Ribosomes in thiostrepton-resistant mutants of Bacillus megaterium lacking a single 50 S subunit protein.

A protein required for the binding of thiostrepton to ribosomes of Bacillus megaterium has been purified and further characterized by immunological techniques. This protein, which does not bind the drug off the ribosome, is serologically-homologous to Escherichia coli ribosomal protein L11 and is designated BM-L11. Ribosomes from certain thiostrepton-resistant mutants of B. megaterium appear to be totally devoid of protein BM-L11 as judged by modified immunoelectrophoresis. Such ribosomes are significantly less sensitive than those from wild-type organisms to the action of thiostrepton in vitro but retain substantial protein synthetic activity. Re-addition of protein BM-L11 to ribosomes from the mutants restores them to wild-type levels of activity and thiostrepton sensitivity. Thus ribosomal protein BM-L11 is involved not only in binding thiostrepton but also in determining the thiostrepton phenotype.     

Thiostrepton-resistant mutants of Thermus thermophilus

Ribosomal protein L11 and its associated binding site on 23S rRNA together comprise one of the principle components that mediate interactions of translation factors with the ribosome. This site is also the target of the antibiotic thiostrepton, which has been proposed to act by preventing important structural transitions that occur in this region of the ribosome during protein synthesis. Here, we describe the isolation and characterization of spontaneous thiostrepton-resistant mutants of the extreme thermophile, Thermus thermophilus. All mutations were found at conserved positions in the flexible N-terminal domain of L11 or at conserved positions in the L11-binding site of 23S rRNA. A number of the mutant ribosomes were affected in in vitro EF-G-dependent GTP hydrolysis but all showed resistance to thiostrepton at levels ranging from high to moderate. Structure probing revealed that some of the mutations in L11 result in enhanced reactivity of adjacent rRNA bases to chemical probes, suggesting a more open conformation of this region. These data suggest that increased flexibility of the factor binding site results in resistance to thiostrepton by counteracting the conformation-stabilizing effect of the antibiotic.     

Molecular and functional analysis of the ribosomal L11 and S12 protein genes (rplK and rpsL) of Streptomyces coelicolor A3(2)

A RelC deletion mutant, KO-100, of Streptomyces coelicolor A3(2) has been isolated from a collection of spontaneous thiostrepton-resistant mutants. KO-100 grows as vigorously as the parent strain and possesses a 6-bp deletion within the rplK, previously termed relC. When the wild-type rplK gene was propagated on a low-copy-number vector in mutant KO-100, the ability to produce ppGpp, actinorhodin and undecylprodigiosin, which had been lost in the RelC mutant, was completely restored. Allele replacement by gene homogenotization demonstrated that the RelC mutation is responsible for the resistance to thiostrepton and the inactivation of ppGpp, actinorhodin and undecylprodigiosin production. Western blotting showed that ribosomes from the RelC mutant KO-100 contain only one-eighth the amount of L11 protein found in ribosomes of the parent strain. The impairment of antibiotic production in KO-100 could be rescued by the introduction of mutations that confer resistance to streptomycin (str), which result in alteration of Lys-88 in ribosomal protein S12 to Glu or Arg. No accompanying restoration of ppGpp synthesis was detected in these RelC str double mutants.     

Molecular and functional analysis of the ribosomal L11 and S12 protein genes ( rplK and rpsL ) of Streptomyces coelicolor A3(2)

A RelC deletion mutant, KO-100, of Streptomyces coelicolor A3(2) has been isolated from a collection of spontaneous thiostrepton-resistant mutants. KO-100 grows as vigorously as the parent strain and possesses a 6-bp deletion within the rplK, previously termed relC. When the wild-type rplK gene was propagated on a low-copy-number vector in mutant KO-100, the ability to produce ppGpp, actinorhodin and undecylprodigiosin, which had been lost in the RelC mutant, was completely restored. Allele replacement by gene homogenotization demonstrated that the RelC mutation is responsible for the resistance to thiostrepton and the inactivation of ppGpp, actinorhodin and undecylprodigiosin production. Western blotting showed that ribosomes from the RelC mutant KO-100 contain only one-eighth the amount of L11 protein found in ribosomes of the parent strain. The impairment of antibiotic production in KO-100 could be rescued by the introduction of mutations that confer resistance to streptomycin (str), which result in alteration of Lys-88 in ribosomal protein S12 to Glu or Arg. No accompanying restoration of ppGpp synthesis was detected in these RelC str double mutants. Received: 12 May 1997 / Accepted: 22 July 1997     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Analysis of proteinase A function in yeast

The antibiotic, micrococcin, binds to complexes formed between bacterial 23-S ribosomal RNA and ribosomal protein L11 and, in doing so, inhibits of thiostrepton. In assay systems simulating partial reaction of protein synthesis, micrococcin inhibits a number of processes believed to involve the ribosomal A site while stimulating GTP hydrolysis dependent upon ribosomes and elongation factor EF-G. The latter effect is not observed upon ribosomes lacking a protein homologous with protein L11. Nor is it apparent upon those containing 23-S RNA previously subjected to the action of a specific methylase known to render ribosomes resistant to thiostrepton. It is concluded that stimulation by micrococcin of factor-dependent GTP hydrolysis results from the binding of the drug to its normal target site which involves 23-S RNA and protein L11.     

Replacement of the L11 binding region within E.coli 23S ribosomal RNA with its homologue from yeast: in vivo and in vitro analysis of hybrid ribosomes altered in the GTPase centre.

Replacement of the protein L11 binding domain within Escherichia coli 23S ribosomal RNA (rRNA) by the equivalent region from yeast 26S rRNA appeared to have no effect on the growth rate of E.coli cells harbouring a plasmid carrying the mutated rrnB operon. The hybrid rRNA was correctly processed and assembled into ribosomes, which accumulated normally in polyribosomes. Of the total ribosomal population, < 25% contained wild-type, chromosomally encoded rRNA; the remainder were mutant. The hybrid ribosomes supported GTP hydrolysis dependent upon E.coli elongation factor G, although at a somewhat reduced rate compared with wild-type particles, and were sensitive to the antibiotic, thiostrepton, a potent inhibitor of ribosomal GTPase activity that binds to 23S rRNA within the L11 binding domain. That thiostrepton could indeed bind to the mutant ribosomes, although at a reduced level relative to that seen with wild-type ribosomes, was confirmed in a non-equilibrium assay. The rationale for the ability of the hybrid ribosomes to bind the antibiotic, given that yeast ribosomes do not, was provided when yeast rRNA was shown by equilibrium dialysis to bind thiostrepton only 10-fold less tightly than did E.coli rRNA. The extreme conservation of secondary, but not primary, structure in this region between E.coli and yeast rRNAs allows the hybrid ribosomes to function competently in protein synthesis and also preserves the interaction with thiostrepton.     

The antibiotic micrococcin acts on protein L11 at the ribosomal GTPase centre

Micrococcin-resistant mutants of Bacillus megaterium that carry mutations affecting ribosomal protein L11 have been characterised. The mutants fall into two groups. "L11-minus" strains containing an L11 gene with deletions, insertions or nonsense mutations which grow 2.5-fold slower than the wild-type strain, whereas other mutants carrying single-site substitutions within an 11 amino acid residue segment of the N-terminal domain of L11 grow normally. Protein L11 binds to 23 S rRNA within the ribosomal GTPase centre which regulates GTP hydrolysis on ribosomal factors. Micrococcin binding within the rRNA component of this centre was probed on wild-type and mutant ribosomes, in vivo, using dimethyl sulphate where it generated an rRNA footprint indistinguishable from that produced in vitro, even after the cell growth had been arrested by treatment with either kirromycin or fusidic acid. No drug-rRNA binding was detected in vivo for the L11-minus mutants, while reduced binding (approximately 30-fold) was observed for two single-site mutants P23L and P26L. For the latter, the reduced drug affinity alone did not account for the resistance-phenotype because rapid cell growth occurred even at drug concentrations that would saturate the ribosomes. Micrococcin was also bound to complexes containing an rRNA fragment and wild-type or mutant L11, expressed as fusion proteins, and they were probed with proteinases. The drug produced strong protection effects on the wild-type protein and weak effects on the P23L and P26L mutant proteins. We infer that inhibition of cell growth by micrococcin, as for thiostrepton, results from the imposition of a conformational constraint on protein L11 which, in turn, perturbs the function(s) of the ribosomal factor-guanosine nucleotide complexes.     

Importance of the 5 S rRNA-binding ribosomal proteins for cell viability and translation in Escherichia coli

A specific complex of 5 S rRNA and several ribosomal proteins is an integral part of ribosomes in all living organisms. Here we studied the importance of Escherichia coli genes rplE, rplR and rplY, encoding 5 S rRNA-binding ribosomal proteins L5, L18 and L25, respectively, for cell growth, viability and translation. Using recombineering to create gene replacements in the E. coli chromosome, it was shown that rplE and rplR are essential for cell viability, whereas cells deleted for rplY are viable, but grow noticeably slower than the parental strain. The slow growth of these L25-defective cells can be stimulated by a plasmid expressing the rplY gene and also by a plasmid bearing the gene for homologous to L25 general stress protein CTC from Bacillus subtilis. The rplY mutant ribosomes are physically normal and contain all ribosomal proteins except L25. The ribosomes from L25-defective and parental cells translate in vitro at the same rate either poly(U) or natural mRNA. The difference observed was that the mutant ribosomes synthesized less natural polypeptide, compared to wild-type ribosomes both in vivo and in vitro. We speculate that the defect is at the ribosome recycling step.     

Interaction of thiostrepton and elongation factor-G with the ribosomal protein L11-binding domain

Ribosomal protein L11 and the L11 binding region of ribosomal RNA constitute an important domain involved in active functions of the ribosome during translation. We studied the effects of L11 knock-out and truncation mutations on the structure of the rRNA in this region and on its interactions with a translation elongation factor and the antibiotic thiostrepton. The results indicated that the structure of the L11-binding rRNA becomes conformationally flexible when ribosomes lack the entire L11 protein, but not when the C-terminal domain is present on ribosomes. Probing wild type and mutant ribosomes in the presence of the antibiotic thiostrepton and elongation factor-G (EF-G) rigorously localized the binding cleft of thiostrepton and suggested a role for the rRNA in the L11-binding domain in modulating factor binding. Our results also provide evidence that the structure of the rRNA stabilized by the C-terminal domain of L11 is necessary to stabilize EF-G binding in the post-translocation state, and thiostrepton may modulate this structure in a manner that interferes with the ribosome-EF-G interaction. The implications for recent models of thiostrepton activity and factor interactions are discussed.     

On the biological role of ribosomal protein BM-L11 of Bacillus megaterium, homologous with Escherichia coli ribosomal protein L11.

Ribosomes from a thiostrepton-resistant mutant of Bacillus megaterium lack a protein, BM-L11, which is homologous with Escherichia coli ribosomal protein L11. Such ribosomes retain partial activity in cell-free synthesis of polyphenylalanine and can be restored to full activity by reconstitution with protein BM-L11. Examination of individual steps involved in polypeptide chain elongation suggested a role for protein BM-L11, and by inference for E. coli protein L11, in promoting the ribosomal GTP hydrolysis dependent upon elongation factor EF G. Evidently, however, protein BM-L11 is not indispensable for ribosomal function.     

A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11

We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C. reinhardtii. Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase. Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time. L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit. Thiostrepton-resistant mutants of Bacillus megaterium and B. subtilis lack L11, show reduced ribosome activity, and have slow growth rates. Similarities between the thiostreptonresistant mutants of bacteria and C. reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E. coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.     

A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11

We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C. reinhardtii. Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase. Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time. L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit. Thiostrepton-resistant mutants of Bacillus megaterium and B. subtilis lack L11, show reduced ribosome activity, and have slow growth rates. Similarities between the thiostreptonresistant mutants of bacteria and C. reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E. coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.     

Requirement for ribosomal protein BM-L11 in stringent control of RNA synthesis in Bacillus megaterium.

A spontaneously occurring thiostrepton-resistant mutant of Bacillus megaterium has been shown to yield ribosomes lacking protein BM-L11, a protein immunologically related to Escherichia coli ribosomal protein L11. Here we have demonstrated that the mutant strain has acquired the relaxed phenotype and is unable to synthesise guanosine tetraphosphate and pentaphosphate in vivo. Ribosomes from the mutant strain are unable to support the synthesis of these two compounds in vitro, but this deficiency can be overcome by re-addition of purified protein BM-L11 to the ribosomes. Thus protein BM-L11 appears to be indispensable for the synthesis of guanosine tetraphosphate and pentaphosphate; the implications of this observation are discussed.     

Mutants of Escherichia coli lacking ribosomal protein L11

Three mutants with ribosomes apparently lacking Protein L11, AM68, AM76, and AM77, were investigated using a variety of immunological techniques to determine whether L11 was indeed lacking. Ouchterlony double diffusion, modified immunoelectrophoresis, and dimer formation on sucrose gradients all gave results indicating Protein L11 was missing from the ribosome in these mutants. Electron micrographs of ribosomes of the mutants were indistinguishable from those of wild type. Ribosomes of AM68, AM76, and AM77, did not bind the antibiotic thiostrepton, but binding was recovered upon reconstitution with wild type Protein L11.     

Induction of the chloramphenicol acetyltransferase gene cat-86 through the action of the ribosomal antibiotic amicetin: involvement of a Bacillus subtilis ribosomal component in cat induction.

The plasmid gene cat-86 and the cat gene resident on pC194 each encode chloramphenicol-inducible chloramphenicol acetyltransferase activity in Bacillus subtilis. Chloramphenicol induction has been proposed to result from chloramphenicol binding to ribosomes, which then permits the drug-modified ribosomes to perform events essential to induction. If this proposal were correct, B. subtilis mutants containing chloramphenicol-insensitive ribosomes should not permit chloramphenicol induction of either cat-86 or pC194 cat. However, we and others have been unable to isolate chloramphenicol-resistant ribosomal mutants of B. subtilis 168. We therefore developed a simple procedure for screening other antibiotics for the potential to induce cat-86 expression. One antibiotic, amicetin, was found to be an effective inducer of cat-86 but not of the cat gene on pC194. Amicetin and chloramphenicol each interact with the 50S ribosomal subunit, and the mechanism of cat-86 induction by both drugs may be similar. Amicetin-resistant mutants of B. subtilis were readily isolated, and in none of six mutants tested was cat-86 detectably inducible by amicetin, although the chloramphenicol-inducible phenotype was retained. The ami-1 mutation which is present in one of these amicetin-resistant mutants was mapped by PBS1 transduction to the "ribosomal gene cluster" adjacent to cysA. Additionally, ribosomes from cells harboring the ami-1 mutation contained an altered BL12a protein, as detected in two-dimensional polyacrylamide gel electrophoresis. Lastly, an in vitro protein-synthesizing system that uses ribosomes from an ami-1-containing cell line was more resistant to amicetin than a system that uses ribosomes from an amicetin-sensitive but otherwise isogenic strain. These results indicate that the host mutation, ami-1, which effectively abolished the inducibility of cat-86 by amicetin, altered a ribosomal component.     

In vivo assembling of bacterial ribosomal protein L11 into yeast ribosomes makes the particles sensitive to the prokaryotic specific antibiotic thiostrepton

Eukaryotic ribosomal stalk protein L12 and its bacterial orthologue L11 play a central role on ribosomal conformational changes during translocation. Deletion of the two genes encoding L12 in Saccharomyces cerevisiae resulted in a very slow-growth phenotype. Gene RPL12B, but not the RPL12A, cloned in centromeric plasmids fully restored control protein level and the growth rate when expressed in a L12-deprived strain. The same strain has been transformed to express Escherichia coli protein EcL11 under the control of yeast RPL12B promoter. The bacterial protein has been found in similar amounts in washed ribosomes from the transformed yeast strain and from control E. coli cells, however, EcL11 was unable to restore the defective acidic protein stalk composition caused by the absence of ScL12 in the yeast ribosome. Protein EcL11 induced a 10% increase in L12-defective cell growth rate, although the in vitro polymerizing capacity of the EcL11-containing ribosomes is restored in a higher proportion, and, moreover, the particles became partially sensitive to the prokaryotic specific antibiotic thiostrepton. Molecular dynamic simulations using modelled complexes support the correct assembly of bacterial L11 into the yeast ribosome and confirm its direct implication of its CTD in the binding of thiostrepton to ribosomes.     

Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts.

Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.     

Tiamulin resistance mutations in Escherichia coli.

Forty "two-step" and 13 "three-step" tiamulin-resistant mutants of Escherichia coli PR11 were isolated and tested for alteration of ribosomal proteins. Mutants with altered ribosomal proteins S10, S19, L3, and L4 were detected. The S19, L3, and L4 mutants were studied in detail. The L3 and L4 mutations did not segregate from the resistance character in transductional crosses and therefore seem to be responsible for the resistance. Extracts of these mutants also exhibited an increased in vitro resistance to tiamulin in the polyuridylic acid and phage R17 RNA-dependent polypeptide synthesis systems, and it was demonstrated that this was a property of the 50S subunit. In the case of the S19 mutant, genetic analysis showed segregation between resistance and the S19 alteration and therefore indicated that mutation of a protein other than S19 was responsible for the resistance phenotype. The isolated ribosomes of the S19, L3, and L4 mutants bound radioactive tiamulin with a considerably reduced strength when compared with those of wild-type cells. The association constants were lower by factors ranging from approximately 20 to 200. When heated in the presence of ammonium chloride, these ribosomes partially regained their avidity for tiamulin.     

Loss of ribosomal protein L11 blocks stress activation of the Bacillus subtilis transcription factor sigma(B)

sigma(B), the general stress response sigma factor of Bacillus subtilis, is activated when the cell's energy levels decline or the bacterium is exposed to environmental stress (e.g., heat shock, ethanol). Physical stress activates sigma(B) through a collection of regulatory kinases and phosphatases (the Rsb proteins) which catalyze the release of sigma(B) from an anti-sigma(B) factor inhibitor. The means by which diverse stresses communicate with the Rsb proteins is unknown; however, a role for the ribosome in this process was suggested when several of the upstream members of the sigma(B) stress activation cascade (RsbR, -S, and -T) were found to cofractionate with ribosomes in crude B. subtilis extracts. We now present evidence for the involvement of a ribosome-mediated process in the stress activation of sigma(B). B. subtilis strains resistant to the antibiotic thiostrepton, due to the loss of ribosomal protein L11 (RplK), were found to be blocked in the stress activation of sigma(B). Neither the energy-responsive activation of sigma(B) nor stress-dependent chaperone gene induction (a sigma(B)-independent stress response) was inhibited by the loss of L11. The Rsb proteins required for stress activation of sigma(B) are shown to be active in the RplK(-) strain but fail to be triggered by stress. The data demonstrate that the B. subtilis ribosomes provide an essential input for the stress activation of sigma(B) and suggest that the ribosomes may themselves be the sensors for stress in this system.