Inhibition of Translation and 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells by 11 Different Ketolide Antibiotics

Eleven structurally similar ketolide antibiotics were tested at a concentration of 1 μg/ml for their relative inhibitory effects on growth and ribosome activities in Staphylococcus aureus cells. Ten of the compounds examined had an inhibitory effect on protein synthesis at this concentration and eight of the 11 compounds were also effective inhibitors of the formation of the 50S ribosomal subunit. All of the drugs tested inhibited protein synthesis to a greater extent than they affected 50S subunit formation. The decline in growth rate and cell number was proportional to the effect on ribosome formation and function. The growth of an ermC erythromycin-resistant strain of S. aureus was also significantly inhibited by nine ketolide compounds, suggesting that they were not inducers of methylase gene expression. These inhibitory activities can be related to structural differences between these ketolide antibiotics. Received: 6 May 1998 / Accepted: 27 July 1998     

Erythromycin inhibits the assembly of the large ribosomal subunit in growing Escherichia coli cells

Erythromycin and other macrolide antibiotics have been examined for their effects on ribosome assembly in growing Escherichia coli cells. Formation of the 50S ribosomal subunit was specifically inhibited by erythromycin and azithromycin. Other related compounds tested, including oleandomycin, clarithromycin, spiramycin, and virginiamycin M₁, did not influence assembly. Erythromycin did not promote the breakdown of ribosomes formed in the absence of the drug. Two erythromycin-resistant mutants with alterations in ribosomal proteins L4 and L22 were also examined for an effect on assembly. Subunit assembly was affected in the mutant containing the L22 alteration only at erythromycin concentrations fourfold greater than those needed to stop assembly in wild-type cells. Ribosomal subunit assembly was only marginally affected at the highest drug concentration tested in the cells that contained the altered L4 protein. These novel results indicate that erythromycin has two effects on translation, preventing elongation of the polypeptide chain and also inhibiting the formation of the large ribosomal subunit.     

Preferential Inhibition of Protein Synthesis by Ketolide Antibiotics in <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> Cells

The ketolide antibiotics are semi-synthetic derivatives of erythromycin A with enhanced inhibitory activity in a wide variety of microorganisms. They have significantly lower MICs than the macrolide antibiotics for many Gram-positive organisms. Two ketolides, telithromycin and ABT-773, were tested for growth-inhibitory effects in Haemophilus influenzae. Both antibiotics increased the growth rate and reduced the viable cell number with IC₅₀ values of 1.5 μg/ml. Protein synthesis was inhibited in cells with a similar IC₅₀ concentration (1.25 μg/ml). Macrolide and ketolide antibiotics have been shown to have a second equivalent target for inhibition in cells, which is blocking the assembly of the 50S ribosomal subunit. Pulse and chase labeling assays were conducted to examine the effect of the ketolides on subunit formation in H. influenzae. Surprisingly, both antibiotics inhibited 50S and 30S subunit assembly to the same extent, with no specific effect of the compounds on 50S assembly. Over a range of antibiotic concentrations, 30S particle synthesis was diminished to the same extent as 50S formation. H. influenzae cells seem to have only one significant target for these antibiotics, and this may help to explain why these drugs are not more effective than the macrolides in preventing the growth of this microorganism. Received: 21 February 2002 / Accepted: 30 April 2002     

Inhibition of 50S Ribosomal Subunit Assembly in <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> Cells by Azithromycin and Erythromycin

Azithromycin is an important antibiotic for the treatment of several different Gram-positive and Gram-negative bacterial infections. Erythromycin and clarithromycin are less useful antibiotics against Gram-negative infections. This difference in inhibitory activity was explored by comparing the effects of azithromycin and erythromycin on cellular functions in Haemophilus influenzae cells. Effects of both antibiotics on translation, cell viability, and growth rates have been measured. An IC₅₀ of 0.4 μg/ml was found for the effects of azithromycin on each of these processes. For erythromycin, an IC₅₀ of 1.5 μg/ml was observed, indicating a fourfold lower sensitivity of the organisms to this compound. The features of a second target for macrolide antibiotic inhibition in H. influenzae cells have also been examined. Inhibition of the synthesis of the large 50S ribosomal subunit was measured. Subunit formation was prevented in a concentration dependent fashion, with azithromycin showing a ninefold greater effect on this process compared with erythromycin. Synthesis of the 30S ribosomal subunit was not effected. Pulse and chase labeling kinetics confirmed the slower synthesis rate of the 50S particle in the presence of each antibiotic. The results are discussed in terms of the stronger effect of azithromycin on ribosome biosynthesis in this organism. Received: 24 July 2001 / Accepted: 25 September 2001     

Preferential inhibition of protein synthesis by ketolide antibiotics in Haemophilus influenzae cells

The ketolide antibiotics are semi-synthetic derivatives of erythromycin A with enhanced inhibitory activity in a wide variety of microorganisms. They have significantly lower MICs than the macrolide antibiotics for many Gram-positive organisms. Two ketolides, telithromycin and ABT-773, were tested for growth-inhibitory effects in Haemophilus influenzae. Both antibiotics increased the growth rate and reduced the viable cell number with IC(50) values of 1.5 microgram/ml. Protein synthesis was inhibited in cells with a similar IC(50) concentration (1.25 microgram/ml). Macrolide and ketolide antibiotics have been shown to have a second equivalent target for inhibition in cells, which is blocking the assembly of the 50S ribosomal subunit. Pulse and chase labeling assays were conducted to examine the effect of the ketolides on subunit formation in H. influenzae. Surprisingly, both antibiotics inhibited 50S and 30S subunit assembly to the same extent, with no specific effect of the compounds on 50S assembly. Over a range of antibiotic concentrations, 30S particle synthesis was diminished to the same extent as 50S formation. H. influenzae cells seem to have only one significant target for these antibiotics, and this may help to explain why these drugs are not more effective than the macrolides in preventing the growth of this microorganism.     

Neomycin and Paromomycin Inhibit 30S Ribosomal Subunit Assembly in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

A number of different antibiotics that prevent translation by binding to the 50S ribosomal subunit of bacterial cells have recently been shown to also prevent assembly of this subunit. Antibacterial agents affecting 30S particle activities have not been examined extensively for effects on small subunit formation. The aminoglycoside antibiotics paromomycin and neomycin bind specifically to the 30S ribosomal subunit and inhibit translation. These drugs were examined in Staphylococcus aureus cells to see whether they had a second inhibitory effect on 30S particle assembly. A ³H-uridine pulse and chase assay was used to examine the kinetics of subunit synthesis in the presence and absence of each antibiotic. 30S subunit formation was inhibited by both compounds. At 3 µg/mL each antibiotic reduced the rate of 30S formation by 80% compared with control cells. Both antibiotics showed a concentration-dependent inhibition of particle formation, with a lesser effect on 50S particle formation. For neomycin, the IC₅₀ for 30S particle formation was equal to the IC₅₀ for inhibition of translation. Both antibiotics reduced the viable cell number with an IC₅₀ of 2 µg/mL. They also inhibited protein synthesis in the cells with different IC₅₀ values (2.5 and 1.25 µg/mL). This is the second demonstration of 30S ribosomal subunit-specific antibiotics that prevent assembly of the small subunit.Received: 13 August 2002 / Accepted: 4 November 2002     

Linezolid Is a Specific Inhibitor of 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells

Linezolid is an oxazolidinone compound that has been shown to have impressive antimicrobial activity against a number of Gram-positive bacteria. It inhibits an initiation step of protein synthesis, and its binding site has been shown to be on the 50S ribosomal subunit. Linezolid was tested to see whether would interfere with the formation of the 50S subunit in Staphylococcus aureus cells, since a number of other 50S-specific antibiotics have this second inhibitory function. Linezolid inhibited protein synthesis in S. aureus cells with an IC₅₀ of 0.3 μg/ml. A concentration-dependent decline in cell number with an increase in generation time was found. Pulse-chase labeling studies revealed a specific inhibitory effect on 50S particle formation, with no effect on 30S subunit assembly. The compound inhibited 50S synthesis with an IC₅₀ of 0.6 μg/ ml, indicating an equivalent effect on translation and particle assembly. A postantibiotic effect of 1 h was found when cells were initially treated with the drug at 2 μg/ ml. 50S particle numbers recovered more rapidly than translational capacity, consistent with the increase in viable cell numbers. The inhibitory activities of this novel antimicrobial agent in cells are discussed. Received: 28 June 2001 / Accepted: 27 August 2001     

Neomycin and paromomycin inhibit 30S ribosomal subunit assembly in Staphylococcus aureus

A number of different antibiotics that prevent translation by binding to the 50S ribosomal subunit of bacterial cells have recently been shown to also prevent assembly of this subunit. Antibacterial agents affecting 30S particle activities have not been examined extensively for effects on small subunit formation. The aminoglycoside antibiotics paromomycin and neomycin bind specifically to the 30S ribosomal subunit and inhibit translation. These drugs were examined in Staphylococcus aureus cells to see whether they had a second inhibitory effect on 30S particle assembly. A 3H-uridine pulse and chase assay was used to examine the kinetics of subunit synthesis in the presence and absence of each antibiotic. 30S subunit formation was inhibited by both compounds. At 3 microg/mL each antibiotic reduced the rate of 30S formation by 80% compared with control cells. Both antibiotics showed a concentration-dependent inhibition of particle formation, with a lesser effect on 50S particle formation. For neomycin, the IC50 for 30S particle formation was equal to the IC50 for inhibition of translation. Both antibiotics reduced the viable cell number with an IC50 of 2 microg/mL. They also inhibited protein synthesis in the cells with different IC50 values (2.5 and 1.25 microg/mL). This is the second demonstration of 30S ribosomal subunit-specific antibiotics that prevent assembly of the small subunit.     

Structure–Activity Relationships for Three Macrolide Antibiotics in <Emphasis Type="Italic">Haemophilus influenzae</Emphasis>

A prior study examining differences in the activities of erythromycin and azithromycin on cellular functions in the Gram-negative pathogen, Haemophilus influenzae, revealed a marked difference in their inhibitory activities. The study revealed that protein synthesis and 50S ribosomal subunit assembly were equal targets for inhibition by azithromycin while erythromycin was a preferential inhibitor of translation. This contrast in inhibitory activities stimulated a comparative analysis of three additional antibiotics: clarithromycin, flurithromycin and roxithromycin. Each compound was tested over a concentration range for inhibitory effects on cellular processes. Clarithromycin was the most effective inhibitor of protein synthesis with an IC₅₀ of 5.6 g/mL, followed by flurithromycin at 6 g/mL, and roxithromycin at 9 g/mL. IC₅₀ values for antibiotic effects on viable cell counts and growth rates were similar to those obtained for protein synthesis. Flurithromycin had the strongest effect on 50S ribosomal subunit formation with an IC₅₀ of 8 g/mL, followed by clarithromycin and roxithromycin, at 9.0 g/mL and 12.5 g/mL respectively. 30S ribosomal subunit formation in cells treated with flurithromycin and roxithromycin was also reduced to some extent. Pulse-and-chase labeling kinetics examining subunit assembly rates verified the slower synthesis rate of the subunits in the presence of each macrolide. The results are discussed in terms of structural differences of these macrolides and their differential inhibitory effects on both cellular targets.     

Specific Inhibition of 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells by 16-Membered Macrolide, Lincosamide, and Streptogramin B Antibiotics

The translational functions of the bacterial ribosome are the target for a large number of antimicrobial agents. The 14- and 16-membered macrolides, the lincosamides, and the streptogramin B type antibiotics are thought to share certain inhibitory properties, based on both biochemical and genetic studies. We have shown previously that the 14-membered macrolides, like erythromycin, have an equivalent inhibitory effect on translation and the formation of the 50S ribosomal subunit in growing bacterial cells. To extend this work, we have now tested the 16-membered macrolides spiramycin and tylosin, the lincosamides lincomycin and clindamycin, and 3 streptogramin B compounds pristinamycin I_A, virginiamycin S, and CP37277. Each of these was a specific inhibitor of 50S subunit formation, in addition to having an inhibitory effect on translation. By contrast, two streptogramin A compounds, virginiamycin M1 and CP36926, as well as chloramphenicol, were effective inhibitors of translation without showing a specific effect on the assembly of the large ribosomal subunit. A combination of an A and B type streptogramin (virginiamycin M1 and pristinamycin I_A) demonstrated a synergistic inhibition of protein synthesis without exhibiting a specific inhibition of 50S subunit formation. These results extend our observations on 50S assembly inhibition to the entire class of MLS_B antibiotics and reinforce other suggestions concerning their common ribosome-binding site and inhibitory functions. Received: 13 January 2000 / Accepted 2 March 2000     

Superiority of 11,12 Carbonate Macrolide Antibiotics as Inhibitors of Translation and 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells

Three pairs of related macrolide antibiotics, differing at the 11,12 position of the macrolactone ring, were compared for effects on growth rate, cell viability, protein synthesis, and 50S ribosomal subunit formation in Staphylococcus aureus cells. For each parameter measured, the 11,12 carbonate–derivatized compound was more inhibitory compared with the corresponding 11,12-hydroxy antibiotic. Substitution at the 3-position of the ring was also important in the relative inhibition observed. The degree of inhibition found in two different growth media was proportional to the generation time of the cells. Inhibition of both protein synthesis and 50S subunit formation by each drug correlated well with the inhibition of cell viability. The results indicate that closure of the 11,12-hydroxyl groups in macrolide antibiotics with a carbonate substitution generates a more effective antimicrobial agent. Received: 11 January 1999 / Accepted: 9 February 1999     

Structure–Activity Relationships for Six Ketolide Antibiotics

Six structurally related 3-keto-substituted macrolide antibiotics (ketolides) were compared for concentration-dependent inhibitory effects on growth rate, viable cell number, and protein synthesis rates in Staphylococcus aureus cells. Inhibitory effects on 50S ribosomal subunit formation were also examined, as this is a second target for these antibiotics. A concentration range of 0.01 to 0.1 microg/ml was tested. An IC50 for inhibition of translation and 50S synthesis was measured for each compound, to relate structural features to inhibitory activity. ABT-773 was the most effective of the six compounds tested with an IC50 = 0.035 microg/ml. HMR 3004 was almost as effective with an IC50 = 0.05 microg/ml. Two 2-fluoroketolides (HMR 3562 and HMR 3787) were equivalent in their inhibitory activity with an IC50 = 0.06 microg/ml. Telithromycin (HMR 3647) had an IC50 = 0.08 microg/ml, and HMR 3832 was least effective with an IC50 = 0.11 microg/ml. Each antibiotic had an equivalent inhibitory effect on translation and 50S subunit formation. These results indicate specific structural features of these antimicrobial agents, which contribute to defined inhibitory activities against susceptible organisms.     

The ketolide antibiotic ABT-773 is a specific inhibitor of translation and 50S ribosomal subunit formation in Streptococcus pneumoniae cells

ABT-773 is a new 3-keto macrolide antibiotic that has been shown to be very effective against infections by Gram-positive microorganisms. This work examines its inhibitory effects in cells of Streptococcus pneumoniae. ABT-773 caused a proportional decline in cell growth rates and viability with an IC₅₀ of 5 ng/ml. Protein synthesis in these cells was reduced by 50% at an antibiotic concentration of 2.5 ng/ml. This compound was also found to be a very effective inhibitor of the formation of the 50S ribosomal subunit in growing cells. Pulse and chase labeling assays revealed a reduced rate of 50S synthesis in antibiotic-treated cells. At 2 ng/ml, the rate was reduced to 33% of the control synthesis rate. An IC₅₀ of 5 ng/ml was found for the effect on this process, indicating an equal effect of the drug on translation and assembly. Synthesis of the 30S ribosomal subunit was unaffected by this antibiotic. The effects of ABT-773 in S. pneumoniae are compared with those of the related ketolide antibiotic telithromycin in S. pneumoniae and in Staphylococcus aureus. Received: 6 November 2001 / Accepted: 14 December 2001     

Kinetics of ribosome synthesis during a nutritional shift-up in Escherischia coli K-12.

Summary The rates of total protein synthesis, polyribosome formation and 70S ribosome accumulation were measured following a nutritional shift-up ofEscherichia coli K-12. Changes in ribosome content and distribution during the shift-up were measured by examining the total cellular content of free and polysome-associated ribosomes using a sensitive double isotope labeling method. The kinetics of ribosomal subunit formation and the biosynthesis of subunit protein and RNA species were also defined. The results indicated that a pre-shift population of ribosomal subunits was utilized for the immediate post shift increase in both total and ribosomal-specific protein synthesis. An assembly time for new subunits of about 3 min was observed. The formation of certain ribosomal proteins during the shift suggested that new subunit assembly was limited by the rate of synthesis of particular ribosomal proteins during this growth transition.     

The chemistry of protein synthesis and voyage through the ribosomal tunnel

High-resolution structures of ribosomal subunits and their complexes with substrates and antibiotics have revealed fundamental principles of template-directed protein synthesis. Mechanistic questions regarding ribosome function and catalysis can now be addressed with structure-based experiments. Recent studies have investigated the mechanism of peptide bond formation catalyzed by the large ribosomal subunit, the mode of protein synthesis inhibition by macrolide antibiotics, the interaction of nascent polypeptides with the ribosomal exit tunnel, and the role of ribosomal proteins in the recruitment of accessory factors that assist protein folding and targeting.     

Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly

Two strains of Saccharomyces cerevisiae were constructed that are conditional for synthesis of the 60S ribosomal subunit protein, L16, or the 40S ribosomal subunit protein, rp59. These strains were used to determine the effects of depriving cells of either of these ribosomal proteins on ribosome assembly and on the synthesis and stability of other ribosomal proteins and ribosomal RNAs. Termination of synthesis of either protein leads to diminished accumulation of the subunit into which it normally assembles. Depletion of L16 or rp59 has no effect on synthesis of most other ribosomal proteins or ribosomal RNAs. However, most ribosomal proteins and ribosomal RNAs that are components of the same subunit as L16 or rp59 are rapidly degraded upon depletion of L16 or rp59, presumably resulting from abortive assembly of the subunit. Depletion of L16 has no effect on the stability of most components of the 40S subunit. Conversely, termination of synthesis of rp59 has no effect on the stability of most 60S subunit components. The implications of these findings for control of ribosome assembly and the order of assembly of ribosomal proteins into the ribosome are discussed.     

TAN-1057A: A Translational Inhibitor with a Specific Inhibitory Effect on 50S Ribosomal Subunit Formation

The inhibitory activities of a novel antibiotic compound have been investigated. A synthetic version of the natural product TAN-1057A was examined for its effects on translation and ribosomal subunit formation. The antibiotic at 6 μg/ml reduced the growth rate of wild-type Staphylococcus aureus cells by 50%. The IC₅₀ for inhibition of protein synthesis in these cells was 4.5 μg/ml. Pulse and chase labeling kinetics showed a strong inhibitory effect on 50S ribosomal subunit formation as well. The IC₅₀ for this process was 9 μg/ml, indicating an equivalent inhibitory effect of the antibiotic on translation and 50S synthesis. The post-antibiotic effect of the drug was investigated. Protein synthesis resumed rapidly after removal of the drug from cells, but full recovery of the normal 50S subunit complement in treated cells required 1.5 h. The dual inhibitory effects of this compound are compared with other antimicrobial agents having similar effects on cell growth. Received: 27 December 2000 / Accepted: 22 March 2001     

An Examination of the Differential Sensitivity to Ketolide Antibiotics in <Emphasis Type="Italic">ermB</Emphasis> Strains of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Several reports in the literature have described a differential sensitivity to ketolide antibiotics in ermB strains of Streptococcus pyogenes and Streptococcus pneumoniae resistant to erythromycin. Strains of S. pyogenes and S. pneumoniae carrying different erm gene alleles were examined for their susceptibility to the ketolide antibiotics cethromycin (ABT-773) and telithromycin. The effect of the antibiotics on cell growth and viability was assessed as were effects on protein synthesis and 50S ribosomal subunit formation. The susceptibility of wild-type strains of both organisms was compared with effects in strains containing the ermA and ermB methyltransferase genes. A wild-type antibiotic-susceptible strain of S. pyogenes was comparable to an ermA strain of the organism in its ketolide sensitivity, with IC₅₀ values for 50% inhibition of protein synthesis and 50S ribosomal subunit formation of 10 ng/mL for cethromycin and 16 ng/mL for telithromycin. An S. pneumoniae strain with the ermB gene and an S. pyogenes strain with the ermA gene were also similar in their sensitivity to ketolide inhibition. IC₅₀ values for inhibition of translation and subunit formation in S. pneumoniae (ermB) were 30 ng/mL and 55 ng/mL and for the ermA strain of S. pyogenes they were 15 ng/mL and 35 ng/mL respectively. By contrast, an S. pyogenes ermB strain was significantly more resistant to both ketolides, with IC₅₀ values for inhibition of 50S synthesis of 215 and 380 ng/mL for the two ketolides. Experiments were conducted to examine ribosome synthesis and translational activity in the two ermB strains at intervals during growth in the presence of each antibiotic. Cell viability and 50S subunit formation were dramatically reduced in the S. pneumoniae strain during continued growth with either drug. By contrast, the ketolides had little effect on the S. pyogenes strain growing with the antibiotics. The results indicate that ketolides have a reduced inhibitory effect on translation and 50S subunit synthesis in S. pyogenes with the ermB gene compared with the other strains examined.     

Chloroplast ribosomal protein L-18 in Chlamydomonas reinhardtii is processed during ribosome assembly

Summary Chloroplast ribosomal protein L-18 is made in the cytoplasm as a precursor, imported into the chloroplast, and processed to the mature form in two steps. We report here that the intermediate produced following the first processing step associates specifically with a ribosomal complex migrating with the chloroplast ribosome large subunit peak in sucrose gradients, and is then processed into mature L-18. This processing event is slowed down in mutant cells deficient in synthesis of non-ribosomal proteins in the chloroplast. Thus the second processing step of L-18 occurs during ribosome assembly, depends on one or more nonribosomal proteins made in the chloroplast, and may be required for the maturation of the 50 S ribosome subunit. The mature L-18 protein shows extensive sequence homology at its amino-terminus to Escherichia coli ribosomal protein L27, which is located at the interface, between 30 S and 50 S subunits and is involved in the formation of the peptidyl-tRNA binding site.     

Lack of complete cooperativity of ribosome assembly in vitro and its possible relevance to in vivo ribosome assembly and the regulation of ribosomal gene expression.

Earlier studies have shown that the reconstitution of Escherichia coli 50S as well as 30S ribosomal subunits from component rRNA and ribosomal protein (r-protein) molecules in vitro is not completely cooperative and binding of more than one r-protein to a single 16S rRNA (or 23S rRNA) molecule is required to initiate a successful 30S (or 50S) ribosome assembly reaction. We first confirmed this conclusion by carrying out 30S subunit reconstitution in the presence of a constant amount of 16S rRNA together with various amounts of total 30S r-proteins (TP30) and by analyzing the physical state of reconstituted particles rather than by assaying protein synthesizing activity of the particles as was done in the earlier studies. As expected, under conditions of excess rRNA, the efficiency of 30S subunit reconstitution per unit amount of TP30 decreased greatly with the decrease in the ratio of TP30 to rRNA, indicating the lack of complete cooperativity in the assembly reaction. We then asked the question whether the cooperativity of ribosome assembly is complete in vivo. We treated exponentially growing E coli cells with low concentrations of chloramphenicol which is known to inhibit protein synthesis without inhibiting rRNA synthesis, creating conditions of excess synthesis of rRNA relative to r-proteins. Several concentrations of chloramphenicol (ranging from 0.4 to 4.0 micrograms/ml) were used so that inhibition of protein synthesis ranged from 40 to 95%. Under these conditions, we examined the synthesis of RNA, ribosomal proteins and 50S ribosomal subunits as well as the synthesis of total protein. We found that the synthesis of 50S subunits was not inhibited as much as the synthesis of total protein at lower concentrations of chloramphenicol, but the degree of inhibition of 50S subunit synthesis increased sharply with increasing concentrations of chloramphenicol and was in fact greater than the degree of inhibition of total protein synthesis at chloramphenicol concentrations of 2 micrograms/ml or higher. The inhibition of 50S subunit synthesis was significantly greater than the inhibition of r-protein synthesis at all chloramphenicol concentrations examined. These data are consistent with the hypothesis that the cooperativity of ribosome assembly in vivo is also not complete as is the case for in vitro ribosome reconstitution, but are difficult, if not impossible, to explain on the basis of the complete cooperativity model.(ABSTRACT TRUNCATED AT 400 WORDS)