Rapid microscale procedure for visualizing intracellular plasmid DNA by electron microscopy

PLASMID

9, 8-16 (1983)

Rapid Microscale Procedure for Visualizing Intracellular Plasmid DNA by Electron Microscopy TAKAHIRO KUNISADA AND HIDEO YAMAGISHI’ Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606. Japan

Received June 1, 1982 A rapid microscale procedure is described that releases plasmid DNA in situ from the bacterial cell and that allows selective observation of the plasmid bound to celhtlar components. The released plasmid DNA was adsorbed preferentially on mica in a divalent cation-free medium then processedfor electron microscopy. The plasmid DNAs studied were pA03 (1683 base pairs (b$)), XdvO21(3505 bp), pTSOll8 (4000 bp), ~A065 (4786 bp), ColEl (6500 bp), and RSF2 124 ( 11,400 bp). These DNAs were seen as supercoiled circles or as relaxed circles of corresponding length. Occasionally an internal loop of replicating DNA was present. One micrometer of measured length corresponded to 3 100 bp.

Large genetic material in cells has been visualized by Miller and his co-workers (Miller and Beatty, 1969; Miller and Hamkalo, 1972; Miller and Bakken, 1972). Small genetic material such as plasmids, however, could not be observed by their procedure (Yamagishi and Okamoto, 1978; Okamoto and Yamagishi, 1981). We have adsorbed plasmids released from cells directly onto a mica sheet in order to make their observation possible. In the current method of DNA adsorption onto mica, divalent cations are indispensable as ligands leading to a net positive charge on the DNA molecules (Portmann and Koller, 1976; Koller and Delius, 1980). We found, however, that DNA/protein complexes may be adsorbed on mica without the use of a divalent cation and exploited this selective adsorption to develop a procedure by which the plasmid/protein complex released from a cell can be seen with electron microscope. (A preliminary account of this work has already been presented [H. Yamagishi and

T. Kunisada, Japan. J. Genet. 56, 651, 19811.) MATERIALS

AND METHODS

Bacterial Strains and Plasmids All the bacterial strains used were derivatives of Escherichia coli K- 12. The strains and the plasmid carried are listed in Table 1. Bacterial Growth Plasmid-carrying E. coli cells were grown under shaking at 37°C to a concentration of about 4 X 1O*cells/ml in L broth (Nomura et al., 1978) for Km723 (Xdv021) and in E broth (Oka et al., 1979) for the rest of the strains. For cells carrying ColE 1 or RSF2 124, chloramphenicol was added to 180 pg/ml, and incubation was continued for 3 h.

Lysis Procedures and Preparation of Plasmid DNA for Electron Microscopy (Mica-Press-Adsorption Method) Koller et al. (1974) developed an electron microscopy method with which to visualize DNA molecules. DNA molecules are ad’ To whom all correspondence should be addressed: sorbed on mica and then shadowed with platDepartment of Biophysics, Faculty of Science, Kyoto inum, after which they are picked up on carUniversity, Salcyo-lot, Kyoto 606, Japan. ’ Abbreviations used: bp, base pairs; kb, kilobases or bon film and examined with an electron microscope. We have modified their basic 1000 bp. 0147-619X/83/010008-09$03.00/0 Copyright 6 1983 by Academic Press,Inc. AU rights of reproduction in any form -cd.

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TABLE

DNA

1

BACTERIAL STRAINS AND PLASMIDS

StlZlin

Plasmid carried

C600 Km723

pAO3; small ColEl XdvO2 1

AE770 C600 w3110 HfrH5 C6OO

pTSOl18 ~A065 pTSO169 ColEl RSF2124

Plasmid length (bP) 1,683 3,505 4,ooo 4,786 5,374 6,500 11,400

method so that intracellular plasmid DNA molecules can be observed. Our current procedure is as follows: (1) Cells are collected by centrifugation and suspended in 0.1 vol of ice-cold B medium (150 mM NaCl, 50 mM Tris-HCl, pH 7.5). (2) A 1-cclsample of T4 lysozyme solution (2 mg/ml) is added to 10 ~1 of the cell suspension, and this mixture is kept on crushed ice for 3 min. (3) Ten-microliter droplets of redistilled water are placed on a fresh sheet of Parahlm, then OS-l.0 ~1 of the lysozyme-treated cell suspension is blown into the droplets. A piece of freshly cleaved mica (0.5 X lcm; Ladd Research Industries, Burlington, Vt.) is immediately placed on the surface of each droplet so that the entire mica surface is in contact with the solution. The top of the mica sheet then is pressed down with the back of a pair of forceps and left for l-3 min. This treatment ruptures the bacterial cells which releasesthe cell contents, the DNA molecules being adsorbed on the mica. (4) Excess solution is removed by touching the mica to the surface of redistilled water. The mica sheet then is washed with 1 M NaCl for 5- 10 s after which it is rinsed and immersed in redistilled water for 1 h with the DNA surface up. (5) This preparation is dehydrated with 99% ethanol for a few seconds and then airdried and rotary-shadowed with platinum at

Source, reference A. Oka; Oka et al. (1979) K. Mats&am; Chow et al. ( 1974); Mats&am (1981) A. Oka, Sugimoto et al. (1979) A. Oka; Oka et al. (1978) A. Oka, Oka et al. (1980) A. Oka; Bazaml and Helinski ( 1968) A. Oka, So et nl. (1975)

an angle of 1:15 through a 3-mm-wide slit. The slit minimizes heat damage and the resulting contamination caused by resistance heating. (6) About 10 nm of carbon is evaporated onto the mica sheetto produce a replica. This replica then is floated off the mica onto the surface of the distilled water. Carbon film is prepared by indirect evaporation. Samples are picked up on 180- or 400-mesh grids. As an alternative, platinum-carbon shadowing and carbon deposition can be performed with an electron beam evaporator, such as the JPD7000, JEOL, equipped with ion deflection plates. Sharp contrasts of shadowing are obtained by electron beam evaporation. The contour widths of DNA are about 30 A with platinum-carbon shadowing and 60 A with resistance heating of platinum. (7) Electron micrographs are usually taken with a JEM 200CX electron microscope at a magnification of 20,000 and traced as lofold-enlarged images with a YokogawaHewlett-Packard 9864A digitizer. The actual magnification was determined by photographing a line-grating replica of 2000 lines/mm. RESULTS

Egect of Divalent Cation on DNA Adsorption on Mica Covalently closed circular DNA molecules of plasmid pTSOl69 were prepared from

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FIG. 1. Plasmid pTS0 169 DNA purified by centrifugations in an ethidium bromide-c&urn chloride gradient, adsorbed on mica in the presence (a) or absence (b) of a divalent cation, Mg’+, then processed for electron microscopy. Electron-dense particles are indicated by an arrowhead. Samples were rotary shadowed by electron beam evaporation of platinum-carbon. The bar represents 0.5 pm.

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FIG. 2. Electron micrographs of plasmid DNAs prepared by the mica-press-adsorption method. Plasmids are pAO3 (a), pTSOll8 (b), ~A065 (c), ColEl (d), and RSM124 (e). Internal loop (open arrow), electron-dense structure (arrowhead), and polyribosomes (solid arrow) are shown. Samples a to d were rotary shadowed by the resistance heating of platinum and sample e by electron beam evaporation of platinum-carbon. The bar represents 0.3 pm.

cleared lysates (Vapnek and Rupp, 1971) and purified by dye-cesium chloride density gradient centrifugation. With the technique of

Portmann and Koller (1976), plasmid DNAs could be directly adsorbed on mica from the surface of a solution containing 17 pg/ml

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FIG. 3. Electron micrographs of Xdv plasmid DNA prepared by the mica-press-adsorption method. Monomer and trimer plasmids (a) and their replicative intermediates showing an internal loop (open arrow) with (b) and without (c) the electron-dense structure (arrowhead) are present. Polyribosomes are indicated by a solid arrow. Samples were rotary-shadowed by the resistance heating of platinum. The bar represents 0.3 pm.

DNA, 3 mM MgCl,, and 10 mM Tris-HCl, pH 8.5, and then processed for electron microscopy. The grids were densely covered with circular DNAs (Fig. la). The molecules

were well separated and there was virtually no aggregation. In addition to pure DNA molecules, spindle-shaped circular DNA bound to an electron-dense particle was also

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present. The pure DNA molecules were distributed homogeneously over the mica surface, but the particle-bound DNA seemed to be attached to the mica at the binding site of the particle. In the absenceof divalent cation, only particle-bound DNA was present; no pure DNA was adsorbed (Fig. 1b). The electron-dense particles seen as “dots” or “blobs” may represent protein complexes or membrane material.

Visualization of Plasmid DNAs Released from the Carrier-Cell When bacterial cells with plasmids were lysed and adsorbed on mica in the presence of a divalent cation, whole pieces of intracellular genetic material were held on the mica as aggregatesand no individual plasmid DNA molecules could be distinguished (data not shown). As suggested above, mica may preferentially adsorb the intracellular plasmid DNA bound to protein complexes or membrane material in the absence of divalent cations. Bacterial cells that carry various plasmids of known length can be ruptured and adsorbed on mica in the absence of divalent cations and then processed for electron microscopy (Figs. 2 and 3). Among the extruded contents, the circular DNA of plasmids associated with the electron-dense structures (round “dots” or “blobs”) that may represent protein complexes or membrane material, free “dots” or “blobs” and polyribosomes are most common. Circular forms are either relaxed or twisted. Repeated washing in 1 M NaCl served to remove the “dots” or “blobs” from the DNA molecules (Figs. 2a and 3c) and to relax the twisted structures. Occasionally, linear duplex DNAs bound to a protein-like structure were seen as artifacts of DNA shearing. No large aggregates of bacterial chromosomes were retained on the mica, probably because the mica could not hold such a large mass. Replicating molecules with an internal loop were also present (Figs. 2b, c and 3b, c). Most branches of the loop were bound to

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a protein-like structure at either end. The frequency of the replicative intermediates detected among all the molecules inspected was 10-l to 10d2.

Contour Length Distributions of Plasmid DNAs The distributions of the contour lengths of the circular DNA molecules are shown by the histogram (Fig. 4). All have a single peak except for ~A065 and RSF2124. A single colony carrying oligomeric plasmids of ~A065 or RSF2 124 may have been isolated. The main peaks in the histogram correspond to the known base-pair lengths of plasmid DNA monomers. The lengths of plasmid DNAs with unimodal distribution are 1.14 f 0.10 pm for XdvO21, 1.30 + 0.10 pm for pTSOll8, and 2.09 + 0.07 pm for ColEl. The mean length of fdRFI1 DNA was measured as 2.13 + 0.12 pm (56 molecules) with the original mica adsorption method (Keller and Delius, 1980) and 2.13 f 0.07 pm with the cytochrome c monolayer method (Yamagishi et al., 1976). The mean value of the measuredlengths is plotted against the known base-pair length in Fig. 5. There is a linear relationship between the measured lengths and the known base-pair lengths; lprn of measured length corresponds to 3 100 bp. There are no significant differences in variations in length among the three different spreading techniques, except for the standard deviation for the measurement of length. Thus our mica-press-adsorption method is useful for the in situ spreading of plasmid DNA for electron microscopy. DISCUSSION

Both DNA molecules and mica surfaces carry negative charges. Therefore, divalent cations serve as ligands that lead to a net positive charge for DNA molecules and mediate their adsorption on mica surfaces(Portmann and Koller, 1976). Recent evidence shows that intracellular DNA molecules are present in a complex formed with proteins

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or membrane material (Miller and Hamkalo, 1972; Yamagishi and Okamoto, 1978; Tsuda et al., 1980; Okamoto and Yamagishi, 1981). These DNA-binding proteins, or the cellular components, may carry a weak positive charge and thereby mediate the binding between plasmid DNA molecules and the mica surface. No large mass of chromosomal genetic material is retained on the mica probably because of its poor holding character. But, even after the removal of the bound protein complexes by washes with 1 M NaCl, pure exposed plasmid DNA molecules remain attached to the mica surface (Figs. 2a and 3~). The small proteins that are essential for mediating DNA-mica binding may not be removed by this treatment. Alternatively, once attached to the mica surface by a protein linker, whole stretches of DNA molecules may be attracted to the weak positive charge of potassium ions dispersed on the negatively charged mica surface. ,

I

FIG. 4. Histograms of length measurements of plasmid DNAs. Each column represents pAO3 (a), XdvO21 (b), pTSO118 (c), ~A065 (d), ColEl (e), and RSF2124 (f). An arrowhead indicates the length of the monomer plasmid estimated from the base&r length (Table 1) and the ratio of 3100 bp/pm (Fig. 5).

Base-pair

length

( kb)

FIG. 5. Plot of the mean contour lengths measured by the mica-press-adsorption method versus the basepair lengths. The points (circles) include Xdv021, pTSO118, and ColEl. For reference, fdRFI1 DNA (square)prepared by the original mica adsorption method or by the protein monolayer spreading method are included. The base-pair length of fdRFI1 DNA is 6408 bp (Beck ef al., 1978).The oblique line represents the linear relationship.

A rapid microscale technique has been developed for the isolation of plasmid DNA from a small broth culture within 3 to 4 h (Klein et al., 1980; Kado and Liu, 1981). We here have described a protocol for observing plasmid DNA within the least possible time (2 h) from the least possible culture volume (0.1 ml). The copy number of pTSOll8 that contained the replication origin of the E. coli chromosome is small, about 0.8 per bacterial genome (Sugimoto et al., 1979). Thus, the concentration of plasmid DNAs (4000 bp) released from cells at a density of 4 X lOa/ ml was estimated as a low lop3 @ml. For the surface spreading used to visualize the DNA (Kleinschmidt and Zahn, 1959; Yamagishi et al., 1976), however, a solution containing at least 0.3 &ml of DNA had to be prepared. The visualization of pTSOll8 plasmid DNA of low copy number must have been made possible by the preferential adsorption of the plasmid/protein complex on the mica. In addition, replicative intermediates of the plasmid that had an internal loop structure were found at a frequency of at least 10w2.According to Meijer and Messer (I 980), the frequency of replicative intermediates of the minichromosome plasmid that contains

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the E. coli replication origin was as low as 1Oe4.The replicative intermediates may have been enriched on the mica sheet because the protein-like structure detected at either end of the branch of the internal loop may serve as an efficient linker which mediates the adsorption of plasmid DNA on mica. Physical mapping of the replication origin appears to be feasible in combination with in situ digestion by restriction endonuclease (Miwa et al., 1979). Electron microscopy of disrupted E. coli cells revealed a condensed DNA fiber of 120 A in diameter (Griffith, 1976). In the micapress-adsorption method, no condensed fiber was observed, but it may have been stretched out by the washings in 1 M NaCl and redistilled water. Rhodes and Klug ( 1980) showed the regular helical periodicity of pure DNA immobilized on a mica surface. In this method, the stretching and distribution of plasmid DNA is as even as in the current protein monolayer film technique (Fig. 5). This technique is rapid and useful for in situ visualization of plasmids of small quantity. ACKNOWLEDGMENTS We thank Dr. H. Ozeki for his continued interest in and pertinent criticisms of this work. We also thank Dr. Y. Fujiyoshi for his help with the electron microscopic study and Drs. A. Oka and K. Matsubara for gifts of bacterial strains. This work was supported by a Grantin-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

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