JouRNAL oF BACTzRIOLOGY, June 1979, p. 878-883 0021-9193/79/06-0878/06$02.00/0

Vol. 138, No. 3

Asymmetric Transcription of R Plasmid NR1 in Proteus mirabilis EDWARD R. APPELBAUMt AND ROBERT H. ROWND* Laboratory of Molecular Biology and Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 28 December 1978

The composite R plasmid NR1, its resistance transfer factor which specifies resistance to tetracycline (RTF-Tc component), and its r-determinants component were each denatured and centrifuged to equilibrium in CsCl density gradients containing polyuridylic acid-polyguanidylic acid. The complementary deoxyribonucleic acid strands of NR1 and the complementary strands of the RTF-Tc component could be separated by this technique because of a threefold difference in polyuridylic acid-polyguanidylic acid binding to the strands of the RTF-Tc component. The two strands of the r-determinants component bound equal amounts of polyuridylic acid-polyguanidylic acid. Hybridization of single strands of plasmid deoxyribonucleic acid with in vivo-labeled ribonucleic acid from Proteus mirabilis containing NR1 indicated that transcription within the RTFTc component is from the NR1 strand which preferentially binds polyuridylic acid-polyguanidylic acid, whereas transcription within the r-determinants component is predominantly from the complementary strand. The R plasmid NR1 (also known as R100 or R222) is a composite molecule consisting of two components: a resistance transfer factor which specifies resistance to tetracycline (RTF-Tc component) and an r-determinants component which specifies resistance to chloramphenicol (Cm), fusidic acid (Fus), streptomycin/spectinomycin (Sm/Sp), sulfonamides (Su), and mercuric ions (Mer). The structure and replication of this plasmid in Proteus mirabilis has been intensively studied (4, 5, 10, 15-17). To obtain information on the transcription of NR1 in this host, the complementary DNA strands of NR1 and of the RTF-Tc component were separated and hybridized with pulse-labeled RNA extracted from P. mirabilis harboring NR1. Transcription within the RTF-Tc component is from only one strand of the R-plasmid DNA, whereas transcription within the r-determinants component is predominantly from the complementary strand, although both strands of r-determinants are transcribed. MATERIALS AND METHODS Bacterial strains and plasmids. P. mirabilis

strains Pml5 and 4S-38 were derived from strain Pml and have been described previously (9, 12, 13). Escherichia coli K-12 strain CR34B requires threonine, leucine, thiamine, and thymine for growth and is lactose and maltose negative and streptomycin resistant. t Present address: National Cancer Institute, Bethesda, MD 20205.

CR34-3 is a spontaneous nalidixic acid-resistant mutant of CR34B which was isolated by D. Taylor. These E. coli strains are missing a cryptic plasmid observed in some CR34 stocks. R plasmid NR1 and its RTF-Tc derivative pRR3 have been described previously (13, 25). R100-1 is a mutant of NR1 which is derepressed for R-plasmid

transfer (7). Isolation of "4C-labeled NR1, RTF-Tc, and rdeterminants DNA. CR34B(NR1) and CR343(pRR3) were grown to stationary phase at 37°C in Penassay broth (Difco Laboratories) supplemented with 3 jig of unlabeled thymine per ml and 0.08 ,uCi of ['4C]thymine (approximately 50 mCi/mmol; New England Nuclear Corp.) per ml. Covalently closed circular plasmid DNA was prepared by nitrocellulose chromatography (2, 20) followed by centrifugation in CsCl gradients containing ethidium bromide. The specific activity of the pooled plasmid closed circular fraction was approximately 103 cpm/,ug of DNA. To isolate rdeterminants DNA, a "transitioned" culture of Pml5(NRl), in which the r-determinants component had undergone amplification, was prepared by repeated subculturing of the cells in medium containing chloramphenicol (14-17). A stationary-phase culture labeled with ['4C]thymine as described above was lysed and centrifuged to equilibrium in a CsCl gradient, and the highest-density (1.716 to 1.718 g/ml) plasmid DNA fractions (containing linear fragments of poly-r-determinants molecules) were pooled (14). Analytical CeCi gradient centrifugation. DNA samples were centrifuged for at least 17 h at 44,000 rpm in a Spinco model E analytical ultracentrifuge, and photographic negatives of each sample were traced in a Joyce-Loebel microdensitometer (12).

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TRANSCRIPTION OF NR1 IN P. MIRABILIS

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Separation of plasmid DNA strands. (i) Ana- beled RNA was incubated in solution with saturating lytical strand separations. Pooled NR1 or pRR3 amounts of isolated single strands of plasmid DNA, DNA samples were stored for several days at 4°C in and the hybrid was collected on nitrocellulose filters. the dark in the presence of CsCl and ethidium bromide The filters were treated with RNase to reduce nonspeto allow random nicking of covalently closed circular cific or partial binding of RNA to DNA or to the filters molecules. The DNA was then extracted at least three and then washed, dried, and counted. The percentage times with equal volumes of isoamyl alcohol (Baker of the 3H label bound to the filters was corrected for Chemical Co.) to remove ethidium bromide. NR1 or nonspecific binding of RNA to filters by subtracting pRR3 DNA smples prepared in this manner or tran- the percent bound in assays where no DNA was added sitioned NR1 DNA samples in CsCl solution were then to the reaction mixture. The DNA was labeled with dialyzed against 15 mM NaCl-1.5 mM trisodium cit- "C so that recovery of the plasmid DNA during filtrarate-1.0 mM EDTA (pH 8.0). The DNA was heated tion could be monitored. The hybridization reaction and the assay of hybrids to 100°C for 4 min, rapidly cooled in ice water, and centrifuged in a Spinco model E ultracentrifuge in were performed as described in detail by B0vre et al. CsCl gradients containing 2.5 ug of DNA per ml and (3), with the following modifications. The hybridizavarious concentrations of polyuridylic acid-polyguan- tion reaction was carried out in 2x SSC (pH 7.0; lx idylic acid [poly(U-G)] (Miles Research Products; ra- SSC = 0.15 M NaCl plus 0.015 M sodium citrate) in tio of uridylic acid to guanidylic acid, approximately tightly capped, siliconized 1-dram vials. The 0.5-ml 1). Random nicking at neutral pH and heat denatur- hybridization solution contained 1 x 105 to 2 x 105 ation were used in these analytical strand separations cpm of [3H]RNA extract, and either 0.1 jig of selfin place of the alkaline nicking and denaturation de- annealed L strand, 0.1 jig of self-annealed H strand, or scribed below for preparative separations, because CsCl solution containing no DNA (blank). The vials small variations in pH can affect the densities of the were gently agitated for 18 h in a 67°C water bath. separated strands in CsCl-poly(U-G) gradients (19). Saturation curves showed that the concentration of (ii) Preparative strand separations. Freshly pre- RNA could be increased at least threefold over the pared covalently closed circular NR1 or pRR3 DNA concentration used in the hybridization experiments was extracted with isoamyl alcohol and dialyzed as without increasing the percentage of [3H]RNA or label above. NaOH was added to a final concentration of which was hybridized. 0.10 M, and the solution was heated for 50 s at 1000C and then rapidly cooled in order to nick and denature RESULTS plasmid DNA. Approximately 30 jig of denatured DNA of Separation complementary DNA and 30 ug of poly(U-G) were centrifuged in CsCl gradients for 60 h at 35,000 rpm and 23°C in a 50 Ti strands. The complementary DNA strands of rotor. Fractions were collected in sificonized (Siliclad; various plasmids and phages have been sepaBecton, Dickinson & Co.) glass tubes. Samples (5 ,l) rated by centrifugation of denatured DNA in were removed from each fraction and counted (14). CsCl density gradients containing polyribonuThe two peaks in each gradient were separately pooled cleotides which bind differentially to the two (see Fig. 2) and self-annealed to renature contaminat- strands (19, 21, 22). Equilibrium centrifugation ing complementary DNA as described by B0vre et al. of denatured, nicked NR1 DNA with an equiv(3). Labeling and isolation of RNA. R+ and R- alent quantity of poly(U-G) in an analytical CsCl strains of Pml5 were cultured in medium containing gradient revealed two broad plasmid DNA M9 salts (1), 1 mM MgSO4, 0.01 mM CaC12, 0.2% bands with densities of 1.781 and 1.752 g/ml glucose, 0.03% Casamino Acids, 10 ug of nicotinic acid (Fig. 1A). The higher-density peak was desigper ml, and 20 ug each of thymine, tryptophan, and nated NRl-H, and the lower-density peak was leucine per ml. The doubling time of the cells was 50 designated NR1-L. Both peaks had densities to 60 min. At a turbidity (optical density at 650 nm) of higher than that of denatured NR1 DNA sedi0.30, 25 ml was removed for analytical CsCl gradient mented in CsCl gradients lacking poly(U-G) centrifugation of extracted DNA. At a turbidity of 0.35 (1.727 g/ml) (data not shown). The densities of (approximately 4 x 108 cells per ml), 1.0 mCi of both NR1-H and NR1-L were increased further [5-3H]uracil (New England Nuclear Corp.; 16.0 Ci/ mmol) was added to the remaining 125 ml. Growth when higher concentrations of poly(U-G) were was terminated after 4 min by adding 10 mM sodium used, up to a maximum of 1.795 and 1.761 g/ml, azide and then immediately pouring the cells into a respectively, when the weight ratio of poly(U-G) flask containing 30 ml of medium with azide which to NR1 DNA was three or greater. At all weight had been previously frozen at -70°C. Cells were har- ratios of poly(U-G) which were used (0.5 to 4.0) vested and resuspended in 4.0 ml of 0.15 M NaCl-1 the increase in H-strand density due to poly(UmM Tris-10 mM EDTA (pH 7.7), and RNA was G) was twofold greater than the isolated by sodium dodecyl sulfate lysis and phenol increaseapproximately in L-strand density. extraction (at 60°C in the presence of macaloid), as An even greater strand separation was obdescribed by Bovre et al. (3). The 4-ml extracts contained approximately 107 trichloroacetic acid-precipi- served when denatured pRR3 (RTF-Tc) DNA table cpm per ml. The specific activity was 2 x 104 was centrifuged in analytical CsCl-poly(U-G) gradients (Fig. 1B). Both DNA strands were cpm per ,ug of RNA. DNA-RNA hybridization in solution. 'H-la- increased in density relative to the density (1.725

APPELBAUM AND ROWND

880

1788

w

-

0

(I)

17 12 MARKER

C r-det 1

1,753

DENSITY

FIG. 1. Centrifugation of denatured R plasmtid DNA in analytical CsCl gradients containingpoly(UG). Each gradient contained 2.5 pg of denatured R plasmid DNA per ml and 2.5 pg ofpoly(U-G) per ml. Microdensitometer tracings of photographic negatives of each gradient at equilibrium are shown here. (A) NRI DNA. Undenatured P. mirabilis chromosomal DNA was added as a reference (density, 1.700 g/ml). (B) pRR3 DNA and P. mirabilis chromosomal reference DNA. (C) r-determinants DNA and undenatured NRI DNA added as a reference (density, 1.712 glml).

g/ml) of denatured pRR3 DNA in gradients without poly(U-G). The increase in pRR3 Hstrand density owing to poly(U-G) binding was threefold greater than the increase in pRR3 Lstrand density. However, the strands of plasmid DNA from a transitioned Pm15/NR1 culture, in which most of the mass of the plasmid DNA was r-determinants, could not be separated by this procedure (Fig. 1C); both strands increased in density by approximately the same amount relative to the density of the denatured r-determinants DNA in gradients lacking poly(U-G) (1.733 g/ml). These results indicate that within the RTF-Tc component of NRl there is a threefold difference in poly(U-G) binding to the two strands, whereas within the r-determinants component there is no detectable difference. A minor portion of the plasmid DNA in Fig. 1A and B had the density of native NRl DNA (1.712 g/ml) or native pRR3 DNA (1.710 g/ml). This double-stranded DNA fraction presumably consisted of covalently closed circular plasmid molecules which were not nicked during purifi-

J. BACTERIOL.

cation, storage, and heating, and, therefore, renatured after the heating step. In comparison, heat denaturation of the linear 1.717-g/ml r-determinants DNA preparation was complete (Fig. 1C). The presence of covalently closed circular molecules in the NR1 and pRR3 preparations indicates that plasmid DNA molecules were not extensively degraded before strand separation. Hybridization of in vivo-labeled RNA to separated plasmid DNA -strands. Pulse-labeled RNA was extracted from exponential cultures of R+ and R- strains of P. mirabilis and hybridized with saturating amounts of the separated H and L DNA strands of NR1 DNA isolated from preparative CsCl-poly(U-G) gradients (Fig. 2). All of the RNA extracts from R+ strains contained [3H]RNA which hybridized to NR1 DNA. No hybridizable [3H]RNA could be detected in extracts of the R- strains Pml5 or 4S-38 (Table 1). [3H]RNA isolated from Pml5(NRl) or from 4S-38(R100-1) (R100-1 is a mutant of NR1 which is derepressed for R-plasmid transfer) hybridized to both strands of NRl (Table 1). Approximately 75% of the hybridizable [3H]RNA was hybridized to the NR1-H strand, and 25% was hybridized to the NR1-L strand. Thus, both

200,

A

NRI L

1000wA 20 2

40

60

80

a

60 40 FRACTION NUMKER

FIG. 2. Preparative CsCl-poly(U-G) centrifugation of " C-labeled denaturedplasmid DNA. (A) NRI DNA. (B) pRR3 DNA. The shaded fractions were pooled and self-annealed for use in RNA-DNA hybridization studies.

TRANSCRIPTION OF NR1 IN P. MIRABILIS

VOL. 138, 1979

TABLE 1. Hybridization ofpulse-labeled [3H]RNA to the separated strands of NRl and pRR3 % of

hybrid Source of [3H]RNA

DNA

% of 3H hy-

bridizeda

bound to

each strand

Pml5 R4S-38 RPml5(NRl)

4OS-38(R100-1) Pml5(pRR3)

NR1-L 0.01 (+.07) NR1-H -0.05 (±.08) NR1-L 0.03 (+.03) NR1-H 0.03 (+.03) NR1-L 0.34 (+.06) NR1-H 0.82 (±.06) NR1-L 0.30 (±.05) NR1-H 1.00 (±.18) NRl-L 0.05 (±.05) NR1-H 0.56 (±.08) NR1-L 0.70 (±.19) NR1-H 0.47 (±.18) pRR3-L 0.00 (±.01) pRR3-H 0.19 (±.01) NR1-L 0.56 (±.10) NR1-H 0.28 (±.01)

29 71 23 77 8 92 60 40