Molec. gem Genet. 162, 43-50 (1978) © by Springer-Verlag 1978

Pressure Dissociation of Bacterial Ribosomes and Reassociation of Ribosomal Subunits Carlo Cocito Department of Microbiology,Institute of Cell Pathology,Universityof LouvainMedicalSchool, Brussels 1200, Belgium

Summary. Disruption of multiplying bacteria by pressure release produces the dissociation of polysomes and the formation of ribosomal subunits, which quickly reassociate into 70S ribosomes. Most reassociated particles are stable when centrifuged at high speed in neutral sucrose gradients containing 10 mM Mg + +. Several environmental factors prevent the reassociation of pressure-dissociated ribosomes: pH 50 raM, N H 4 C 1> 100 mM, Mg + + < 6 raM, and glutaraldehyde > 0.1%. When cellular growth is blocked either by nutrient exhaustion (stationary phase, starvation of auxotrophs) or by prolonged incubation with antibiotics (transcriptional and translational inhibitors) most cytoplasmic polysomes disappear and ribosomes which accumulate, upon pressure-dissociation, yield subunits displaying a normal reassociation kinetics. Such reassociated particles, however, dissociate again in the course of high-speed centrifugation unless fixed with glutaraldehyde. Polysomes from multiplying bacteria, upon incubation with RNase and French Press treatment, yield subunits reassociating into monosomes that are labile to low-speed centrifugation: this effect is still prevented by glutaraldehyde fixation. Ribosomes produced under different experimental conditions can, thus, be classified according to the 2 criteria of (a) reassociation capacity after pressure dissociation, and (b) resistance of reassociated particles to high- and low-speed centrifugation, into four distinct groups (non reassociating subunits, and subunits forming low- speed-labile, high-speed-labile and centrifugation-resistant ribosomes),

Introduction Ribosomes of exponentially multiplying bacteria undergo a periodical dissociation into 50S and 30S sub-

units, and a reassociation into 70S particles, "ribosomal cycle", during the process of protein synthesis (Schlessinger and Apirion, 1969; Schlessinger, 1969 ; Davis, 1971; Kaempfer, 1974; Kaempfer and Meselson, 1969). It remains undecided whether termination of peptide chains produces monosomes or subunits, and whether free subunits transiently associate into monosomes that split again at the onset of initiation (Davis, 1974). Since initiation and termination are stepwise processes involving the periodical binding to ribosomes, and the detachment from ribosomes, of different RNA and protein factors, different species of monosomes and subunits are expected to be present in cells kept under different experimental conditions: they might be distinguishable on the basis of their biophysical and biochemical properties. The hydrodynamic dissociation of mammalian cell ribosomes in high gravitational fields was originally discovered by Infante and Baierlein (Infante and Baierlein, 1971). Bacterial ribosomes prepared under given experimental conditions were also found to split into their subunits during centrifugations in sucrose gradients, and to form a 60S peak of dissociated particles travelling behind that of stable 70S monosomes (Spirin, 1971; Subramanian and Davis, 1971; Van Diggelen, Heinsius, Kalowsek and Bosch, 1971; Cocito, 1973). In the present work, another type of in vitro dissociation of ribosomal particles is described. It will be shown that bacterial ribosomes from whole cells and purified polysomes, when submitted to compression and sudden pressure release, split into subunits, which quickly reassociate into 70S ribosomes. Some of the reassociated particles are sensitive and others are resistant to further centrifugal dissociation, depending on their origin, mode of preparation and environmental factors. These two criteria: capacity of reassociation after pressure release, and centrifugal stability of reassociated particles, allow the recognition of 4 types of

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particles, which are produced either by in vivo treatment of bacteria with several inhibitors, or after in vitro incubation of cytoplasm with different chemicals.

Materials and Methods Microorganisms. The following strains of B. subtilis 168 were used: 168/6 w.t., 168/2 tryp- leuc , M~S~ sensitive to virginiamycin M and S (Cocito, 1969). They were grown in low- and high-phosphate synthetic media supplemented with an enzymatic hydrolysate of casein, and labeled with either (32p)phosphate or (~H)uracil (Cocito and Fraselle, 1973). Cultures were kept in shaking water baths at 37 ° C. Polyribosomes. Labeled cells were incubated with lysozyme (100 gg/ 108 cells/ml, 10 rain, 37 ° C) in 35% (w/v) sucrose. Protoplasts sedimented at 10,000 rpm were shocked by suspension in 0.06 M KC1, 0.01 M Mg-acetate, 0.006 M/~-mercaptoethanol, 0.01 M Tris-HCl pH 7.4 (NB buffer). Membranes were pelleted by centrifugation at 12,000 rpm for 10 rain, and supernatants were layered over 0.5 to 28.5% sucrose gradients in NB buffer, and centrifuged at 40,000 rpm for 30 rain in swing-out rotors (SW 50.1 Spinco): A26o, m and radioactivity of the gradients were monitored. In some experiments, polysomes were harvested by centrifugation of membrane free cytoplasm at 50,000 rpm for 20 min at 4 ° C, before analysis in density gradients. Ribosomes and Subunits. For concentration of ribosomes and subunits, protoplast lysates previously freed of polysomes were centrifuged at 50,000 rpm for 75 min at 4 ° C. Sedimented particles were layered over 0.5 to 28.5% sucrose gradients, and centrifuged at 50,000 rpm for 60 min at 4 ° C. Pressure Disruption and Fixation. Ice-cold cells and particles were disrupted by compression at 8,000 psi in a French Pressure Cell (Aminco Instr., Silver Spring, Md) operated by a Wabash hydraulic press (Wabash, Indiana) and sudden release. Samples were fixed by addition at 0.5 to 2.5% (w/v) glutaraldehyde at different times after the compression-release treatment, and processed without delay. Chemicals. Glutaraldehyde was a product of BDH Chemicals (Pool, England): aqueous solutions of this reagent were freshly prepared and neutralized with KOH just before use. Chloramphenicol and actinomycin D were purchased from Serva Feinbiochemica (Heidelberg, W. Germany). Crystalline preparations of virginiamycin M and S (Cocito and Kaji, 1971) were obtained by fractionation of crude fermentation batches of the antibiotic, and fresh aqueous solutions of both inhibitors were used in all experiments.

Results

Kinetic of Reassociation of Pressure-Dissociated Ribosomes When exponentially multiplying bacteria were disrupted by compression and sudden release in a French pressure cell, a dissociation of polysomes occurred with production of ribosomal subunits. Split 50S and 30S particles joined and formed 70S ribo-

C. Cocito : Pressure Dissociation of Bacterial Ribosomes

somes according to a very fast kinetics, practically unmeasurable at room temperature. This process was followed by slowing down the reaction (low temperature + NAN3), and fixing the particles with glutaraldehyde at increasing intervals. As shown in Figure 1, essentially all the ribosomes were dissociated into subunits by the French Press treatment, and most of the subunits reassociated into ribosomes within 30 s. Note that similar results were obtained when intact bacterial cells, purified polyribosomes, or ribosomes isolated from shocked protoplasts were submitted to pressure dissociation (v.i.).

Reassociation Capacity of Ribosomes Incubated in vitro with Different Concentrations of Inorganic Ions It is well established that incubation of ribosomes at low temperature with hypertonic solutions of salts frees the particles of protein factors transiently associated during different steps of peptide chain formation. Such treatment might also impair the reassociation capacity and stability of pressure-dissociated ribosomes. Polyribosomes obtained from growing cells were incubated with 2 concentrations of NH4C1 , and submitted to compression in a French pressure cell. Dissociated particles were fixed with glutaraldehyde without delay (time 0), or after 1 min, and centrifuged in density gradients. Reassociation velocity slowed down, upon incubation of polysomes with 50 mM NH4C1 (Fig. 2C and 2D), as compared to the controls (Fig. 2A and 2B). When particles were incubated with 500 mM NH4C1, pressure dissociation was followed by little or no reassociation (Fig. 2E and F), Similar results were obtained by incubating polysomes with decreasing concentrations of Mg acetate, increasing amounts of Na azide, and low pH (Table 1).

Lability of Reassociated Ribosomes from Non-Growing Bacteria In exponentially multiplying B. subtilis most ribosomal particles were present as polysomes; in addition, there were prominent peaks of 100S and 70S particles, and negligible amounts of free ("native") subunits (Fig. 3A). When bacterial growth was blocked either by exhaustion of nutrients, or by treatment with some antibiotics, polysomes disappeared and monosomes and subunits accumulated in different proportions. Thus, e.g., stationary cultures showed, in addition to polysome depletion, an accumulation of 100S and 70S particles (Fig. 3 B); whereas in auxo-

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Fig. 1A-F. Kinetics of subunit reassociation after pressure disruption of bacterial cells. M~S1 cells labeled with (3H) uracil and suspended in NB buffer containing 50 m M NaN3 were disrupted in a French pressure cell. Glutaraldehyde (2.5%) was added after 0 (A), 10 (B), 20 (C), and 30 s (D) or after 5 rain (E); (F) is the control without glutaraldehyde. Samples layered over 0.5 to 28.5% sucrose gradients were centrifuged at 50,000 rpm for 60 rain

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