PLASMID 1, 584-588 (1978)

A Rapid Method for the Identification of Plasmid Desoxyribonucleic Acid in Bacteria

THOMASECKHARDT

Department of Microbiology, New York University Srhool of Medicine, New York, New York 10016

Accepted June 14, 1978

A fast and very sensitive procedure is described for detecting plasmids in bacterial strains. The size of plasmids is determined by agarose gel electrophoresis. Plasmids present in one or more copies per cell with a molecular mass ranging from 2 to over 150 megadaltons may be identified.

Currently two types of rapid screening techniques for plasmid desoxyribonucleic acid (DNA) are used (1,4,5,7). One type re- quires little starting material, but subjects the DNA to considerable stress during lysis (5,7) or during separation of plasmid DNA from chromosomal DNA (1) and is therefore not suitable for large plasmids [greater than 50 megadaltons (Md)] or small plasmids with a low copy number. The other type (4) is time consuming, requiring several steps for plasmid isolation and separation from the chromosomal DNA.

The aim in the design of the technique presented here was to combine the advan- tages of the above-mentioned techniques and to avoid the disadvantages inherent in them. A gentle lysis procedure and minimiza- tion of the manipulations of DNA lysate were used to develop a very sensitive tech- nique with a good yield of circular covalently closed (CCC) plasmid DNA. The bacteria (between lo7 and lo* cells from a liquid cul- ture or one to two single colonies) are lysed directly in the slots of an agarose gel. The chromosomal and plasmid DNA are then separated by electrophoresis. Since the bulk of the chromosomal DNA is intact, it is ex- cluded from the gel under the conditions used; usually less than 0.5% of the total chromosomal DNA migrates into the gel as a band of linear DNA. Plasmids with a mole-

cular mass ranging from 2 to over 150 Md and present in one or more copies per cell can be visualized within 3 to 4 h. The pro- cedure has been carried out with a variety of bacterial species including Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas phaseolicola, Pseudomonas pseudoalcaligenes, and Neis- seria ghonorrhea.

MATERIALS AND METHODS

The conditions used to lyse gram-negative bacteria are slightly different from those for gram-positive bacteria (B. subtilis) and are therefore described separately.

Lysozyme mixture for gram-negative bac- teria . The lysozyme mixture for gram-nega- tive bacteria was composed of lysozyme (Calbiochem, 7500 U/ml), ribonuclease I (Worthington, 0.3 U/ml), and 0.05% brom- phenol blue in Tris-borate buffer (pH 8.2; 89 mM Tris base, 12.5 mM disodium EDTA, and 8.9 mM boric acid), and 20% Ficoll 400,000 (Sigma). The ribonuclease is first dissolved in 0.4 M sodium acetate buffer, pH 4.0, at 10 mg/ml and heated for 2 min at 98°C before diluting it into the rest of the lysozyme mixture.

Lysozyme mixture for gram-positive bac- teria. This mixture was similar to the one for gram-negative bacteria but contained

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75,000 U/ml lysozyme, 50 mM disodium EDTA (pH 8.0), and 0.1 M sodium chloride. Both lysozyme mixtures are stable for sev- eral months at room temperature.

SDS mixture for gram-negative bacteria. This mixture was composed of 0.2% sodium dodecyl sulfate (SDS) in Tris-borate buffer (89 mM Tris base, 2.5 mM disodium EDTA, and 8.9 mM boric acid) in 10% Ficoll400,OOO.

SDS mixture for gram-positive bacteria. This mixture was similar to the one for gram- negative bacteria but contained 2% SDS.

Overlay mixture for gram-negative and gram-positive bacteria. The overlay mix- ture was the same as the SDS mixture for gram-negative bacteria except it contained 5% Ficoll 400,000.

Electrophoresis procedure. For electro- phoresis a standard vertical slab gel appara- tus (gel dimensions: 110 x 140 x 2.5 mm) with 12 slots (6.5 x 15 x 2.5 mm) is used. Gel concentrations vary from 0.75 to 1.2% Agarose (Seakem, type ME, Marine Col- loids, Inc.) in electrophoresis buffer (89 mM Tris base, 2.5 mM disodium EDTA, and 8.9 mM boric acid).

Loading and lysis procedure. Either single colonies from plates (24 to 72 h old) or liquid cultures are used as sources of plasmid DNA. The procedure involves the follow- ing steps.

(i) One or two single colonies (10’ to IO8 cells) are picked with the flat end of a tooth- pick and resuspended in 15 ~1 of the lysozyme mixture which had been previously put into the empty slots of the mounted gel. The suspension becomes slightly turbid. If cells from a liquid culture are used, a suitable volume (0.1 to 0.5 ml) is centrifuged and resuspended in 10~1 of electrophoresis buffer with 20% Ficoll 400,000 and the cell sus- pension is mixed with the lysozyme mix- ture in the gel slot. Gram-negative bacteria are left for 2 to 5 min at room temperature, and gram-positive bacteria are left for 30 to 45 min.

(ii) Thirty microliters of the SDS mixture is carefully layered on top of the bacteria-

lysozyme mixture and the two layers are gently mixed with a toothpick, moving it from side to side (not more than twice for gram-negative bacteria). Complete mixing should be avoided and the two layers should still be distinguishable.

(iii) One hundred microliters of the over- lay mixture is layered on top of the SDS- lysozyme layers without disturbing the now viscous DNA lysate.

(iv) The slots are sealed with hot agarose (SO’C) and both chambers are filled with electrophoresis buffer. The plasmid DNA is electrophoresed for 60 min at 2 mA and then for 60 to 150 min at 40 mA (depending on the plasmid size and the separation de- sired). The gel is stained with ethidium bro- mide (0.4 pg/ml) in electrophoresis buffer for 15 min and then photographed under uv light using a short-wave transilluminator (type C61 from Ultraviolet Products, Inc.) and Polaroid type 55 or 57 film and a red filter (Wratten number 25).

Notes on the procedure. It is important that in Step (i) that the gel is not overloaded with cells, since this reduces the yield of intact plasmid DNA and simultaneously in- creases the chromosomal background dras- tically. Different amounts of liquid culture should be used in initial trials to get an idea of the optimal amount of cells required. For the lysis of B. subtilis it is important to use vegetative cells. Therefore an exponentially growing culture was used routinely. The cells were resuspended directly in the lysozyme mixture and the whole mixture was transferred into an empty slot of the gel. This is feasible since B. subtifis cells are not lysed immediately by the lysozyme mixture, in contrast to the gram-negative bacteria. Incomplete mixing in Step (ii) was found to be necessary in all cases to obtain an optimal yield of plasmid DNA. Pre- sumably partial lysis occurs at this stage and lysis is completed by migration of the sodium dodecyl sulfate through the bac- teria-lysozyme layer during electrophoresis. Adding the overlay mixture in Step (iii) al-

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FIG. 1. Agarose gel, 0.78% CCC plasmid DNA (O), OC plasmid DNA (X); the position of chromosomal DNA is indicated by an arrow at the left. (1) pE194cop6; (2) and (3) pE194, 10” and 2 x 10’ cells used, re- spectively; (4) pSClO1; (5) pBR322; (6) pEC125; (7) pEC124 and F’lac; (8) R 1; and (9) F’ser. For a detailed description of the strains see the text. Electrophoresis conditions: 1 h at 2 mA, followed by 2 h at 40 mA. Migra- tion of the chromosomal band was 5.9 cm from the top (anode); the bromphehol blue marker migrated out of the gel.

lows the stirred up DNA to settle again and of the gel by an arrow. The size and nature avoids smearing of DNA in the gel. of this DNA were verified by using non-

RESULTS AND DISCUSSION plasmid-bearing strains and linear molecular mass standards. Slots 1 to 3 show B. subtifis

Typical gel patterns of plasmids of various plasmids, and slots 4 to 9 illustrate plasmids sizes are shown in Figs. 1 and 2. Figure 1 in E. coli. Slot 1 shows a copy number shows the migration pattern in a 0.78% mutant of plasmid pE194, pE194cop6 (2 Md), agarose gel, and Fig. 2 shows the same strains with the starting material being IO7 cells. in a 1% agarose gel. The two different gel The CCC plasmid DNA migrates at both concentrations are used to distinguish gel concentrations in front of the chromo- whether a given DNA band is formed either somal DNA band. Between the CCC DNA by linear DNA or by open circular (OC) or and the chromosomal band two or three in- CCC plasmid DNA, since the higher gel con- termediate bands are visible. It is not known centration retards the migration of OC and whether they represent OC DNA or multi- CCC plasmid DNA more than that of the mers of pE 194~0~6. Slots 2 and 3 show the linear (e.g. chromosomal) DNA (5). CCC parental plasmid pE194, present in only one DNA is indicated on the left side of the slot copy per cell (L. Mindich, personal com- by 0 , and OC DNA by X. The position munication). In slot 2 lo* cells were used, of the chromosomal linear DNA in the range and in slot 3, 2 x 10’ cells were used. Even of 15 to 20 Md is indicated on the left side at the lower cell concentration enough plas-

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FIG. 2. Agarose gel, 1%. For explanation of the symbols see the legend to Fig. 1. The electrophoresis c :on- ditions were the same as those given in the legend in Fig. 1. Migration of the chromosomal band was 4.7 cm from the top.

mid DNA is released to form a visible band. The patterns of the bands in slots 1,2, and 3 are similar. Slot 4 shows plasmid pSC 101, a 5.6-Md plasmid with a low copy number of about 7 (3). At the lower gel concentra- tion the CCC plasmid DNA migrates in front of the chromosomal DNA, and at the higher gel concentration the CCC DNA migrates behind it. Slot 5 shows pBR322, a plasmid of 2.6 Md with high copy number (over 20) [see Ref. (2)] which is frequently used as a cloning vehicle for recombinant DNA. Slot 6 shows pEC125, a recombinant plasmid with a molecular mass of 7.1 Md derived from pBR322. At the lower gel concentra- tion (Fig. 1) the CCC plasmid DNA comi- grates with the chromosomal band; at 1% agarose concentration the CCC plasmid DNA moves behind the band. Slot 7 shows the plasmids F’lac [96 Md, see Ref. (6)] and pEC124 (4 Md) present in the same strain. pEC124 is a recombinant plasmid derived

from pBR322. The multiple CCC DNA bands of pEC124 observed at the higher get con- centration are an artifact due to the high plasmid concentration at this agarose con- centration; they are not found at the lower concentration. A comparison of the migra- tion rate at the two gel concentrations shows that the smaller molecule is relatively more retarded at the higher gel concentration than the larger F’lac. Slot 8 shows plasmid R 1 [61 Md, see Ref. (2)]. The band moving in front of the chromosomal DNA has the same relative migration rate relative to the chromosomal DNA at both gel concentra- tions and is therefore not CCC or OC plas- mid DNA. Bands of this type are char- acteristic for some of the larger plasmids (see slot 9) and they disappear if the plasmid is absent from the host strain. The origin of this band is not known. Slot 9 shows a F’ser plasmid (over 140 Md). It gives rise to seg- regant plasmids of smaller size, as indicated

588 SHORT COMMUNICATIONS

by the two fainter bands appearing below the CCC plasmid DNA.

The technique described here is useful for many purposes because of its sensitivity, simplicity, and the variety of bacterial species it can be applied to. The migration rate was found to be inversely related to the logarithm of the plasmid mass in the 2- to 50-Md range in a 0.8% agarose gel as noted elsewhere (4) (data not shown). The technique has been used to identify plasmids present in a given strain, to screen mixtures of recombinant plasmids for those of a de- sired size, and to investigate the fate of un- stable plasmids in different host back- grounds.

L. Mindich and Dr. D. Dubnau for providing some of the strains and data prior to publication. This work was supported by USPHS Grant 5RO GM 06048 to W. Maas.

REFERENCES

I. BARNES, W. M., Science 195, 393-395 (1977). 2. BUKHARI, A. I., SHAPIRO, J. A., AND ADHYA,

S. L., “DNA Insertion Elements, Plasmids, and Episomes,” Appendix B, pp 607-670. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1977.

3. CABELLO, F., TIMMIS, K., AND COHEN, S. N., Nature (London) 259, 285-290 (1976).

4. MEYERS, T. A., SANCHEZ, D., ELWELL, L. P., AND FALKOW, S., .I. Bacterial. 127, 1529- 1537 (1976).

5. MICKEL, S., ARENA, V., AND BAUER, W., Nucleic Acid Res. 4, 1465-1482 (1977).

ACKNOWLEDGMENTS 6. PALCHAUDHURI, S., AND MAAS, W. K. Mol. Gen. Genet. 146, 215-231 (1976).

I am very grateful to Dr. Werner Maas for support 7. TELFORD, T., POSELEY, P., SCHAFFNER, W., AND and helpful discussion of the manuscript and to Dr. BIRNSTIEL, M., Science 195, 391-392 (1977).