APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1992, p. 2723-27290099-2240/92/092723-07$02.00/0Copyright © 1992, American Society for Microbiology

Physiological Properties of a Pseudomonas Strain WhichGrows with p-Xylene in a Two-Phase

(Organic-Aqueous) MediumDIANA L. CRUDEN,1 JAMES H. WOLFRAM,2 ROBERT D. ROGERS,2 AND DAVID T. GIBSON'*

Department of Microbiology and Center for Biocatalysis and Bioprocessing, The Universityof Iowa, Iowa City, Iowa 52242,1 and Idaho Research Center, Idaho National

Engineering Laboratory, Idaho Falls, Idaho 834152

Received 3 February 1992/Accepted 11 June 1992

Pseudomonas putda Idaho utilizes toluene, m-xylene, p-xylene, 1,2,4-trimethylbenzene, and 3-ethyltolueneas growth substrates when these hydrocarbons are provided in a two-phase system at 5 to 50%o (vol/vol).Growth also occurs on Luria-Bertani medium in the presence of a wide range of organic solvents. The abilityof the organism to grow in the presence of organic solvents is correlated with the logarithm of the octanol-waterpartition coefficient, with dimethylphthalate (log POCT = 2.3) being the most polar solvent tolerated. Duringgrowth with p-xylene (20%o [vol/voll), there was an initial lag period accompanied by cell death, which was

followed by a period of exponential growth. The stationary phase of growth was characterized by a dramaticdecrease in cell viability, although cell dry weight and turbidity measurements slowly increased. Electronmicrographs revealed that during growth in the presence of p-xylene, the outer cell membrane becomesconvoluted and membrane fragments are shed into the culture medium. At the same time, the cytoplasmicmembrane invaginates, forming vesicles, and becomes disorganized. Electron-dense intracellular inclusionswere observed in cells grown with p-xylene (20%o [vol/vol]) and p-xylene vapors, which are not present in cellsgrown with succinate. Attempts to demonstrate the presence of plasmid DNA in P. putida Idaho were negative.However, polarographic studies indicated that the organism utilizes the same pathway for the degradation oftoluene, m-xylene, and p-xylene as that used by P. putida mt-2 which contains the TOL plasmid pWWO.Southern hybridization experiments showed that P. putida Idaho contains regions of DNA that are homologousto genes encoding enzymes of the upper operon in the TOL plasmid pWWO.

There is considerable interest in the ability of microorgan-isms to grow in two-phase (organic-aqueous) systems wherethe major component is an organic solvent. Much of thiswork is directed towards the development of technology forthe production of value-added chemicals from hydrophobicsubstrates (5, 9, 10, 21, 28). Additional interest stems fromthe fact that many hydrophobic solvents are classified asenvironmental pollutants and their degradation by microor-ganisms is the focus of the emerging bioremediation indus-try.

Volatile aromatic hydrocarbons fall into both of the abovecategories. For example, bacterial oxygenases convert ben-zene (2, 11, 22), toluene (12, 16), and p-xylene (34, 35) intouseful chemical intermediates that are not readily availableby conventional synthetic chemistry. Benzene, toluene, andxylenes are also components of gasolines and are found ingroundwater contaminated by leakage of liquid fuels fromunderground storage tanks (19, 25).Aromatic hydrocarbons are usually toxic to microorgan-

isms when they are present in a two-phase system, andtoluene is frequently used to inhibit the growth of microor-ganisms. Toxicity is apparently due to the interaction oftoluene with the cytoplasmic membrane, leading to the lossof the cations Mg2+ and Ca2+, as well as other smallmolecules (8).

It is clear from the above observations that bacterialstrains resistant to the toxic effects of low-molecular-weightaromatic hydrocarbons are desirable for biotransformation

* Corresponding author.

and bioremediation studies. Recent reports have shown thatcertain strains and mutants ofPseudomonasputida can growin the presence of more than 50% (vol/vol) toluene whenother sources of carbon and energy are available for growth(17, 18, 30). In addition, mutant strains of Escherichia coliK-12 have been isolated that can grow in complex media inthe presence of 10% (vol/vol) p-xylene (1). None of theseorganisms can utilize toluene orp-xylene as a transformationor growth substrate.We now report some physiological properties of a strain of

P. putida that can grow with p-xylene, toluene, and otheralkylbenzenes as sole sources of carbon and energy whenthese aromatic hydrocarbons are provided at 5% to morethan 50% (vol/vol) in the culture medium.

MATERIALS AND METHODS

Organisms and growth conditions. Strains of bacteria andplasmids used are listed in Table 1. P. putida Idaho has beendeposited with the Northern Regional Research Center,U.S. Department of Agriculture, Peoria, Ill., with the acces-

sion number NRRL B-18435 (37). All bacteria were stored at-70°C in Luria-Bertani (LB) medium (1% [wt/vol] trypti-case, 0.5% [wt/vol] yeast extract, 0.5% [wt/vol] NaCl) con-

taining 25% [wt/vol] glycerol. Cultures of P. putida Idahowere routinely grown in mineral salts basal (MSB) medium(33) withp-xylene or toluene added directly to the medium at5% (vol/vol) or on plates of MSB medium solidified with1.5% agar flooded with 5.0 ml ofp-xylene or toluene.Growth in the presence of organic solvents was measured

in LB medium with 25% (vol/vol) solvent. The total volume

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TABLE 1. Bacterial strains and plasmids

Strain or plasmid Relevant characteristics Reference orderivation

BacteriaP. putida Idaho Utilizes p-xylene or toluene in a 37(NRRL B- two-phase system where the18435) hydrocarbons are present at 5

to 50% (vol/vol)P. putida Fl Oxidizes toluene through cis- 13

toluene dihydrodiol (dihy-drodiol pathway)

P. putida mt-2 Oxidizes toluene through ben- 36zyl alcohol (TOL plasmidpathway)

P. mendocina KR1 Oxidizes toluene through 34p-cresol (toluene 4-monoxy-genase pathway)

Plasmid pED3306 Apr; pBR322 hybrid plasmid 24containing the upper pathwayoperon promoter and thexy1C, xylM, and xyLA genesfrom the TOL plasmidpWWO

of medium plus solvent was 25% of the culture flask. Eachflask was sealed with a butyl rubber stopper and incubated at30°C on a shaker operating at 200 rpm. Growth was mea-

sured by the increase in turbidity at 600 nm, the total numberof viable cells per milliliter as determined by the most-probable-number procedure on LB agar plates, and by celldry weight measurements.DNA procedures. Pseudomonas total DNA was purified by

the method described by Davis et al. (7). E. coli plasmidDNA was purified by the method of Birnboim and Doly (4).The procedures of Hansen and Olsen (14), Kado and Liu(20), and Connors and Barnsley (6) were used in attempts todetect plasmid DNA in P. putida Idaho. Pseudomonas sp.strain NCIB 9816, which contains an 81-kb catabolic plasmiddesignated pDTG1 (29), was used as a positive control. DNAwas analyzed by electrophoresis in 0.75% agarose gels byusing standard procedures (23). Southern hybridization ex-

periments were performed by using the Genius nonradioac-tive DNA labeling and detection kit (Boehringer MannheimBiochemicals, Indianapolis, Ind.). Probes were prepared byrandom primed labeling with digoxigenin according to themanufacturer's instructions. Hybridization was performedovernight at 42°C in the presence of 50% formamide. Filters(Nytron transfer membranes; Schleicher & Schuell, Dassell,Germany) were washed under conditions of high stringency,twice for 15 min at room temperature in 2x SSC (lx SSC is0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodiumdodecyl sulfate (SDS) and twice for 15 min at 68°C in 0.1 xSSC-0.1% SDS.

Substrate oxidation. Oxygen uptake was measured polaro-graphically at 30°C with a Rank Brothers Oxygraph. Cellswere grown to mid-log phase, harvested by centrifugation,washed with 50 mM potassium phosphate buffer (pH 7.2),and resuspended in the same buffer to give a turbidity of 1.0at 600 nm. Reaction mixtures containing 0.9 ml of 50 mMpotassium phosphate buffer (pH 7.2) and 0.1 ml of cellsuspension were equilibrated at 30°C before the addition ofsaturating amounts of substrate dissolved in 10 ,ul of H20 or

dimethylformamide. Results reported are corrected for

endogenous oxygen consumption in the absence of sub-strate.

Electron microscopy. Cells of P. putida Idaho grownovernight with 20 mM succinate or p-xylene vapors or inmedium containing 20% p-xylene (vol/vol) were harvestedand quickly washed with 50 mM potassium phosphate buffer(pH 7.2). They were then fixed with 2.5% glutaraldehyde andpostfixed with osmium, both in the presence of 0.15%ruthenium red, and embedded in Eponate 12. Sections werepoststained with uranyl acetate and lead and examined in aHitachi 7000 transmission electron microscope at an accel-erating voltage of 75 kV.

Protein concentrations. The bicinchoninic acid methoddescribed by Smith et al. (32) was used to determine theprotein concentration of washed cell suspensions. The pro-tein reagents and standard bovine serum albumin wereobtained from Pierce (Rockford, Ill.).

Materials. All solvents and chemicals used were of thehighest purity commercially available. cis-(1S,2R)-Dihy-droxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydro-diol) was prepared as described previously (12). Restrictionendonucleases were obtained from Bethesda Research Lab-oratories (Gaithersburg, Md.) and used according to themanufacturer's instructions.

RESULTS

Growth studies. P. putida Idaho can grow in minimal saltsmedium whenp-xylene is supplied in the vapor phase or in atwo-phase system where p-xylene is present at 5% to morethan 50% (vol/vol). Figure 1 shows typical growth parame-ters during growth of P. putida Idaho with 20% (vol/vol)p-xylene. The lag phase, which is often accompanied by adecrease in the number of viable cells (data not shown), isfollowed by a period of exponential growth with a celldoubling time of 1.64 h. After cells reach the stationaryphase, there is a dramatic decrease in the numbers of viablecells. Turbidity and cell dry weight measurements parallelthe increase in viable cells during the lag and exponentialphases of growth but continue to increase after the cellsreach the stationary phase even though the number of viablecells decreases. The maximum growth rates and yields ofviable cells in batch culture at the end of the exponentialgrowth phase are the same when p-xylene is present at 5 to50% (vol/vol). The yield of viable cells varies inversely withtemperature, and the highest values observed were 5 x 10'cells per ml at 18°C. Growth rates and cell yields are alsodependent on agitation and were considerably lower whenthe organism was grown in baffled flasks.Growth of P. putida Idaho with benzoate, p-xylene, or

toluene, when the hydrocarbons were provided in the vaporphase, followed conventional patterns, with turbidity, celldry weight, and viable cell measurements remaining parallelthroughout growth. A doubling time of 1.45 h and a maxi-mum viable cell count of 5 x 109 cells per ml was observedduring growth of P. putida Idaho with 25 mM benzoate.Growth withp-xylene or toluene, when supplied in the vaporphase, is slower because of the rate-limiting diffusion of thevolatile substrate into the medium. Typical doubling times of3.4 h were observed for both p-xylene and toluene vapors,with viable cell yields in the range of 109 cells per ml.

Ultrastructure ofP. putida Idaho when grown with p-xylene.Thin sections of succinate-grown cells of P. putida Idaho(Fig. 2A) show the ultrastructure typical of pseudomonads.The outer and cytoplasmic membranes are close together(Fig. 2A, arrowheads), and well-defined ribosomes are

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10

0

00(0ci60

.1

.01

.001

108

1074

1.4

C.-C)

106

105

1.2 r-

1.0 I-i

-0.8

E0) 0.6

* 0.4

r 0.2

0.00 10 20 30 40 50

Time (h)FIG. 1. Growth of P. putida Idaho with 20% (vollvol)p-xylene as measured by turbidity at 600 nm (-), viable count (in cells per milliliter)

(A), and dry weight (in milligrams per milliliter) (A).

present. Inclusion bodies (Fig. 2A, arrow) probably consistof glycogen. When cells are grown with p-xylene vapors(Fig. 2B), the outer membrane is convoluted and, in places,clearly separated from the cytoplasmic membrane (arrow-heads). Inclusion bodies (Fig. 2B, arrows), different fromthose seen in succinate-grown cells, are present. The largerof these are electron dense, while the smaller ones have anemulsionlike appearance. When cells are grown with 20%(vollvol)p-xylene (Fig. 2C and D), the outer and cytoplasmicmembranes are separated and convoluted and the cytoplas-mic membrane, especially, appears disorganized. There areelectron-transparent regions in the cells resulting from in-vaginations of the cytoplasmic membrane (Fig. 2C and D,arrowheads) as well as intracellular vesicles. Electron-denseinclusion bodies are still present (Fig. 2C and D, arrows),and fragments of outer membrane appear to be shed into themedium (Fig. 2D, paired arrowheads). Cells of P. putidaIdaho grown with toluene, as described above forp-xylene,have the same ultrastructural features seen in Fig. 2B, C,and D. However, more disruption of the membranes isapparent with vapors and with 20% toluene than withp-xylene.

Solvent tolerance. The ability ofP. putida Idaho to grow onLB medium in the presence of a range of organic solventswas determined (Table 2). Solvents are listed by decreasinghydrophobicity expressed as the logarithm of the octanol-water partition coefficient (log PocT [21]). Of the solventslisted, only p-xylene and toluene could be used as growthsubstrates. Abundant growth was also observed on LBplates when the surface of the agar was flooded with solventsthat have a log Pocr equal to or greater than 2.3.Four Pseudomonas species which can metabolize toluene

were examined for their tolerance of selected organic sol-vents. The results (Table 3) show that Pseudomonas men-docina KR1 and P. putida mt-2 cannot grow in the presenceof 20% (vol/vol) p-xylene or toluene even though bothorganisms can grow with both hydrocarbons at lower sub-strate concentrations (34, 38). P. putida Fl grows well withtoluene when the solvent is provided in the vapor phase (12).However, significant growth was not observed when thisstrain was grown with LB medium in the presence of 20%(vol/vol) toluene. None of the strains tested was able totolerate organic solvents to the same extent as P. putidaIdaho.

Metabolic studies. Aromatic compounds that can serve as

growth substrates for P. putida Idaho include toluene,m-xylene,p-xylene, 1,2,4-trimethylbenzene, 3-ethyltoluene,benzylalcohol, benzoic acid, m-toluic acid, p-toluic acid,p-hydroxybenzyl alcohol, m-cresol, and p-cresol. Thosecompounds that showed no growth were benzene, o-xylene,ethylbenzene, propylbenzene, isopropylbenzene, 4-ethyltol-uene, styrene, chlorobenzene, biphenyl, naphthalene, phe-nol, and o-cresol. (Aromatic hydrocarbons were provided inthe vapor phase with the exception of biphenyl and naph-thalene, which were added directly to the mineral saltsmedium, and water-soluble substrates, which were used at aconcentration of 5 mM.) Growth was also observed on solidmedia when the surface of the agar was overlaid with 5.0 mlof toluene, m-xylene, p-xylene, 1,2,4-trimethylbenzene, or3-ethyltoluene. These results suggest that P. putida Idahoutilizes the same metabolic pathway as P. putida mt-2 for thedegradation of alkyl-substituted aromatic hydrocarbons.Further evidence was provided by polarographic experi-ments with cells of P. putida Idaho grown with toluene,m-xylene, or p-xylene. The results obtained (Table 4) showthat cells grown with any one of these aromatic hydrocar-bons are capable of oxidizing each hydrocarbon substrateand the corresponding benzyl alcohols and carboxylic acidsformed by methyl group oxidation. These results are similarto those reported for P. putida mt-2 which contains the TOLplasmid pWWO (38). Toluene-grown cells of P. putida Idahodid not oxidize cis-toluene dihydrodiol or o-, m-, orp-cresol(data not shown).

Location of the genes encoding p-xylene degradation. At-tempts to detect the presence of a catabolic plasmid in P.putida Idaho by the procedures of Hansen and Olsen (14),Kado and Liu (20), and Connors and Barnsley (6) were notsuccessful. In control experiments, these procedures gavegood yields of the 81-kb plasmid which carries the genes fornaphthalene degradation in P. putida NCIB 9816 (29). Re-peated transfers of P. putida Idaho in medium with benzoateas the sole carbon and energy source did not yield cells witha stable Xyl- phenotype as has been reported for the TOLplasmid pWWO in P. putida mt-2 (3). These observationssuggest that the genes encoding enzymes for the degradationof toluene, m-xylene, and p-xylene are located on the P.putida Idaho chromosome. To test this hypothesis, totalDNA from P. putida Idaho was purified and digested withHindIII. DNA fragments were separated by gel electropho-resis, blotted to a nylon membrane, and hybridized under

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FIG. 2 Effect of p-xylene on the ultrastructure of P putida Idaho. Cells were grown with succinate (A), p-xylene vapors (B), and 20%(vol/vol) p xylene (C andD)p Bars, 200nae

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TABLE 2. Growth of P. putida Idaho in the presence oforganic solventsa

Solvent Log Po0b Growthc

n-Decane 5.6 +++n-Octane 4.5 +++Cyclooctane 4.1 +++n-Heptane 4.0 +++1-Octene 3.7 +++Propylbenzene 3.6 +++n-Hexane 3.5 +++Diethylphthalate 3.3 + + +Cyclohexane 3.2 +++Ethylbenzene 3.1 +++p-Xylene 3.1 +++n-Pentane 3.0 + +Styrene 3.0 +++1-Octanol 2.9 +++Cyclopentane 2.5 +++Toluene 2.5 +++1,5-Hexadiene 2.5 + +1-Heptanol 2.4 +++Dimethylphthalate 2.3 + + +Fluorobenzene 2.2Benzene 2.0Chloroform 2.01-Hexanol 1.81-Butanol 0.8

a Cells were grown in LB medium in the presence of 25% (vol/vol) solventas described in Materials and Methods.

b Calculated from hydrophobic fragmental constants in Laane et al. (21) andfrom values in Rekker and deKort (27).

c Symbols indicating turbidity values at 600 nm: + + +, > 1 after 24 h; + +,

0.7 to 1.0 after 48 h; -, <0.2 after 48 h.

conditions of high stringency with a labeled probe preparedfrom the plasmid pED3306 which contains the upper path-way operon promoter and the xylCAM genes from the TOLplasmid pWWO. The results obtained (Fig. 3) indicate thatsequences homologous to the pWWO fragment cloned inpED3306 are present on the P. putida Idaho chromosome.

DISCUSSION

There have been many reports on the ability of bacteria togrow with volatile aromatic hydrocarbons as the sole sourceof carbon and energy when the hydrocarbon is provided atlow concentrations. P. putida Idaho appears to be unique in

TABLE 3. Growth of selected pseudomonas strains in thepresence of organic solventsa

GrowthbSolvent LogtPocr P. putida P. mendocina P. putida P. putida

Idaho KR1 mt-2 Fl

Decane 5.6 +++ +++ +++ +++Hexane 3.5 +++ +++ +++ +++Cyclohexane 3.2 +++ +++ +++ +++Pentane 3.0 +++ +++ +++ +++p-Xylene 3.1 +++ - - +++Cyclopentane 2.5 +++ - - +++Toluene 2.5 +++1-Heptanol 2.4 +++Benzene 2.0

a The experimental conditions were the same as those described in Table 2,footnote a.

b For explanation of symbols, see Table 2, footnote c.

TABLE 4. Oxidation of TOL substrates by P. putida Idahoa

Oxygen consumption" by cellsAssay substrate grown with:

Toluene m-Xylene p-Xylene

Toluene 132 60 55Benzyl alcohol 190 200 28Benzoate 100 175 50Catechol 2,150 1,890 1,300m-Xylene 55 240 130m-Methylbenzyl alcohol 130 150 300m-Toluate 80 115 303-Methylcatechol 1,680 3,300 600p-Xylene 125 120 250p-Methylbenzyl alcohol 75 200 120p-Toluate 75 75 304-Methylcatechol 770 2,500 1,600

a Reaction mixtures contained the following in 1.0 ml: 50 pmol of potassiumphosphate buffer (pH 7.2); substrates, 1.0 pLmol in 10 p.l of dimethylformamide(toluene, m-xylene, p-xylene) or distilled water (all other substrates); and cellsuspension in 0.1 ml of 0.05 M potassium phosphate buffer (pH 7.2).

b Results are expressed as nanomoles minute-l milligram of protein1 andare corrected for endogenous activity in the absence of substrate.

its ability to grow in minimal medium with p-xylene andtoluene when these substrates are present at 5% to morethan 50% (vol/vol). During exponential growth in a batchsystem with p-xylene (20% [vol/vol]) or benzoate (10 mM),doubling times of 1.64 and 1.45 h, respectively, were ob-served. However, striking differences were noticed whenturbidity, cell dry weight, and numbers of viable cells wereused as growth parameters. After 48 h, turbidity and dry cellweight measurements for cells grown with 20% (vol/vol)p-xylene were almost twice the values observed with ben-zoate. In contrast, the number of viable cells present incultures grown with p-xylene was 2.2 x 106 cells per mlcompared with 4.8 x 10 cells per ml for cells grown withbenzoate. The number of viable cells present during expo-nential growth with p-xylene (20% [vol/vol]) gave values inthe range of 5 x 105 to 5 x 108 cells per ml. Variable resultswere obtained, depending on the incubation temperature andthe type and rate of agitation. The increased disruption ofcells grown in the presence of solvents with increasedagitation and temperature has been noted previously (15).Nevertheless, cell viability decreases in all cases after cellsenter the stationary phase even though cell mass continuesto increase. Similar results were reported for the growth ofPseudomonas oleovorans in a two-phase system with n-oc-tane and 1-octene (9). A 98 to 99% decrease in cell viabilitywas also observed when a recombinant strain of E. coli,containing the alk genes from P. oleovorans, was grown withglucose in the presence of 20% (vol/vol) n-octane. Thedecrease in cell viability occurred 4 to 5 h after the cellsentered the stationary phase of growth (10).

Transmission electron microscopy of thin sections pre-pared from cells grown with p-xylene revealed distinctchanges in structure consistent with profound damage to themembranes and increased membrane synthesis. De Smet etal. showed that membranes of E. coli were disrupted bytoluene (8), and the membranes of P. oleovorans (9) and E.coli (10) are damaged by extended growth in octane. Al-though ultrastructure was examined by freeze fracture inthese studies and we used transmission electron microscopyof thin sections, the changes are similar: more damageoccurs to the cytoplasmic membrane than the outer mem-brane. Intracellular and extracellular vesicles are formed

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FIG. 3. Homology between chromosomal DNA from P. putidaIdaho and plasmid DNA containing the xyl genes from the upperoperon of the TOL plasmid pWWO. Plasmid DNA and chromo-somal DNA were cleaved with restriction endonucleases, andfragments were separated by electrophoresis in an 0.75% agarosegel. Restriction fragments were probed with the 10.1-kb fragment ofpED3306, which contains the upper pathway promoter, and thexyI4, xylM, and xylC genes from the TOL plasmid pWWO. Lanecontents: 1 and 3, pED3306 cleaved with Hindll; 2, P. putida Idahototal DNA cleaved with HindIII. The arrow indicates the 10.1-kbfragment from pED3306. Only the relevant portion of the gel isshown.

from the cytoplasmic membranes, and the outer membranesappear convoluted, with increased surface area relative tothe total cell surface than is observed in cells grown in theabsence of solvents.

P. oleovorans grown on octane accumulates polyal-kanoates as intracellular inclusion bodies but does not incor-porate substrate into the membranes (9). We do not know ifthe inclusion bodies which appear in P. putida Idaho duringgrowth with aromatic compounds consist of modified orunmodified substrate. Both P. oleovorans (9) and E. coliwith cloned genes for octane metabolism (10) show damageto the cells after reaching the stationary phase when grownwith octane or octene. Even though these substrates are farless polar (log Pocr = 4.5 for octane and 3.7 for octene) thantoluene andp-xylene (log Pocr = 2.5 for toluene and 3.1 forp-xylene), the damage is similar.

The usual limits for bioconversions are at log Pocr valuesgreater than 4.0, with activity at lower values occurring onlywhen a water layer around the biocatalyst is stabilized bysome hydrophilic support (21). The lower limit of solventpolarity for growth of P. putida Idaho is similar to thatreported by Inoue and Horikoshi (17) for their strain of P.putida and occurs between a log Pocr of 2.4 (1-heptanol) and

a log Pocr of 2.0 (benzene). Favre-Bulle et al. (10) report alog PocT of 3.7 as the lowest limit for growth of their strainof E. coli with cloned alkane genes under optimized condi-tions of temperature and agitation. The resistance of organ-isms such as P. putida Idaho to solvents may be due to anability to synthesize membranes rapidly to compensate forthose damaged by the solvent or to some biochemicaldifference in the cytoplasmic membrane which makes itmore stable in the presence of solvent. The increased dryweights of cultures relative to viable counts of P. putidaIdaho may be due to the membranous material shed into theculture fluid seen in electron micrographs as well as tocontinued growth of a small proportion of the cells after themajority of the population enters stationary phase or is killedby the solvent.Except for its high tolerance to solvents, P. putida Idaho

appears to be very similar to P. putida mt-2. Oxygen uptakestudies by cells of P. putida Idaho, grown with TOL sub-strates, indicate that the metabolic pathways used by P.putida Idaho for the degradation of toluene, m-xylene, andp-xylene are the same as those utilized by P. putida mt-2. Inaddition, Southern hybridization experiments indicate signif-icant homology between fragments of P. putida Idaho DNAand a fragment of P. putida mt-2 DNA known to contain thexylMA4B genes. These genes encode the first two enzymes ofthe TOL catabolic pathway.The apparent chromosomal location of the genes for the

degradation of toluene by the TOL plasmid pathway is notuncommon. Sinclair et al. (31) and Polissi et al. (26) havedescribed similar strains. In P. putida MW1000 (31), the tolgenes are very similar to the toluene-catabolizing genes ofthe TOL plasmid of P. putida mt-2 but are physically linkedto chromosomal markers. However, they also behave as a56-kb transposon, leading to the speculation that they mayhave a plasmid origin. The ability of P. putida Idaho todegrade toluene and p-xylene is stable through numeroustransfers on nonselective media, as is the resistance tosolvents, and it is possible that the chromosomal location ofthe genes in P. putida Idaho may make the ability to degradearomatic hydrocarbons a more stable character. Furtherstudies are required to identify the genetic componentsresponsible for solvent resistance in P. putida Idaho.

ACKNOWLEDGMENTS

This work was supported by grant C87-101334 from the IdahoNational Engineering Laboratory, U.S. Department of Energy.We thank Joanne M. Horn for supplying the plasmid pED3306

used as a probe in Southern hybridization experiments, Hong Yi ofthe University of Iowa Central Electron Microscopy Facility forpreparation of the samples for electron microscopy, Heather Baileyfor technical assistance, and Sharon Gaffney for assistance inpreparing the manuscript.

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