Pseudomonas putida - Journal of Bacteriology - American Society for ...

JOURNAL OF BACTERIOLOGY, Apr. 1976, p. 410-416 Copyright © 1976 American Society for Microbiology

Vol. 126, No. 1 Printed in U.S.A.

Isolation of Plasmid Deoxyribonucleic Acid from Pseudomonas putida SUNIL PALCHAUDHURI* AND A. CHAKRABARTY Department of Microbiology, New York University School of Medicine, New York, New York 10016,* and General Electric Research and Development Center, Schenectady, New York 12301 Received for publication 5 November 1975

Conditions suitable for reproducible recovery of covalently closed circular deoxyribonucleic acid from strains of Pseudomonas putida containing degradative plasmids (CAM, SAL, OCT, etc.) have been defined. These degradative plasmids could not be isolated by the usual procedure, whereas RP1, an R factor of the P group, present in the isogenic strain of P. putida, was isolated equally well by either the usual procedure or the modified procedure. Characterization by electron microscopy of RP1 deoxyribonucleic acid confirmed the molecular weight (about 40 x 106) previously determined by sucrose gradient centrifugation. The existence of plasmid deoxyribonucleic acid (DNA) in different bacterial species may be established by genetic and physical methods. Pseudomonas putida has been shown by genetic means to carry transmissible plasmids, which contain genes for many of the naturally occurring hydrocarbon degradative pathways (degradative plasmids). Until now there was no physical evidence for the extrachromosomal nature of these plasmids (7). Most of the available techniques employed for the isolation of plasmid DNA depend upon the fact that the plasmids exist as covalently closed circular (CCC) DNA in the cytoplasm of the cell and do not sediment with the cellular membrane and chromosome. Therefore, in all these techniques the cellular membrane and chromosomal DNA are removed by centrifugation or by preferential precipitation in the presence of high NaCl concentration (2, 4, 6). Applying this technique, we have isolated F-prime factors (molecular weight, 60 x 10" to 180 x 10"), R factors (molecular weight, 2 x 10" to 78 x 10"), and Col factors (molecular weight, 4.2 x 10" to 78 x 10") from Escherichia coli, R factors from Pseudomonas, and cryptic plasmids from Neisseria gonorrhoeae (10, 12). The same technique did not work when applied to Pseudomonas strains containing SAL, OCT, or CAM plasmids but could easily recover the R factor DNA present in the same parent strain. We, therefore, thought of two major modifications of our usual procedure. The harvested cells were not washed with buffer containing ethylenediaminetetraacetic acid (EDTA), because these strains showed partial lysis even in the presence of 5 mM EDTA.

Secondly, the cellular membrane and chromosome were not removed by centrifugation or by high salt precipitation like the usual method. Changes introduced in our usual procedure to isolate these degradative plasmids are described in the present communication. MATERIALS AND METHODS The bacterial strains (ac 34-1, ac 35, and ac 36) used were derivatives of P. putida PpGl (7). The adenine-requiring mutant (ac 34) was originally isolated in I. Gunsalus' laboratory after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Strain ac 35 is the same as ac 34, except that it carries R factor RP1; ac 36 is also the same as ac 34, except that it carries a degradative plasmid, SAL. During this study ac 34-1 was derived from ac 36 after curing the SAL plasmid by treatment with mitomycin C (1). The RP1 factor that carries a group of genes specifying resistance to carbenicillin, neomycin, kanamycin, and tetracycline originated in a strain of P. aeruginosa (5) and was transferred to P. putida (ac 34) by conjugation. The adenine-requiring male strain of P. aeruginosa (JC9005 FP2+) was obtained from J. M. Pemberton (13). All of these strains were tested for their genetic character, including plasmid-borne traits, before being used for the isolation of plasmids. Media. AF medium of Novick and Maas, without vitamins (9), was used for growing bacteria for the isolation of plasmid DNA. Luria broth, consisting of 1 liter of distilled water, 10 g of tryptone (Difco), 5 g of yeast extract (Difco), and 0.5 g of NaCl, was also used. Preparation of cleared lysates. Overnight cultures of adenine auxotrophs of P. putida (ac 34-1, ac 35, and ac 36) derived from single colonies were diluted 50-fold in 500 ml of modified M9 medium (3) containing 50 ,g of adenine per ml. When the cell density had reached 5 x 108 cells/ml, the bacteria 410

VOL. 126, 1976 were harvested at 4 C, suspended in 25 ml of cold sucrose solution (25% sucrose in 0.05 M tris(hydroxymethyl)aminomethane [Tris], pH 8.0), and frozen at -20 C. Crude lysates were prepared from these strains as previously described by Palchaudhuri et al. (10). The crude lysates were diluted about twofold to reduce the viscosity and then spun at 49,000 x g in a model L ultracentrifuge at 2 C for 25 min in a fixed-angle 30 rotor. This separated out most of the chromosomal DNA and most of the membrane in the pellet. The supernatant is referred to as "cleared lysate," and the supercoiled DNA present in this lysate was purified by the dye-buoyant procedure of Radloff et al. (14). Preparation of labeled DNA for zone sedimentation in an alkaline sucrose gradient. Overnight cultures were diluted 50-fold in 9.8 ml of AF medium containing glucose (0.5%) and adenine (10 ug/ml).

[2-3H]adenine (specific activity, 22 Ci/mmol;

Schwarz/Mann) was added to a final concentration of 25 .,uCi/ml, and cells were grown at 30 C for about 6 h (more than three generations), until cell density had reached 3 x 108 cells/ml, before they were harvested. Cleared lysates, prepared in the same way as described above, were layered on a 5 to 20% alkaline sucrose gradient and centrifuged at 20 C for 30 min at 30,000 rpm in a Spinco model L centrifuge, SW50 rotor (11). Immediately thereafter, the centrifuge tube was punctured at the bottom, and 3-drop fractions were collected directly onto Whatman no. 1 filter disks (3 MM). The procedures used for washing and counting these disks of the radioactivity have been described (12). Modified procedure for the preparation and purification of plasmid DNA. Cells were grown in 500 ml of AF medium supplemented with adenine (final concentration, 50 Ag/ml) at 30 C to a density of 5 x 108/ml and harvested by centrifugation at 2 C. The pellet was suspended in 25 ml of a 25% sucrose solution (in 0.05 M Tris, pH 8.0) and frozen at -20 C. Washing with TES buffer (Tris, 0.05 M; EDTA, 0.005 M; and NaCl, 0.05 M; pH 8.0) was avoided because there was extensive lysis in the presence of EDTA. After thawing, 2.5 ml of lysozyme (5 mg/ml in 0.25 M Tris, pH 8.0) and 2.5 ml of Na2 EDTA (pH 8.0) were added and mixed gently, and the mixture was incubated at 30 C until the entire cell suspension appeared viscous. Twenty-five milliliters of 2% sodium lauryl sarkosinate containing 1.5 M NaCl and 0.05 M Tris, pH 8.0, prewarmed to 30 C, was added slowly. Lysis occurred almost immediately, resulting in a clear and uniform viscous mass. The viscous lysate was sheared by passing two to three times through a 35-ml disposable syringe (without needle) to reduce its viscosity and titrated to pH 12.0 by dropwise addition of 4 N NaOH. It is difflcult to adjust the pH of the viscous solution, and, therefore, stirring was carried out by simultaneous use of a magnetic stirrer and a glass rod during titration. After reaching pH 12.0 the solution was immediately brought back to pH 8.0 by stepwise addition of solid Tris-hydrochloride (Sigma); longer incubation at pH 12.0 will also degrade the covalently closed plasmid DNA. To remove the denatured fragments of DNA, 25 g of nitro-

PLASMID FROM P. PUTIDA

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cellulose powder (Hercules, 0.25 s cubed), which had been washed four times with 1 liter of 0.3 M NaCl and 0.03 M sodium citrate, was added. The mixture of DNA and nitrocellulose was then centrifuged at 5,000 rpm for about 10 min in the cold, and the supernatant was collected discarding most of the nitrocellulose, which had adsorbed the denatured DNA as sediment. DNA (plasmid and chromosomal) was concentrated by layering the supernatant on a 2.5-ml bed of saturated CsCl in a polymer tube and centrifuging for 14 h at 14,000 rpm in a fixed-angle 30 rotor at 2 C. After the run, about 25 ml of liquid was discarded from the top without disturbing the remainder (because the bed of CsCl gets diluted during the run and DNA sediments into the lower part of the tube). The lower part of the liquid (about 9 ml) was mixed well with the pellet by homogenizing with a glass rod and was filtered through glass wool to remove most of the remaining nitrocellulose. This is termed "concentrated lysate." Small crystals of CsCl (about 5.7 g) were added to the concentrated lysate and dissolved slowly to avoid foaming, and the refractive index was adjusted to 1.399. The rest of the steps were carried out in the dark. A 0.3-ml amount of ethidium bromide (stock solution, about 5 mg of 0.05 M Tris per ml, pH 8.0) was added to the above solution of DNA, mixed well by repeated inversion of the tube, and centrifuged for 40 h in a Spinco fixed-angle rotor 50 at 105,000 x g at 12 C. After the run two well-separated bands were clearly seen with an ultraviolet lamp; the upper band comprising linear and open circular DNA was removed from the top with a capillary micropipette before collection of the lower band to avoid the possibility of mixing, because the whole of the lower band was collected as one fraction by puncturing the bottom of the cellulose nitrate tubes. The satellite bands (lower) from several tubes of the same rotor were pooled and run again in an SW50.1 rotor for 20 h at 40,000 rpm, 12 C, without any adjustment of the refractive index. Again two bands were seen, but the lower satellite band contained most of the material and pure plasmid DNA in CCC form. The minor upper band was mostly the single-strand fragments, which derived during denaturation and escaped removal during collection of the satellite band after the first centrifugation. The DNA of the lower band was stored at 4 C in the dark in the original solution of CsCl-ethidium bromide after collection from the ultracentrifuge tube. Our concentration of ethidium bromide is much less than that of Radloff et al. (14) and also CCC-DNA binds less dye. The removal of dye was, therefore, not essential for molecular weight determination by electron microscopy (12). Instead, the stored DNA could be used directly. Electron microscopy of plasmid DNA. Stored DNA bands (see above) were examined with the electron microscope as described previously (10). About 95% of the DNA molecules present in the lower bands appeared as supertwisted circles (form I) without contamination of single strands or doublestranded fragments. Supertwisted molecules (form I) were converted to the open circular form (form II) by X-ray doses ranging from 50 to 200 rads given at

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room temperature. The aqueous technique for DNA spreading was used for making grids and mounting the DNA molecules for the study of size distribution and homogeneity. Staining, shadowing, and measuring were carried out as described previously (10). The electron microscope (Siemens 101 Elmiskop) used for the analysis was calibrated for a set of exposures at the same operational magnification using a grating replica (2,160 lines/mm; Fullam, Inc.). Contour lengths were measured by projecting electron micrograph negatives on a screen and tracing on paper. Lengths were determined from tracings of molecules with well-defined contours with a curvimeter and dividing by total magnification (electron microscope and projector).

J. BACTERIOL.

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were exposed to X-irradiation as described in Materials and Methods and examined under the electron microscope. About 50% of the circular molecules was in the open circular form and the rest was in CCC form. Only those molecules having well-defined contours were used for length measurements. Data on RP1 are presented in Table 1. RP1 contains a single molecular species (Fig. 2) and has an average molecular weight of 39.5 x 10'i, which is in good agreement with the value obtained previously by neutral sucrose gradient analysis (5). The recovery of supertwisted DNA molecules from 1 liter of exponential-phase culture (5 x 108 cells/ml) of strain ac 35 was about 20

RESULTS /g. "Cleared lysates" containing plasmid DNA The same amount of cells containing the SAL and fragments of chromosomal DNA of P. pu- plasmid (ac 36) gave a higher yield of plasmid tida strains ac 34-1, ac 35, and ac 36 were ana- DNA. The majority of these molecules had conlyzed by the dye-cesium chloride gradient tour lengths of 27 ± 0.7 ,um (Fig. 3), and about method (14). A minor satellite component of 5% were shorter (18 ± 0.5 akm). Complete charDNA separate from the major component and acterization of the SAL, OCT, and CAM plasvisible in ultraviolet light was present only in mids will be published elsewhere. Also the sex strain ac 35. Examination by electron micros- factor, FP2 of P. aeruginosa (JC9005), was copy of the heavy minor component showed isolated by this modified procedure. Its molecuthat the majority of these DNA molecules were lar weight, determined from contour length supercoiled. In the case of strains ac 34-1 and ac (58.2 ± 1 x 10'i, micrograph not shown), was in 36, no corresponding satellite band was seen; good agreement with the published value, 59 x the only visible bands (one band in both cases) 1 0" (13). were collected and examined with the electron microscope. None of the molecules of these DISCUSSION bands obtained from strains ac 34-1 and ac 36 Previous attempts by Williams and Murray were circular; they presumably represented (15), and others (personal communication) to sheared fragments of chromosomal DNA. isolate the plasmids of P. putida have failed. To confirm the above interpretation, cleared We had similar failures when we applied our lysates of these three strains (ac 34-1, ac 35, and usual isolation procedure (12) for isolating ac 36) grown in a radioactive medium, as de- closed circular DNA from the strains of Pseuscribed in Materials and Methods, were pre- domonas containing SAL (ac 36), OCT, or CAM pared. [2-3H]adenine-labeled DNA present in plasmids, but we could isolate the R factor presthe lysates was examined by sedimentation ent in the same parent strain (ac 35). We, through alkaline sucrose gradients (Fig. lA, B, therefore, introduced some changes in our usand C). In the case of ac 35, a fast-sedimenting ual isolation procedure on the assumption of peak occupied the characteristic position of co- different kinds of association of these degradavalently closed circular DNA molecules (Fig. tive plasmids with host chromosome and memlB), but no such peak was seen in the case of ac brane. The usual method fails because the ini34-1 and ac 36 (Fig. lA and C). tial step in isolating CCC-DNA involves reConcentrated lysates of all three strains were moval of chromosomal DNA as part of a large prepared by the modified procedure described complex of membranes, and apparently the in Materials and Methods and analyzed again degradative plasmid DNA is a part of this comby the dye-buoyant density method (14). plex. The modified method avoids this problem Strains of P. putida carrying the resistance by removing the cellular DNA and cell debris elements RP1 (ac 35) and SAL (ac 36) each in a different fashion: denaturation, followed by showed a satellite component well separated adsorption of the denatured fragments to nitrofrom the major component. The breadth of the cellulose. The CCC-DNA is renatured at neutwo bands appeared to be same, and the cured tral pH and hence is not adsorbed by the nitrostrain (ac 34-1) showed no such satellite band. cellulose. For the measurement of their contour lengths Association (not linear insertion) of superthe satellite bands, after recentrifugation as twisted plasmid DNA with folded host DNA

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TABLE 1. Mean contour lengths and estimtated molecular weights of the circular DNA molecules present in strain ac 35 (RPJ) Isolation technique

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isolating the plasmids (cryptic and known) of P. tious Diseases to W. K. Maas. S. Palchaudhuri is a Career aeruginosa (Palchaudhuri, unpublished data). Scientist under the Irma T. Hirschl Trust. Because of its good recovery, the satellite LITERATURE CITED plasmid band in an ethidium bromide-CsCl 1. Chakrabarty, A. M. 1972. Genetic basis of the biodegragradient is easily visible; thus its collection is dation of salicylate in Pseudomonas. J. Bacteriol. very simple. Further, the use of AF medium 112:815-823. 2. Clewell, D. B., and D. R. Helinski. 1969. Supercoiled (9) increased the yield of plasmid DNA of E. circular DNA-protein complex inEscherichia coli: pucoli and Pseudomonas (two- to fourfold; Palrification and induced conversion to an open circular chaudhuri, unpublished data). form. Proc. Natl. Acad. Sci. U.S.A. 62:1159-1166. One of the reasons of higher recovery of SAL 3. Cohen, S. N., and C. A. Miller. 1970. Non-chromosomal antibiotic resistance in E. coli: molecular nature of R plasmids, starting with the same number of factors isolated from Proteus mirabilis and Eschecells and identical conditions of isolation, may richia coli. J. Mol. Biol. 50:671-687. be the greater number of SAL plasmid copies 4. Freifelder, D. 1970. Isolation of extrachromosomal DNA from bacteria, p. 153-163. In L. Grossman and present per cell. However, quantitative experiK. Moldave (ed.), Methods in enzymology, vol. 21. ments have not been carried out to estimate the Academic Press Inc., New York. number of copies of plasmids per cell. It can be 5. Grinsted, J., J. R. Saunders, L. C. Ingram, R. C. Sykes, concluded from our findings that it is not approand M. H. Richmond. 1972. Properties of an R factor priate to generalize about the types of plasmids which originated in Pseudomonas aeruginosa 1822. J. Bacteriol. 110:529-537. (cryptic or known) present in a particular orgaP., D. J. Leblanc, and S. Falkow. 1973. Gennism on the basis of usually employed isolation 6. Guerry, eral method for the isolation of plasmid deoxyribonumind in keep to procedures, and it is necessary cleic acid. J. Bacteriol. 116:1064-1066. the possible loss in the DNA membrane sedi- 7. Gunsalus, I., M. Herman, W. A. Toscano, Jr., D. Katz, ment. ACKNOWLE DGMENTS We thank Werner K. Maas for his constructive criticism and continued encouragement. This work was supported by Public Health Service grant Al 09079 from the National Institute of Allergy and Infec-

and G. K. Garg. 1975. Plasmids and metabolic diversity, p. 207-212. In D. Schlessinger (ed.), Microbiology - 1974. American Society for Microbiology, Washington, D.C. 8. Kline, B. C., and J. R. Miller. 1975. Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosome of E. coli: physicochemical ap-

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10.

11.

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proach to studying the unit of segregation. J. Bacteriol. 121:165-172. Novick, R., and W. K. Maas. 1961. Control by endogenously synthesized arginine of the formation of ornithine transcarbamylase in Escherichia coli. J. Bacteriol. 81:236-240. Paichaudhuri, S., E. Bell, and M. R. J. Salton. 1975. Electron microscopy of plasmid deoxyribonucleic acid from Neisseria gonorrhoeae. Infect. Immun. 11:11411146. Palchaudhuri, S., and V. N. Iyer. 1971. Compatibility between F' factors in an E. coli strain bearing a chromosomal mutation affecting DNA synthesis. J. Mol. Biol. 57:319-333. Paichaudhuri, S., A. Mazaitis, W. K. Maas, and A. K.

J. BACTERIOL. Kleinschmidt. 1972. Characterization by electron microscopy of fused F' prime factors in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 69:1873-1876. 13. Pemberton, J. M., and A. J. Clark. 1973. Detection and characterization of plasmid in Pseudomonas strain PAO. J. Bacteriol. 114:424-433. 14. Radloff, R., W. Bauer, and J. Vinograd. 1967. A dyebuoyant density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1514-1521. 15. Williams, P. A., and K. Murray. 1974. Metabolism of benzoate and the methylbenzoate by P. putida (arvilla) mt-2: evidence for the existence of a TOL plasmid. J. Bacteriol. 120:416-423.