Vol. 171, No. 9

Plasmid Control of the Pseudomonas aeruginosa and Pseudomonasputida Phenotypes and of Linalool and p-Cymene Oxidation

MARIE-JOSE DE SMET,t* MARK B. FRIEDMAN, AND IRWIN C. GUNSALUSt

La Jolla Biological Laboratories, The Salk Institute, San Diego, California 92138

Received 1 September 1988/Accepted 19 June 1989

Two Pseudomonas strains (PpG777 and PaG158) were derived from the parent isolate Pseudomonasincognita (putida). Strain PpG777 resembles the parental culture in growth on linalool as a source of carbon andslight growth on p-cymene, whereas PaG158 grows well on p-cymene, but not on linalool or other terpenestested, and has a P. aeruginosa phenotype. Curing studies indicate that linalool metabolism is controlled by an

extrachromosomal element whose loss forms a stable strain PaG158 with the p-cymene growth and P.aeruginosa phenotype characters. The plasmid can be transferred by PpG777 to both P. putida and P.aeruginosa strains. Surprisingly, the latter assume the P. putida phenotype. We conclude that the geneticpotential to oxidize p-cymene is inherent in PpG777 but expression is repressed. Similarly, this observationimplies that support of linalool oxidation effectively conceals the P. aeruginosa character.

Members of the genus Pseudomonas play a major role inthe metabolism of hydrocarbon compounds (12, 30). Extra-chromosomal DNA elements were first shown to be involvedin the oxidation of the terpene camphor (33), and laternaphthalene (9), salicylate (3), and octane (5, 13). Subse-quently, plasmids were shown to carry genes for a variety ofstructures, including xylene (11), toluene (37-40), and thealkaloid nicotine (35). Most of these catabolic plasmids are

conjugative, while others depend on transfer genes found on

separate plasmids, i.e., fertility factor K in Pseudomonasputida (4). There are indications that discrete DNA segmentsfrom these plasmids, which carry some or all of the hydro-carbon degradative genes, are transposable (6, 18). Thetransposition of catabolic capacity parallels the well-charac-terized transposition of antibiotic resistance (22). The con-

veyance of hydrocarbon catabolic genes by transpositionmay contribute to the evolution of catabolic pathways andmay explain the broad nutritional versatility of these bacte-ria. Transposition can thus be described as an efficientmolecular mechanism for rapid, multidimensional environ-mental adaptation.While our genetic knowledge of camphor catabolism is

well developed (23; see 12 and The Bacteria X), the geneticsof other terpene pathways is relatively unknown. The pri-mary progress has resulted from advances in the chemistryof the metabolic pathways and the enzymology of theoxygenases (12; The Bacteria X).

In this study, we focused on the linalool and p-cymene

pathways. Several Pseudomonas spp. capable of oxidizingp-cymene have been isolated (12). One of these pseudomo-nads, known as P. putida, oxidizes a- and 3-pinenes,limonene, A1-p-menthene, and p-cymene (7, 24). The catab-olism of p-cymene by this strain is shown in Fig. 1. Bhatta-charyya and colleagues (25) isolated a soil pseudomonadidentified as P. incognita which is capable of utilizing

* Corresponding author.t Present address: Gist-Brocades Research and Development,

Wateringseweg 1, 2611 XT Delft, The Netherlands.t Present address: Roger Adams Laboratory, Department of

Biochemistry, University of Illinois at Urbana-Champaign, Urbana,IL 61801.

various acyclic monoterpenes, e.g., linalool. They providedevidence for the catabolic reaction sequence shown in Fig. 2.We are interested in the following two areas: (i) the

relationship, if any, between terpene catabolism pathways inmicroorganisms and (ii) the evolution of catabolic pathwaysin microorganisms. To better understand these problems, weisolated two Pseudomonas strains. One strain, Pseudomo-nas sp. strain PpG777, utilizes a variety of monoterpenes,e.g., linalool, but demonstrates weak growth on p-cymene.The other Pseudomonas strain, designated PaG158, utilizesp-cymene as the sole carbon source but grows on no otherterpenes.

In this study, we showed that the linalool and p-cymenepathways are closely related and that the pathways interferewith expression of phenotypic characteristics. These conclu-sions are supported by the findings that (i) loss of linalooloxidation capacity by strain PpG777 was accompanied byexpression of the p-cymene pathway and (ii) p-cymeneoxidation was accompanied by conversion of the strain tothe PaG158 phenotype. Since strain PpG777 was identifiedas a strain of P. putida and strain PaG158 has propertiescharacteristic of P. aeruginosa, these findings imply that thegenetic elements controlling the linalool pathway mask therobust phenotypic characteristics of P. aeruginosa.

MATERIALS AND METHODS

Bacterial strains. The derivation and genotypic and phe-notypic properties of the strains used in this study are listedin Table 1. The parent P. incognita was a gift from P. D.Bhattacharyya (Indian Institute of Science, Bangalore, In-dia), from which P. Harder isolated and characterized thestable strains, PpG777 and PaG158. P. aeruginosa PA0303was supplied by T. Gingeras (SIBIA, San Diego, Calif.). P.incognita was purified extensively as described below beforeuse.Media and growth conditions. LB medium (27) was rou-

tinely used as the complete medium. Phosphate ammoniumsalts (PAS) medium (5) was used as the minimal medium.PAS medium was supplemented with carbon sources at 5 to10 mM. Linalool, however, was added to a concentration of3 mM. Casamino Acids (0.02% [wt/vol]) and yeast extract(0.02% [wt/vol]) were added to media whenever p-cymenewas used as the sole carbon source. Amino acids were added

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p-cymene

cytochromeP450-containing I

hydroxylaseCH20H

p-cumicalcohol

I

CHO

p-cumicaldehyde

I

COOH

p-cumicacid

FIG. 1. Metabolic pathwayputida PL (7).

COOHOHX 2,3-dihydroxy-I p-cumic acid

OHOH

ll'0o

i

COOH2-oxo-pent-4-enoate

I

COOH

isobutyrate

HO04-hydroxy-2-oxo-

CO HpentanoateCOOH

I

CHO CH30

OH3 COOHfor oxidation of p-cymene by P.

to a final concentration of 20 pug/ml to media for auxotrophicmutants, with the exception of arginine, which was added toa final concentration of 100 ,ug/ml. Production of pyocyanineand other phenazine pigments was detected by using King Amedium (21). When applicable, agar was added to a concen-

tration of 1.5% (wt/vol).Liquid cultures (5 ml) in test tubes (18 by 150 mm) on

300-ml shake flasks (10 to 100 ml) were incubated withagitation at 250 rpm. P. putida strains were incubated at30°C, and P. aeruginosa strains were incubated at 37°Cunless otherwise indicated. Growth was determined bymeasuring the A450. By using a calibration curve giving therelationship between the absorbance and the dry weight, thecell density in milligrams of cell dry mass per milliliter wasderived (39).

Capacities for growth on various compounds as solesources of carbon and energy were tested in liquid medium.Minimal medium cultures supplemented with the indicatedcarbon source were inoculated with early stationary cells ata density of 105 cells per ml. The latter cells originated fromcultures in PAS medium supplemented with linalool, p-

cymene, or glutamate. Final cell densities were measuredafter incubation at 30°C for 20 h.Growth rates were determined by inoculating cultures

containing LB or PAS medium supplemented with linalool,p-cymene, or glutamate with 105 cells per ml from theovernight cultures described above. Cultures were incubatedat 30, 37, or 42°C as required.

Antimicrobic susceptibility testing. Sensitivity disks (DifcoLaboratories, Detroit, Mich.) were used to test bacteria forresistance to the following antibiotics: ampicillin, carbenicil-lin, chloramphenicol, clindamycin, erythromycin, gentami-cin, kanamycin, lincomycin, nalidixic acid, penicillin G,

OHcytochrome P450 -containing hydroxylase hydroxylase

X,0s X CH20HJ<OH linalool )9OH8-hydroxy- [nr 10-hydroxy-linalool linalool

CH20H

CHObOH lOH

linalool-8- linalool- 10-aldehyde aldehyde

CHO

COOHJOH l

linalool-8- tlinalool- 10-carboxylic L carboxylicacid acid

COOH

COOH

oleuropeicm acid

OOH

COOH

J perillic acid

C02 + H20

FIG. 2. Metabolic pathways for oxidation of linalool by P. in-cognita (8, 25, 32).

polymyxin B, rifampin, streptomycin, and tetracycline. Theprocedures used were suggested by the manufacturer.

Purification of colonies. Cells from a PAS plate supple-mented with the appropriate carbon source and amino acids(with auxotrophic mutants) were suspended in 0.9% (wt/vol)NaCl up to 109/ml, and after serial dilution in 0.9% (wt/vol)NaCl (107-fold), they were plated on LB medium. About 10isolated colonies were picked and streaked on PAS supple-mented with the appropriate carbon source and amino acids.After incubation at 30°C for 48 to 72 h, the procedure wasrepeated. This procedure was repeated at least seven times.The colonies (n = 100) from the LB plate were routinelychecked for pigment formation on King A medium, growthat 420C, and resistance to kanamycin and carbenicillin. Inaddition, the suspensions were examined by phase-contrastmicroscopy to ensure that most of the cells occurred singly.

Genetic experiments. Mutants were prepared by treatingcell suspensions with N-methyl-N'-nitro-N-nitrosoguanidine(NG) as described by Miller (27). Mutants were detected andcharacterized as previously described (2). Alternative at-tempts were made to prepare mutants by treating cells withthe acridine half-mustard compound ICR-191 at 50 to 100,ug/ml as previously described (2).Plasmid curing. The following three methods were used to

eliminate plasmid-mediated phenotypes: (i) growth with mi-tomycin C as described by Rheinwald et al. (33), (ii) growthunder nonselective conditions at 30°C (PAS medium supple-mented with glucose [20 mM] was inoculated with 104 early

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LINALOOL AND p-CYMENE CATABOLISM IN PSEUDOMONAS SPP. 5157

TABLE 1. Pseudomonas strains used in this study

Stock no. Relevant phenotype' Genotypea Derivation or sourceb Reference

Wild typesPpG777 Lin' Cym- WT; LIN, CYM Linalool enrichment 25PaG158 Cym+ WT; CYM p-Cymene enrichment P. Harder and this

paper

MutantsPpG273 Trp- Cam' trpB615(CAM) PpG1, PC 33PpG277 Trp- Cam- trpB615(ACAM) PpG273, MC 33PpG379c Leu- Nah+ leu-801(NAH) PpG378, NG 9PA0303 Arg- argB18, chl-2 FP- 17D104 llv- Lin' Cym- ilv-801(LIN, CYM) PpG777, NGD122 Leu- Lin' Cym- leu-803(LIN, CYM) PpG777, NGD208 Met- Cym+ met-899(CYM) PaG158, NGD312 Pgb+ Cym- pgb-801(ACYM) PaG158, NG

Strains obtained fromconjugations

D500 Pgb+ Lin' Cym- pgb-801(LIN, ACYM) PpG777 x D312D503 llv- Lin' Cym- Cam ilv-801(LIN, CYM, CAM) pG273 x D104D504 llv- Lin' Cym- Nah+ ilv-801(LIN, CYM, NAH) PpG379 x D104D507 Arg+ Lin' chl-2(LIN) D122 x PA0303D508 Trp+ Lin+ LIN D104 x PpG277D800 Cym+ Cam+ CYM, CAM PpG273 x PaG158D804 Cym+ Nah+ CYM, NAH D504 x PaG158D805 Met- Cym+ Cam+ met-899(CYM, CAM) PpG273 x D208D806 Met- Cym+ Nah+ met-899(CYM, NAH) D504 x D208

a Marker abbreviations: WT, wild type; arg, arginine; chl, chloramphenicol resistance; pgb, brown pigment production; trp, tryptophan. Phenotype symbols:Cam, camphor utilization; Cym, p-cymene utilization; Lin, linalool utilization; Nah, naphthalene utilization. LIN, CYM, CAM, and NAH are plasmid (pathway)designations; A, deletion of pathway or plasmid.

b NG, Nitrosoguanidine; x, conjugation; MC, mitomycin C; PC, penicillin-cycloserine.c High-frequency donor for plasmid markers in conjugation.

stationary phase cells per ml and incubated for 18 h. Thecultures were then serially diluted in LB medium, inoculatedin minimal medium, and allowed to grow for 18 h. Thestarting cell concentration in the final medium was 103/ml.Following each incubation, loss of the Lin' or Cym+phenotype was determined as described below), and (iii)growth under nonselective conditions at elevated tempera-tures (37°C for strain PpG777 and 42 to 45°C for strainPaG158). The procedures used were previously described(2). The medium used contained PAS supplemented withglucose (20 mM).Loss of the Lin' and Cym+ phenotypes was determined

as follows. Cultures were serially diluted in LB medium andplated onto LB plates. The plates were incubated at 30°C for18 h, after which 100 colonies were transferred to platescontaining minimal salts medium (PAS) supplemented withthe appropriate hydrocarbon. Plates containing glucose (20mM) were used as controls. The plates were incubated at30°C for 72 h. Growth was examined every 24 h.

Conjugation procedure. Plate matings were performed bythe method of Dunn and Gunsalus (9). When a PpG777mutant was the donor, another procedure was used. Expo-nentially growing cultures of donor and recipient cells wereconcentrated 10-fold to a density of ca. 1010/ml. A 100-,ulsample of donor cells and a 10-1l sample of recipient cellswere then mixed on LB medium. After incubation at 30°C for6 h, cells were harvested in 0.9% (wt/vol) NaCl and plated onselective medium. Viability counts were determined onappropriately supplemented PAS medium. Donor parentswere eliminated by selection against auxotrophic markers,except in one case (the cross PpG777 x D312), in whichtransconjugants were detected by production of a telltale

brown pigment on LB medium. The donor markers usedreverted to prototrophy at a frequency of <10-9. Transcon-jugants were purified as described above.

RESULTS

Characterization of Pseudomonas sp. strains PpG777 andPaG158. Two Pseudomonas strains designated PpG777 andPaG158 were derived from a culture of P. incognita andcharacterized with respect to growth substrates, pigmentformation, temperature range, and antimicrobial resistance(Table 2). By the criteria of Bergey's Manual of SystematicBacteriology (30), PpG777 would be classified as a strain ofP. putida and PaG158 would be classified as a strain of P.aeruginosa.Both PpG777 and PaG158 grew well at 30 and 37°C in LB

medium and in PAS medium supplemented with glutamate,linalool, or p-cymene as the sole source of carbon andenergy (Table 3). PpG777 did not grow in any of the mediatested when incubated at 42°C, whereas PaG158 grew well inboth LB and PAS supplemented with glutamate. PaG158 didnot grow in PAS supplemented with p-cymene at 42°C. TheCym+ phenotype was stably maintained in strain PaG158under nonselective conditions at 42°C (see below). The Lin'phenotype was stably maintained by PpG777 throughout thegrowth experiments.

Stability of the Lin' and Cym+ phenotypes in PpG777 andPaG158, respectively. To encourage plasmid loss, strainsPpG777 and PaG158 were grown under nonselective condi-tions at elevated temperatures (37°C for PpG777 and 42 to45°C for PaG158) or treated with mitomycin C, acridine halfmustard, or NG (Fig. 3). At different times during incuba-

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TABLE 2. Characteristics of Pseudomonas sp. strains PpG777 and PaG158 and the cured derivative of PpG777

Utilization ofr: Resistance tod:

Strain Genotypea Phenazine Growth CG

u C

PpG777~~~~~~~~WT;-I- -aStrainWT,eWildotype.ua C

-,opigent;+, pimentprdcigmnt at 420CA medum

carbons Ews E(u Q Q u C C

PpG777 WT; LIN -- ± + ± + + + + ± R S S SPaG158 WT;CYM + + + + ± + - - - + S R R RPpG777 curedeCyM + + + + ± + - - - + S R R R

aWT, Wild type.b, No pigment; +, pigment production on King A medium.+,Growth (maximum cell density, 0.4 to 1.5 mg of cell dry mass per ml); ±,weak growth (maximum cell density, 0.1I to 0.3 mg/ml; when p-cymene was the

carbon source, growth was seen only on PAS plates after about 3 days); -, no growth.d R, Resistant; S, sensitive. The amounts of antibiotics in the disks were 100 (carbenicillin), 30 (kanamycin), 10 (streptomycin), and 15 (erythromycin) pg. For

the resistance phenotype, the growth-free zone had a diameter of <5 mm; for the sensitivity phenotype, it was .22 mm.

tion, samples were serially diluted and plated on LB. From50 to 100 colonies were later transferred to PAS mediumsupplemented with linalool orp-cymene to test for the Lin'or Cym+ phenotype.Under these conditions, the Lin+ phenotype was readily

lost from strain PpG777, while the Cym+ phenotype wasstably maintained by strain PaG158. Analysis of curedPpG777 cells indicated complete phenotypic similarity tostrain PaG158 (Table 2).

Transmissibility of the Lin' and Cym' phenotypes. Auxo-trophic mutants of strains PpG777 and PaG158 were gener-ated by NG treatment and used in conjugation experiments.Plate matings were performed with auxotrophic derivativesof PpG777 as donors. Recipient strains included PaG158mutants D312 and D208, P. aeruginosa PA0303, and P.putida PpG277. Selections were made for the Lin+ pheno-type. Donor cells were eliminated by selection against theauxotrophic markers. An exception was the cross PpG777 xD312, for which transconjugants were detected by scoringproduction of brown pigment on LB medium.The Lin+ phenotype was readily transferred to D312

(producing transconjugant D500), D208, PA0303 (producingtransconjugant D507), and PpG277 (producing transconju-gant D508) (Table 4). Of 50 transconjugants tested, all hadlost the auxotrophic marker of the recipient (data notshown), indicating that chromosomal genes were transferredat a high frequency. When selection was made for prototro-phy (on minimal medium supplemented with glucose), all ofthe 50 clones were Lin' (data not shown).

TABLE 3. Growth rates at different temperatures in theexponential phase for strains PpG777 and PaG158 in

cultures with different carbon sources

Range' of specific growth rates

Strain Carbon source (h') at:(concn [mM])300C 37°C 420C

PpG777 LB 0.48-0.75 0.34-0.46 0Glutamate (10) 0.31-0.35 0.28-0.29 0Linalool (3) 0.34-0.42 0.26-0.28 0

PaG158 LB 0.55-0.82 0.6-1.0 0.54-0.67Glutamate (10) 0.47-0.53 0.53-0.56 0.45-0.48p-Cymene (5) 0.32-0.45 0.24-0.28 0

a Experiments were performed three times.

To ensure that conjugation had occurred, transconjugantswere extensively purified as described in Materials andMethods. Pure transconjugants were then subjected to de-tailed analysis (Table 5). Introduction of the Lin' phenotypeto the PaG158 mutant, strain D312, or P. aeruginosaPA0303 resulted in expression of the phenotypic character-istics of strain PpG777.The following observations indicate that the purified col-

onies do not contain either donor or recipient cells. (i) Sixpurified single colonies from transconjugants D500 and D507(similar to PpG777) initially grown in PAS medium supple-mented with linalool were suspended in 0.9% (wt/vol) NaCl.From 100 to 200 cells were then plated on LB medium.Following incubation at 30°C for 40 h, the resulting colonieswere tested for growth at 42°C and pigment production onKing A medium, and D507 was tested for growth on PASmedium supplemented with glucose. From 30 to 50% of thecolonies were Lin- and had reverted to the D312 andPA0303 phenotype, while 50 to 70% were PpG777-like andexpressed Lin'. Twenty percent of the PA0303-like colo-nies were Arg+, and 80% were Arg-. (ii) After being cured,D500 and D507 cells were unable to utilize p-cymene (Table5). (iii) D507 and strain PA0303 were unable to utilizeisobutyric acid (Table 5). In contrast, PpG777 exhibited

FIG. 3. Conversion of strain PpG777 to PaG158. Details of theconditions are described in Materials and Methods. Experimentswere performed four times with similar results (the spread of valuesis indicated).

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TABLE 4. Transfer of the Lin phenotype from strain PpG777to several Pseudomonas spp. by conjugation

Cross TransferSelected Genotype frequency/ Trancon-markers of recipient donor jugant

Donor Recipient cella

PpG777 D312 Lin' Pgb+ pgb-801(CYM) 10-2 D500D122 D208 Lin' Leu+ met-899(CYM) 10-3D122 PA0303 Lin' Leu+ argB18 10-6 D507D104 PpG277 Lin' Ilv trpB615(CAM) io-8 D508

a Defined as the number of transconjugants per viable donor cell. Experi-ments were done twice with identical results.

growth on isobutyric acid (Table 2). (iv) After being cured,strain D507 became streptomycin sensitive (Table 5),whereas cured PpG777 cells, which are identical to PaG158,exhibited streptomycin resistance (Table 2).The results demonstrate that the Lin+ phenotype can be

transferred to P. aeruginosa. A byproduct of this transcon-jugation is expression by P. aeruginosa of phenotypic char-acteristics of P. putida.The Lin' phenotype was rapidly lost by D500 and D507

under nonselective conditions; however, it was stably main-tained in transconjugant D508. D508 retained several prop-erties characteristic of PpG277 (colony morphology andresistance to erythromycin).

Conjugation of the Cym+ phenotype from strain PaG158into strains of P. aeruginosa or P. putida resulted in notransconjugants (data not shown).

Transfer of plasmids NAH7 and CAM to PpG777 andPaG158. To determine whether the Lin' and Cym+ pheno-types are compatible with catabolic plasmids NAH7 andCAM, we attempted to transfer these plasmids to strainsPpG777 and PaG158. Plasmid NAH7 was transferred byconjugation from P. putida PpG379 to PpG777 but not toPaG158 (Table 6). Plasmid NAH7 could be introduced intoPaG158 only after it had first passed to PpG777 (i.e.,crossings D504 x PaG158 and D504 x D208). Stabilitystudies (data not shown) indicated that plasmid NAH7 wasstably maintained in PpG777 mutant D104 (transconjugantD504) but rapidly lost from PaGI58 (transconjugant D804)and D208 (transconjugant D806). D804 and D806 did not losethe p-cymene pathway.

In contrast to plasmid NAH7, plasmid CAM was mobi-lized from P. putida PpG273 into both D104 and PaG158(D208) with high frequency (Table 6). In addition, plasmid

CAM was stably maintained in transconjugants D503, D800,and D805 (data not shown). Thus, the Lin' and Cym+phenotypes appear to be compatible with catabolic plasmidCAM. Plasmid NAH7 is compatible with the Lin' pheno-type but is unstable in strains PaG158 and D208.

Plasmid isolations. Attempts to isolate plasmid DNAs fromstrains PpG777 and PaG158 by using modified versions ofseveral procedures (20, 26, 36) have so far been unsuccess-ful.

DISCUSSION

The results presented here suggest that the genetic infor-mation required for initiation of linalool oxidation by strainPpG777 is plasmid associated. It is unclear whether thisholds true for initiation of p-cymene oxidation by strainPaG158. Strain PpG777 is also capable of utilizing p-cumicalcohol (Table 2). Whether this compound is oxidized via thep-cymene pathway or via a pathway that is related to thelinalool pathway will be the subject of further studies.The following lines of evidence favor the hypothesis that

linalool-oxidizing enzymes are plasmid controlled in strainPpG777: (i) spontaneous and mitomycin C-induced forma-tion of Lin- mutants and (ii) transmissibility of the entirelinalool pathway through conjugation. It was found that thefrequency of transfer of the auxotrophic markers met, arg,and trp with the Lin phenotype was close or equal to 100%.This is possible, since it has been reported that auxotrophicmarkers in both P. putida and P. aeruginosa are confined toa limited region on the chromosome (28, 29). Morgan andDean (28) found that in P. putida PPN, 85% of knownauxotrophic markers, including most met, arg, and trpmarkers, are restricted to 36% of the genetic map. Plasmidswhich mobilize large chromosome fragments are well-known(15j. It is possible that the LIN plasmid is highly efficient inchromosome mobilization.We were unable to detect a plasmid in either PpG777 or

PaGI58 by traditional isolation procedures for plasmid DNA(20, 26, 36). This might be a consequence of the large size ofthe LIN plasmid. Physical demonstration of the large CAMand OCT plasmids by other groups has been very difficult(H. Koga, personal communication; 5, 6). However, Harderand Kunz (14) have isolated and visualized the OCT plasmidon agarose gels. Currently we are developing procedures toisolate bacterial DNA from cells that are immobilized in a gelmatrix. The DNA will be analyzed by pulsed-field gradientgel electrophoresis (34).

TABLE 5. Characteristics of transconjugants D500, D507, and D508 and their cured derivatives

Strain Genotype' Antimicrobial Growth6 Phenazine Growth on the following substrate6:phenotypea at 420C pigment' Cym CumOH Lin Ibu

D500 pgb-801(LIN, CYM) CbF Kms Strs - - + + + +D500 curedd pgb-801(CYM) CbS Kmr Strr + + - - - +D312 pgb-801(CYM) Cbs Kmr Strr + + - - - +D507 LIN Cbr Km Strs - - + + + -D507 cured arge(LIN) Cb5 Kmr StrP + +PA0303 argB18 Cbr Kmr Strs + +D508 LIN Er - - + + + +PpG277 trpB615 Er - - - - - +

a Marker abbreviations: Cb, carbenicillin; E, erythromycin; Km, kanamycin; Str, streptomycin; Ibu, utilization of isobutyrate. The other markers are definedin Table 1, footnote a.

b See Table 2, footnote c; CumOH, p-cumic alcohol.c +, Pigment production; -, no pigment production on King A medium.d Cells were cured by culturing at 37'C in LB during 20 generations.e Ca. 20%o of the cured cells were prototrophs.

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TABLE 6. Transfer of plasmids NAH7 and CAM to PpG777 and PaG158 by conjugation

Cross Phenotype Frequency/donor

Donorb Recipientc Selected markers Nonselected markers cella Transconjugant

PpG379 D104 Nah+ Leu+ Lin' lv- Cbr Kms >10-4 D504PpG379 PaG158 Nah+ Leu+ Cym+ Kmr CbS 0D504 PaG158 Nah+ Ilv+ Cym+ Kmr Cb5 >10-6 D804D504 D208 Nah+ Ilv Cym+ Met- Kmr CbS 10-6 D806PpG273 D104 Cam' Trp+ Lin' lv- Cbr Km' >10-3 D503PpG273 PaG158 Cam' Trp+ Cym+ Kmr CbS >10-3 D800PpG273 D208 Cam' Trp+ Cym+ Met- Kmr Cb5 >10-3 D805

a Number of transconjugants per viable donor cell.bPpG379, leu-801(NAH7); PpG273, trpB6JS(CAM).c D104, NG mutant of PpG777, ilv-801(LIN) Cb' Kms; D208, NG mutant of PaG158, met-899(CYM).

When strain PpG777 lost the Lin' phenotype, the curedcells were phenotypically identical to cells of strain PaG158(Table 2). Specifically, the presence of the Lin' phenotypeprevents growth on p-cymene, growth at 42°C, and produc-tion of phenazine pigments. Cured PpG777 cells exhibitedthe same antimicrobial resistance spectrum as PaG158,further suggesting that the Lin' phenotype interferes withexpression of antibiotic resistance. Reintroduction of theLin' phenotype by conjugation into mutant strains ofPaG158 and P. aeruginosa PA0303 resulted in return of theP. putida phenotype (Table 2).

Evidence has been presented that a TOL-like plasmidfrom Alcaligenes eutrophus controls the expression of achromosomally encoded p-cresol pathway (16). Our resultsagree with this finding, although they are more dramatic inthat a presumed metabolic plasmid masks several robustphenotypical properties characteristic of P. aeruginosa. Thispreliminary finding may have significant medical implica-tions with respect to the identification of important clinicalstrains.While the Cym+ phenotype was stably maintained under

all of the conditions tested, the Lin' phenotype was veryunstable in the Pseudomonas sp. tested.

Recently, Bryan et al. (1) demonstrated that the pheno-type characteristic of P. aeruginosa can be mimicked bygenetic mutants ofPseudomonas stutzeri generated by trans-poson mutagenesis. It is known that the chromosome mapsof P. aeruginosa and P. putida have fundamental similarities(although some regions of the P. putida chromosome areinverted relative to the P. aeruginosa map [28]). Similarphenomena may occur in linalool regulation.The ability of transconjugants D500 and D507 to utilize

p-cumic alcohol as the sole source of carbon and energy(Table 5) may indicate that p-cumic alcohol is oxidized via apathway that is related to the linalool pathway. Cured strainswere incapable of growth on p-cumic alcohol.The Lin' phenotype appears to be compatible with the

CAM and NAH plasmids, and thus it does not belong toeither the IncP2 or IncP9 group (19). The Cym+ phenotypewas compatible with plasmid CAM, but plasmid NAH wasnot stably maintained in strain PaG158. Since plasmid NAHcould be introduced into PaG158 only after it had passedthrough PpG777, a restriction endonuclease barrier mayexist in PaG158.We have generated pathway mutants of strains PpG777

and PaG158 by NG mutagenesis and are currently usingthese mutants to construct a library of linalool and p-cymenegenes to gain insight into their genetic organization andregulation.

ACKNOWLEDGMENTS

We thank J. Cregg, T. Gingeras, S. Kellogg, and W. Wood foruseful comments during preparation and I. Wingate for processingof the manuscript.

This work was supported by Public Health Service grant 5 ROIAM00562 from the National Institutes of Health and grant PCM83-07757 from the National Science Foundation.

ADDENDUM IN PROOF

Studies by Mark Vandeyar with these mutants and avariety of recombinants in the Gunsalus laboratory haveassociated the phenotypic changes with the presence of asmall regulatory plasmid. The DNA structure is being inves-tigated.

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