoclastic bacteria wereable to degrade 2-methyl-branched alkanes, 3-methyl-branched alkanes were attacked by veryfew species. Pirnik demonstrated that the me-tabolism of 2,6-DMD by Brevibacterium ery-throgenes is largely prevented by the productionof an intermediate 3-methyl acyl-CoA species.Such a species is not susceptible to ordinary ,B-oxidation because of the presence of the alkylgroup in the 3 position. Our own recent studieswith a wider range of hydrocarbonoclastic mi-croorganisms, including bacteria and fungi, haveshown that of 27 strains able to utilize n-octaneas a sole carbon source, none was able to assim-ilate 3,6-DMO, a hydrocarbon with an anteiso-substitution pattern at each terminus (22).These studies point to the general conclusionthat a 3-methyl branch represents a recalcitrantstructural feature preventing microbial biodeg-radation.

In the work described here we have attemptedto combine an existing pathway, the citronellolpathway, that is present in certain pseudomon-ads (8) with the alkane oxidation pathway toeffect the degradation of 3-methyl-branched hy-drocarbons. For example, the compound 2,6-di-methyl-2-octene, also a recalcitrant structure(22), appeared to be only one step away fromconversion to citronellol (Fig. 1). That is, ter-minal hydroxylation at the anteiso-terminuswould produce citronellol, which should be de-graded by the existing pathway. We focusedthen on determining whether the alkane-hy-droxylating systems present in various pseudo-monads (7, 19), including P. citronellolis, couldbe capable of this terminal hydroxylation of 2,6-dimethyl-2-octene.Our first approach was to extend the range of

n-alkanes that could be degraded by P. citronel-lolis by insertion of the OCT plasmid. The OCTplasmid has been transferred from the originalhost, P. oleovorans, probably a P. putida strain(9), to a variety of other P. putida strains, con-ferring the ability to metabolize n-alkanes of 6to 10 carbons (6). We were successful in trans-ferring OCT to P. citronellolis, a fluorescentpseudomonad taxonomically similar to but dis-

tinct from P. putida (8), and the exconjugatestrains obtained were able to utilize short- tomedium-chain alkanes of 6 to 10 carbons, as wellas medium- to long-chain alkanes of 11 to 16carbons. Unfortunately, this effort did not pro-duce a strain capable of metabolism of 2,6-di-methyl-2-octene, even when the enzymes of theOCT plasmid were induced by a gratuitous in-ducer. In separate experiments with P. oleovor-ans and P. putida AC4, both harboring OCT,we were unable to observe the oxidation ormetabolism of 3,6-DMO or 2,7-dimethyloctane(22). These results are consistent with the con-clusion that the w-hydroxylase (5, 17) coded forby the OCT plasmid has a substrate-binding sitethat accommodates linear unbranched alkanesof 6 to 10 carbons, but only poorly bindsbranched analogs. A similar conclusion wasreached by workers studying the alkane hydrox-ylase of P. aeruginosa (26).As a second approach to promoting the deg-

radation of 2,6-dimethyl-2-octene by P. citronel-lolis, we considered the possibility that the me-dium- to long-chain alkane oxidation systempresent in the wild-type strain might be able toinitiate the oxidation of the branched hydrocar-bon, if expressed. We set up a selection formutants able to utilize n-decane and readilyisolated several different Dec+ mutants. Thesemutants proved to be constitutive for the oxi-dation of medium- to long-chain alkanes andsimultaneously acquired the ability to assimilate2,6-dimethyl-2-octene or 3,6-DMO as a sole car-bon source, although at a slow rate. Bettergrowth was obtained with 2,6-DMD, a longer-chain hydrocarbon. Possibly 2,6-DMD is a bet-ter substrate for the medium- to long-chain al-kane hydroxylase than 2,6-dimethyl-2-octene or3,6-DMO, perhaps due to its chain length or lesshindered terminus or both. The substrate spec-ificity of this alkane hydroxylase, especially inregard to its hydroxylation of branched alkanes,remains to be investigated. Our results suggestthat it contains a substrate-binding site that willaccommodate at least some methyl-branchedalkanes.The results that we obtained with P. citronel-

lolis F Dec3 on (i) terminal oxidation of 2,6-dimethyl-2-octene to produce citronellol, (ii) in-duction of geranyl-CoA carboxylase, and (iii)detection of f-decarboxymethylation of the hy-drocarbon are all consistent with the view thatthe degradation of 2,6-dimethyl-2-octene by themutant proceeded via a combination of the al-kane and citronellol pathways (Fig. 1). We haverecently described a similar case in spontane-ously arising mutants of P. aeruginosa whichacquire the ability to utilize 2,6-dimethyl-2-oc-

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tene by constitutive expression of a medium- tolong-chain alkane oxidation system combinedwith induction of the citronellol pathway (J.Brown and R. Fall, unpublished observation).Our data on the assimilation of 3,6-DMO and

2,6-DMD by the Dec+ mutants are also consist-ent with the common pathway for 3-methyl-branched alkyl moieties depicted in Fig. 1. Theseare clear examples of how enzyme recruitmentcan lead to the acquisition of new degradativepotential and to the evolution of new metabolicpathways.

ACKNOWLEDGMENTSWe thank A. Chakrabarty for supplying microbial cultures,

Susan Cantwell for excellent technical assistance, RobertShapiro and Robert Sievers for allowing us to use their gaschromatography and mass spectrometry equipment, and Rob-ert Barkley for running the gas chromatograph-mass spec-trometer.

This investigation was supported in part by Public HealthService grant HL 16628 from the National Institutes of Healthand by a grant from the Council on Research and CreativeWork of the University of Colorado.

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