The genes for ribosomal proteins IA and L22 from two erythromycin-resistant mutants of Escherichia coli have been isolated and sequenced. In the L4 mutant, an A-to-G transition in codon 63 predicted a Lys-to-Glu change in the protein. In the L22 strain, a 9-bp deletion removed codons 82 to 84, eliminating the sequence Met-Lys-Arg from the protein. Consistent with these DNA changes, in comparison with wild-type proteins, both mutant proteins had reduced first-dimension mobilities in two-dimensional polyacrylamide gels. Complementation of each mutation by a wild-type gene on a plasmid vector resulted in increased erythromycin sensitivity in the partial-diploid strains. The fraction of ribosomes containing the mutant form of the protein was increased by growth in the presence of erythromycin. Erythromycin binding was increased by the fraction of wild-type protein present in the ribosome population. The strain with the IA mutation was found to be cold sensitive for growth at 200C, and SOS-subunit assembly was impaired at this temperature. The mutated sequences are highly conserved in the corresponding proteins from a number of species. The results indicate the participation of these proteins in the interaction of erythromycin with the ribosome.The interaction of antimicrobial agents with the bacterial ribosome has been a significant area of research for many years because of its relevance to infectious diseases (20, 39). Erythromycin and other macrolide antibiotics in particular have been widely studied for their effects on the functions of the ribosome during translation (12). These compounds bind strongly to the 50S ribosomal subunit of both gram-positive (12, 35) and gram-negative (46) cells and interfere with the elongation of the nascent peptide chain (9, 13,52 (55). Affinity labeling studies with erythromycin derivatives have identified a strong interaction of the drug with protein L22 and weaker associations with proteins L2, IA, and L15 (2). Each of these proteins has been shown to be essential for-reconstitution of the peptidyltransferase activity of the 50S subunit (24). Recent studies have identified the location of several of these proteins at a common region in the 5OS-subunit structure (11).The involvement of rRNA in erythromycin activity has been indicated by a number of recent reports identifying 23S rRNA mutations leading to erythromycin resistance in Escherichia coli (15,18,50) (26,59).Resistance to erythromycin can also result from changes in ribosomal proteins in E. coli (1,(42)(43)(44)(45)58) and in Bacillus species (48,49,56). Whereas the RNA sequence changes have * Corresponding author. Phone: (615) 461-7040. been specifically identified, no sequence information about the alterations in ribosomal proteins leading to erythromycin resistance is presently available. Phenotypic effects of alterations in ribosomal proteins L4 and L22 in two resistant mutants of E. coli were described many years ago (1, 58). We have recently acquired these mutants and have determined the specific DNA sequence c...

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.

The aminoglycosides paromomycin and neomycin were examined in Escherichia coli cells for an inhibitory effect on 30S ribosomal subunit assembly. Both compounds inhibited the growth rate, viable cell number, and protein synthesis rate with similar 50% inhibitory concentrations. Each drug also showed a concentrationdependent inhibition of 30S subunit formation. The inhibitory effect on 30S particle formation was approximately equivalent to the inhibitory effect on translation for these antibiotics.Paromomycin and neomycin are aminoglycoside antibiotics, which are effective against a number of aerobic and facultative anaerobic gram-positive bacilli and staphylococci (9, 11). Paromomycin and neomycin differ in chemical structure by the functional group attached to the CЈ 6 of ring 1. Paromomycin has a hydroxyl group at this position, while neomycin possesses an amino group (20).Both antibiotics bind specifically to the 30S ribosomal subunit (20). They interact with the 16S rRNA of the ribosome within an internal loop of the decoding site (12,16,18). Binding to this region results in a conformational change of the conserved bases within the loop of the A-site, which facilitates high-affinity binding between the rRNA of the internal loop and rings I and II of the aminoglycoside antibiotic (7,8,15). The tightly bound antibiotic contributes to codon misreading and mistranslation of mRNA.Previous work with a number of structurally different inhibitors of 50S subunit function has demonstrated that these antibiotics also inhibit 50S particle formation (reviewed by Champney [2]). These antibiotics halt 50S subunit assembly and cause accumulation of a precursor particle, which later becomes degraded by cellular ribonucleases (19). Inhibition of assembly is equivalent to inhibition of translation for most of these drugs (3-5). The present investigation examines the effects of paromomycin and neomycin on growing Escherichia coli cells. The results indicate that these aminoglycosides also have two inhibitory activities, preventing protein synthesis and 30S particle assembly in cells.Studies were conducted with E. coli strain SK901 (1). Cells were grown at 37°C in tryptic soy broth (TSB) in the presence and absence of each antibiotic as described previously. Growth rates, cell viability, and 35 S-labeled amino acid incorporation into proteins were determined as described previously (6). The previous methods for [ 3 H]uridine labeling of cells and sucrose gradient sedimentation of ribosomal subunits were used, except the centrifugation time was 5.25 h (6). The MIC of each antibiotic was measured in E. coli cells growing in TSB. The MIC of paromomycin was 10 g/ml, and that of neomycin was 15 g/ml. The compounds were used at subinhibitory concentrations to investigate their effects on cell growth and protein synthesis. Each antibiotic increased the growth rate and reduced the cell viability in a similar fashion. Figure 1A and B show the concentration-dependent inhibition of the growth rate and cell number by these drugs. Paromomyci...

The development of microbial resistance to practically all currently used antimicrobial agents has spurred efforts to develop new antibiotics and to identify novel targets in bacterial cells. This review summarizes the evidence for inhibition of bacterial ribosomal subunit formation as a target for many antibiotics distinct from their well-known inhibition of translation. Features of a model to explain this activity are explored. Results are presented to show the accumulation of both 30S and 50S ribosomal subunit precursors in antibiotic inhibited cells. These precursors have been characterized and are shown to bind radio-labeled drugs. Pulse and chase labeling studies have revealed the slower rates of subunit synthesis in drug treated cells compared with uninhibited controls. Resynthesis of subunits after antibiotic removal precedes recovery of control protein synthesis capacity, consistent with the model presented. Also certain mutant strains defective in different ribonuclease activities are more susceptible to antibiotic inhibition of assembly as predicted. Results indicating the equivalence of assembly inhibition and translational inhibition are described. Lastly, the identification of a 50S subunit precursor particle as a substrate for rRNA methyltransferase activity is shown. The weight of evidence presented clearly indicates that ribosomal antibiotics have a second target in cells. Inhibition of cell growth and subsequent cell death results from the activity of these antibiotics on the combined targets. The possibility of designing assembly specific inhibitors is discussed.

SummaryThe effects of erythromycin on the formation of ribosomal subunits were examined in wild-type Escherichia coli cells and in an RNase E mutant strain. Pulse±chase labelling kinetics revealed a reduced rate of 50S subunit formation in both strains compared with 30S synthesis, which was unaffected by the antibiotic. Growth of cells in the presence of [ 14 C]-erythromycin showed drug binding to 50S particles and to a 50S subunit precursor sedimenting at about 30S in sucrose gradients. Antibiotic binding to the precursor correlated with the decline in 50S formation in both strains. Erythromycin binding to the precursor showed the same 1:1 stoichiometry as binding to the 50S particle. Gel electrophoresis of rRNA from antibiotic-treated organisms revealed the presence of both 23S and 5S rRNAs in the 30S region of sucrose gradients. Hybridization with a 23S rRNAspecific probe confirmed the presence of this species of rRNA in the precursor. Eighteen 50S ribosomal proteins were associated with the precursor particle. A model is presented to account for erythromycin inhibition of 50S formation.