Vol. 140, No. 3

JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 1017-1022

0021-9193/79/12-1017/06$02.00/0

Resistance Plasmids in Pseudomonas cepacia 4G9 JOHN A. WILLIAMS,* JAMES P. YEGGY, CHRISTOPHER C. FIELD,t AND A. J. MARKOVETZ Department of Microbiology, University of Iowa, Iowa City, Iowa 52242 Received for publication 1 October 1979

Pseudomonas cepacia 4G9 utilizes 2-tridecanone as its sole carbon source and has been shown to be resistant to a variety of antibiotics. To ascertain whether any of these characteristics were plasmid mediated, Escherichia coli HB101 was transformed with plasmid DNA isolated from Pseudomonas cepacia 4G9. No 2tridecanone-utilizing transformants were obtained. Tetracycline (Tc)- and ampicillin (Ap)- resistant transformants were obtained at a low frequency. Plasmid deoxyribonucleic acid from antibiotic-resistant E. coli HB101 transformants had molecular weights of 2.9 x 106 for pJW2 Tcr and 5.4 x 10 for pJW3 Apr as determined by electron microscopy. Electron microscopy of plasmid deoxyribonucleic acid from P. cepacia 4G9 revealed a single plasmid species, pJW1 of 1.78 x 10'. Tetracycline resistance in both P. cepacia 4G9 and E. coli HB101(pJW2) was inducible, whereas ampicillin resistance in P. cepacia 4G9 was constitutive. The level of ampicillin resistance coded by pJW3 was lower in P. cepacia 4G9 than in the transformant E. coli HB101(pJW3). agarose electrophoresis patterns due to nucleases active in the presence of the detergents Brij 58 and sodium dodecyl sulfate (J. A. Williams and A. J. Markovetz, Abstr. Annu. Meet. Am. Soc. Microbiol., 1979, H102, p. 136). This report describes the isolation of plasmid DNA from P. cepacia 4G9 and transformation of plasmid DNA into Escherichia coli with selection for antibiotic resistance or 2-TDO utilization. Three small, nonconjugative plasmids have been sible for the initial oxidation and dehydrogena- identified. tion reactions are coded by the OCT plasmid MATERIALS AND METHODS (16). The similarity of the ketone and alkane degradative pathways raised the possibility that Bacterial strains and media. The bacterial the 2-TDO monooxygenase and undecyl acetate strains used are listed in Table 1. Minimal agar plates esterase from P. cepacia 4G9 could be plasmid and L-broth were made as described (23). Amino acid coded. Furthermore, a study of plasmids in P. supplements were used at a concentration of 1 mM. inhibitory concentrations (MIC) of antibiotcepacia 4G9 could establish whether resistance Minimal were determined on Mueller-Hinton agar (Difco of this organism to some antibiotics is due to cell ics Laboratories, Detroit, Mich.). Transformants were sewall impermeability analogous to that in P. lected on antibiotic-supplemented Mueller-Hinton aeruginosa (12, 32) or to specific resistance plas- agar. mids. This aspect would be of interest as P. Assay for ability to macerate onion tissue. The cepacia has been identified in several clinical outer scales were removed from yellow onions before cutting transverse slices 3 to 5 mm thick with an infections (30, 31). Recently, plasmids have been isolated from alcohol-flamed knife. The slices were placed on moistseveral strains of P. cepacia of plant, soil, and ened sterile filter paper in petri dishes. Organisms human origin (15). No phenotypic function was grown overnight in L-broth were pelleted, washed in suspended in saline. These cultures were assigned to any of these plasmids. Plasmids were saline, and to a Klett value of 100 units by using a Klettpreviously found in P. cepacia 4G9 (J. A. Wil- adjusted colorimeter with a green filter (no. 54). liams and A. J. Markovetz, Abstr. Annu. Meet. Summerson Onion slices were inoculated with 0.1-ml portions of Am. Soc. Microbiol., 1978, H40, p. 110). Isolation various dilutions spread over the surface of the slices of plasmids from this strain resulted in variable and incubated for 72 h at 30°C. The degree of macer-

Pseudomonas cepacia 4G9 (Table 1) is able to utilize methyl ketones as its sole carbon source by a subterminal oxidation pathway (14). A specific methyl ketone, 2-tridecanone (2TDO), is metabolized via undecyl acetate to undecanol and acetate (13). Enzymes catalyzing both of these reactions have been isolated, purified, and characterized (4, 27). Alkanes are utilized by P. putida as the sole carbon source by terminal oxidation (1). The enzymes respon-

t Present address: Southwest Missouri State University, Springfield, MO 65802.

ation was estimated by pricking the slices with a toothpick. Uninoculated slices served as controls and were scored as 0, whereas the slices inoculated with a

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WILLIAMS ET AL.

J. BACTERIOL.

TABLE 1. Bacterial strains Strain

P. cepacia 4G9 P. cepacia P. multivoransb P. kingiib

Origin

Relevant characteristics

Plasmiid

Genotype

Soil Onion Soil Bronchial washings

Reference

Phenotype

2-TDO+a

E. coli K-12 HB101 proA, leuB, recA RR1 pBR322 GS245 pheA, glyA, rpsL 1485-1 RSF1030 C600 pMB8 Utilizes 2-TDO as the sole carbon source. b Synonyms for P. cepacia (29). 'Department of Microbiology, University of Iowa.

HsdR- HsdMTcr, Apr Apr

Rfrneoor source

14 ATCC 25416 ATCC 17759 ATCC 25609 3 26 G. V. Staufferc 10 25

a

known plant pathogen which macerates onion tissue were scored as 5 (15). MIC. After overnight growth of the organisms in Lbroth, 5-ml portions were centrifuged, and the pellet was washed twice in sterile saline. The pellets were suspended in saline and diluted to a Klett value of 50 units by using a green filter (no. 54). These standard cultures were diluted 1:20 and spot inoculated with a replicator apparatus onto Mueller-Hinton agar plates containing doubling concentrations (5 to 2,560 Lg ml-') of various antibiotics. The antibiotics tested were tetracycline, ampicillin, kanamycin, neomycin, gentamicin, streptomycin, chloramphenicol, trimethoprim, and sulfadiazine (Sigma Chemical Co., St. Louis, Mo.), carbenicillin (Geopen, Pfizer, Inc., New York, N.Y.), and spectinomycin (Trobicin, The Upjohn Co., Kalamazoo, Mich.); The plates were incubated for 16 to 20 h at 37°C before being scored for growth. Plates without antibiotics were used for controls. The MIC recorded was that concentration of antibiotic which prevented colony formation. Plasmid isolation. Plasmid DNA was isolated by using three different procedures dependent on the organism and the quantity required. (i) Large-scale isolation from P. cepacia 4G9 grown in 1- to 2-liter cultures was followed by using the procedure of Guerry et al. (17). Plasmid DNA was concentrated by using polyethylene glycol before cesium chloride-ethidium bromide equilibrium centrifugation (18). (ii) Plasmid DNA was isolated from E. coli transformants and E. coli RR1 (pBR322) by the Brij 58 "cleared lysate" procedure (8). (iii) Plasmid DNA was isolated from 1-ml cultures of E. coli and P. cepacia 4G9 by a modification of the procedure described by Cameron et al. (5). All centrifugation was done in an Eppendorf model 5412 centrifuge. Cells were pelleted in 1.5-ml polypropylene tubes by centrifuging for 2 min. The supernatant fluid was decanted, and the pellet was suspended in 0.5 ml of 50 mM Tris (pH 8.0)-50 mM EDTA-15% sucrose containing 1 mg of lysozyme per ml. After incubation for 10 min at room temperature, 1 pi of diethylpyrocarbonate was added, and the mixture was vortexed for

1 to 2 s. The spheroplasts were lysed by the addition of 20 pl of a 10% sodium dodecyl sulfate solution. Chromosomal DNA and sodium dodecyl sulfate were precipitated by addition of 50 ul of 5 M potassium acetate. This mixture was kept on ice for 30 min before clearing by centrifugation for 10 min. The supernatant fluid was decanted into another tube, which was then filled with cold 95% ethanol and placed at -20°C for 15 min to precipitate the DNA. The DNA was pelleted by centrifugation for 5 min, and the pellet was washed with 70% ethanol for 15 min at -20°C. After centrifugation for 5 min, the pellet was dried under vacuum for 10 min. The DNA was dissolved in 10 pd of 10 mM Tris (pH 8.0)-i mM EDTA, and contaminating RNA was digested with RNase (5 pl of 10 mg ml-') by incubation at 37°C for 15 min. DNA samples for agarose electrophoresis had 10 pl of electrophoresis buffer (40 mM Tris-5 mM sodium acetate (pH 7.9)-i mM EDTA) added plus 5 pl of 0.5% bromophenol blue in 60% sucrose, whereas samples for transformation were diluted to 200 pl with 50 mM CaCl2. Transformation. Both E. coli HB101 and E. coli GS245 were rendered competent and stored by the cryogenic procedure of Morrison (24). Transformation with plasmid DNA was as described (24), and selection was on Mueller-Hinton agar supplemented with each of the antibiotics (20 uLg ml-'). Selection for 2-TDO utilization was on minimal agar supplemented with the appropriate amino acids and with 20 Atl of 2-TDO on filter paper in the petri dish lid. Randomly selected transformants were screened on minimal agar and appropriately supplemented minimal agar to verify phenotypes. Plasmid DNA was isolated from transformants by procedure iii and assayed by agarose gel

electrophoresis. Agarose gel electrophoresis. Plasmid DNA was electrophoresed in a horizontal 0.7% agarose gel in 40 mM Tris-5 mM sodium acetate (pH 7.9)-i mM EDTA buffer containing 0.2 ,tg of ethidium bromide ml-l. Electrophoresis was at 50 mA and continued until the bromophenol blue marker had migrated approximately 8 cm. The gels were placed on a transilluminator, model C-61 (Ultra-Violet Products, Inc., San Gabriel, Calif.), and photographed with a Polaroid

R PLASMIDS IN P. CEPACIA

VOL. 140, 1979

MPL4 system with Polaroid 665 film and a Tiffen 23A filter. Electron microscopy. Plasmid DNA from E. coli HB101 transformants was isolated by procedure ii and kept at 40C for several days to permit single-strand scissions in some of the covalently closed circular DNA which produce the open circular form. Plasmid DNA from P. cepacia 4G9 was isolated by procedure i. Replicative form (RF) II of 4)X174 was used as a standard and was obtained from New England Biolabs Inc., Beverly, Mass. Preparation of DNA for electron microscopy was as described by Davis et al. (11). The contour lengths were measured on prints (20 by 25 cm) by using a Hewlett-Packard programmable calculator (model 9100B) and digitizer (model 9107A). Induction of antibiotic resistance. The MIC for P. cepacia 4G9, E. coli HB101 (pJW2) and E. coli HB1O1(pJW3) were determined after overnight growth in L-broth or L-broth supplemented with either 20 tg of tetracycline ml-l or 20 tg of ampicillin mnl'. Stability of P. cepacia 4G9 R plasmids in E. coli HB101. The stability of the plasmids was assessed by the method of Chang and Cohen (7). E. coli HB101(pJW2) and E. coli HB101(pJW3) were grown overnight in L-broth supplemented with 20 ug of tetracycline ml-' and 20 ,g of ampicillin ml-', respectively. These cultures were diluted X106 into fresh unsupplemented L-broth and grown to late exponential phase. Similar dilutions were repeated six times, and the final culture was plated on nutrient agar. One hundred randomly selected colonies of each transformant were tested for antibiotic resistance and amino acid requirements to confirm the phenotype. RESULTS

Onion tissue maceration. Strains of P. cepacia isolated from plant, soil, and human origin have been distinguished by several criteria (15), one of which was the ability to macerate onion tissue. As P. cepacia 4G9 had originally been isolated from soil it could have been a plant pathogen. To clarify this point P. cepacia 4G9 together with the three other pseudomonad strains listed in Table 1 were cultured, and their ability to macerate onion tissue was compared. By this criterion P. cepacia 4G9 was not a plant pathogen (data not shown). MIC. The resistance of P. cepacia 4G9 to various antibiotics was determined (Table 2). The MICs for the same antibiotics were established for E. coli HB101 which was to be the recipient in the transformation assays. Plasmid isolation. Plasmid DNA from P. cepacia 4G9 could only be visualized as a band in cesium chloride-ethidium bromide equilibrium density gradients after polyethylene glycol concentration of sodium dodecyl sulfate-sodium chloride lysates. The low yield complicated analysis of the plasmids in P. cepacia 4G9. Transformation. E. coli HB101 was transformed to tetracycline and ampicillin resistance

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TABLE 2. Minimal inhibitory concentration of various antibiotics for P. cepacia 4G9 and E. coli HB1OI MIC

Antibiotic

(fig ml-')

P. cepacia 4G9 E. coli HB101

Tca 80